Heneicosane An Oviposition-attractant Pheromone Of Larval Origin In

HYPOTHESIS 6. Berezinsky, V. S., Nuc. Phys. B (Proc. Suppl.), 1993, 31, 413–427. 7. Watson, A., Science, 1997, 275, 1736– 1738. 8. Kanipe, J., New Sci., 1997, 14. 9. Vermeij, G. J. and Dorritie, D., Science, 1996, 274, 1550. 10. Isozaki, Y., Science, 1997, 276, 235– 238. 11. Isozaki, Y., Science, 1997, 277, 1745. 12. Retallack, G. J. and Holser, W. T.,

Science, 1997, 277, 1745. 13. Zioutas, K., Phys. Lett. B, 1990, 242, 257–264. 14. Collar, J. I, Phys. Lett. B, 1996, 368, 266–269. 15. Smith, D. G. (ed.), Cambridge Encyclopedia of Earth Sciences, Cambridge University Press, Cambridge, 1989, p. 345. 16. Kivelson, M. G., Nature, 1996, 384, 537–541; Robinson, M. S. and Lucey,

P. G., Science, 1997, 275, 197–200. 17. Petuch, E. J., Science, 1995, 270, 275– 277.

Samar Abbas and Shukadev Mohanty are in the Physics Department, Utkal University, Bhubaneshwar 751 004, India; Afsar Abbas* is in the Institute of Physics, Bhubaneswar 751 005, India. *For correspondence (e-mail: [email protected])

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Lightning return stroke electric field escaping out and giving rise to optical and associated emissions In connection with lightning, two new terminologies, namely ‘Sprites1’ and ‘Elves2’ have appeared in many of the papers on the topic. It did attract the attention of readers but the use of the nomenclatures for optical phenomena arising due to the upward propagation of lightning generated electric field continues to be a riddle for workers in this field of research. These phenomena are not at all new and are occasional manifestations of lightning that has been known for over a century3. Scanning through the scientific literature in this area, one finds scattered anecdotal descriptions of lightning popularly known as ‘blue’ or ‘green’ pillars and rocket discharge-like columns of optical emissions4–7 which continueto be an occasionalfeature.Wilson8 discussed the possibility of lightning discharges propagating upwards from the cloud-top and undergoing occasional multiple reflections between the cloud and the ionosphere. Vaughan et al.9 and Bell et al.10 have reported television observations from the space shuttle and showed that a large number of upwarddirected cloud discharges, either ‘red’ or ‘blue’, are observed at altitudes around 60 km and above. Using a low light level All Sky Television System (ASTS), Sentman and Wescott11 recorded a large number of upward-directed optical emission phenomena during NASA’s single airborne DC-flight over thunderstorms in Iowa, Nebraska and Kansas. Initially, they estimated the most probable terminal heights of the events to be 60 km with error bars extending up to 100 km. The duration of this optical phenomenon was found to be 16 m sec or less, and brightness was estimated to be 25–50 kR, 1292

which is almost the same as that of bright aurora. The occurrence rate of this optical phenomenon was found to vary from time to time and region to region, and was estimated to occur once for every 200–300 cloud-to-ground strokes12. In addition, the ‘Red Sprites’ and the ‘Blue Jets’ were also videorecorded13 and their properties studied. Further refinements in the ongoing measurements are being made and the results are being reported. Continued observations have shown that high altitude luminous phenomena do take place and are thought to be arising due to the escaping part of the cloud-toionosphere lightning discharges6,14. The observed phenomenon is simple enough and occasionally it is seen to be generated and extending upwards and, at times, undergoing multiple reflections between the cloud and the ionosphere. A state of nonjudgment prevails when it is observed by aeroplane flights11. The first definite observation with supporting details has been made by using the aeroplane ‘Sprite-94’, although the detailed mechanism of the optical features extending from cloud-totroposphere and ionosphere was not known. The phenomenon of escaping out of the ground-to-cloud generated electric field and the generation of optical emission into the upper atmosphere is confined to the cloud-to-ionosphere12,15 region and is known as ‘Sprites’. Recorded features have shown that many of these events are spatially varying and one event is different from the other. It has been further shown that the ‘Sprites’ appearing at different altitudes15 are ‘Red Sprites’ and ‘Blue Jets’. The escaping out of the

positive cloud-to-ground leader stroke and the geometry of the return stroke is schematically illustrated in Figure 1. The most prevalent lightning phenomenon is the cloud-to-ground discharge in which the negative charge is lowered giving rise to positive return stroke. However, ‘Sprites’ are associated with less frequent phenomena of positive cloud-to-ground discharges. The upward-directed electric field is known to generate this optical emission around 60 km. Depending on the cloud features and details of the cloud-to-ground discharges, the return stroke is not terminated by the cloud-top and the electric field generated in the upper atmosphere is allowed to extend upwards towards the ionosphere. There are various possibilities which depend mainly on relative orientation of the return stroke; (i) strokegenerated electric field may accelerate

Figure 1. Schematic diagram of escaping out cloud-to-ground lightning discharges.

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SCIENTIFIC CORRESPONDENCE charged particles that undergo multiple reflections between the cloud and the ionosphere; (ii) the electric field originating from the cloud-top and the extended return stroke may propagate through the cloud to the ionosphere. Whenever the electric field is large, the field-aligned plasma breaks down, giving rise to optical emissions. If the electric field is further increased, the transient charged particle accelerations may give rise to X-ray and even gamma-ray emissions. Some of these features can be clearly seen from the high altitude records of atmospherics, which are not routinely recorded. The statistical details of the ‘Sprites’ and fractured features of luminous traces known as ‘Elves’ are governed by detailed features of causative lightning and the physical features of the ionized layer above. These details have not been understood as yet. It is quite likely that nonlinear plasma processes may be playing an important role. These upward propagating lightning are recorded by aircraft missions since these are not very common features. The ‘Sprites’ and ‘Elves’ brightness on ground are measured by a chain of photometers16 known as ‘Fly’s Eye’ with weak intensity measuring specification; time resolution of 30 µsec and field of view of 3.5° by 7°. The observed features of upward propagating lightning has drawn the attention of many people. However we find that this phenomenon has not yet fully developed so as to sustain itself scientifically1,2. The upward-directed lightning generated electric field forming ‘Sprites’ and ‘Elves’ freely propagates a much longer path in the lower ionosphere, and under suitable conditions interacts with the upper ionosphere giving rise to these optical effects. The heated optical channels carry a large current and seem to become unstable as a result of modulational instability. The fracturing features of these optical traces into smaller parts are known to result in short duration features known as ‘Elves’. These phenomena can be better investigated in terms of

theoretical models and can be compared with experimentally measured details only if results of such measurements are systemically collected over a long period of time. Although most of the reportings are made by a single major group of workers, one finds inconsistent and varying statements made, at times, about the observed optical phenomena. Without ascertaining the detailed nature of the optical phenomenon, it seems unjustified to name the phenomenon after the aeroplane used for the observing and recording mission. It is not well explained how the terminology ‘Elves’ came into being. This trend is still continuing and the optical traces are being named ‘carrot’, ‘radish’, etc. If this trend continues, we will have many interesting nomenclatures. The phenomenon is known to cover X-rays and gamma-rays. Suggestions and arguments are made that similar phenomena could also be generated during cloud-to-cloud discharges. When some of these suggestions take concrete shape, we will have a better picture. No doubt, the groups have freely and extensively used these terminologies and induced others to use these in their papers. However, it is clearly seen that an in-depth analysis of this phenomenon is still lacking due to absence of precise measurements with desired spatial and temporal resolutions17. The origin of the terminology ‘Elves’ is not adequately explained and seems to be arising from occasional fracturing of Sprites into smaller and smaller traces. If this be true, the notation ‘Elves’ which denotes the plural of ‘ELF’ is hardly justifiable. Since these details have not been given in the reported papers of the group, the reader’s curiosity remains unquenched. It is hoped that the associated and interested research groups would make qualitative and extensive measurements of this phenomenon and leave the terminologies to be decided by the scientific community. ACKNOWLEDGEMENTS.

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This work

was carried out by the author during his tenure as ‘Emeritus Fellow’ of AICTE, New Delhi, operating at C.S.J.M. University, Kanpur. 1. Reising, S. C., Inan, U. S. and Bell, T. F., Geophys. Res. Lett., 1999, 26, 987. 2. Barrington-Leigh, C. P. and Inan, U. S., Geophys. Res. Lett., 1999, 26, 683. 3. Everett, W. H., Nature, 1903, 68, 599. 4. Boys, C. V., Nature, 1926, 118, 749. 5. Malan, D., Acad. Sci., Paris, 1937. 6. Ashmore, S. E., Weather, 1950, 5, 331. 7. Wright, J. B., Weather, 1951, 6, 230. 8. Wilson, C. T. R., Proc. R. Meteorol. Soc. London, 1956, 236, 32D. 9. Vaughan, O. H. Jr., Blakeslee, R., Boeck, W. L., Vonnegut, V., Brook, M. and McKune, Jr., Mon. Weather Rev., 1992, 120, 1459. 10. Bell, T. F., Reising, S. C. and Inan, U. S., Geophys. Res. Lett., 1998, 25, 1285. 11. Sentman, D. D. and Wescott, E. M., Geophys. Res. Lett., 1993, 20, 2857. 12. Sentman, D. D., Wescott, E. M., Osborne, D. L., Hampton, D. L. and Heavner, M. J., Geophys. Res. Lett., 1995, 22, 1205. 13. Sentman, D. D. and Wescott, E. M., Red Sprites an Blue Jets, Geophysical Institute Video Production, University of Alaska, Fairbanks, 9 July, 1994. 14. Wood, C. A., Weather, 1951, 6, 64. 15. Wescott, E. M., Sentman, D., Osborne, D. L., Hampton, D. L. and Heavner, M., Geophys. Res. Lett., 1995, 22, 1209. 16. Cummer, S. A., Inan, U. S., Bell, T. F. and Barrington-Leigh, C. P., Geophys. Res. Lett., 1998, 25, 1281. 17. Pasko, V. P., Inan, U. S. and Bell, T. F., Geophys. Res. Lett., 1999, 26, 1247. Received 10 October 1999; revised accepted 28 February 2000

R. N. SINGH House No. 425, IIT Kanpur, Kanpur 208 016, India (e-mail: [email protected])

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Application of atomic force microscopy in seed surface studies Atomic force microscopy (AFM) is a valuable tool for studying physical and biological structures. Since the initial reports of Binnig et al.1, considerable progress has been made, but applications in the field of biology are scanty. Bustamente and Keller2 reviewed the possibilities of exploring biological structures under conditions in which living organisms exist. AFM is a kind of scanning probe microscope where imaging of the sample is realized by interaction of the probe with the sample surface and no imaging beam (light or electron) is involved in the process. The tip of the probe is mounted on the end of a flexible cantilever. ‘As the sample is scanned beneath the tip, small forces of interaction with the sample cause the cantilever to deflect, revealing the sample’s topography. The most common approach, called an optical lever approach, is to reflect a laser beam off the backside of the cantilever into a 4-segmented photodetector’2. The photodetector generates a measurable signal voltage to make the image. Internet search reveals that no plant materials (including seeds) have so far been studied with the help of this new tool. We therefore attempted AFM imaging of mung bean seeds, a valuable commodity in the food market of India. Seeds (mung bean) were collected from various local geographical sites with and without the seed coats in connection with a project by these authors. We approached with the rationale that of the three modes of AFM operation, contact, non-contact and tapping, the first would be most suitable for the hard surface. As this mode is also likely to offer the best resolution, we tried to scan the surface of the seed coat and the cotyledon surface of different strains of mung bean. (In this microscopy no sample preparation is necessary, the image of the ‘native’ seed is captured.) The cantilever was of the 100 µm wide-legged, triangular type in which an optical lever reflects a laser beam off the backside of the cantilever. The forces selected in this mode of imaging are the capillary force and atomic repulsion force between the tip and the sample surface. The microscope (Digital Instruments) was a standard top view contact AFM, the Nano Scope ESPM system. The image was 1294

simultaneously captured and displayed from the native seed surface at 512 × 512 resolution. The image acquired was stored in the system computer hard disk. Figure 1 a and b shows the cotyledon surface of two strains of mung bean (from Malda and Lalgola in West Bengal, respectively) at the same magnification (scan size). The differences in the strains reflect the different genomic imprint at this ultrastructural level. SEM micrographs even at

Figure 1.

the highest magnification do not reveal some of these features. Figure 2, the highest magnification that we obtained, shows 5–6 structural units (of the seed coat) within 20 Å (2 nm). Thus each unit size is ~ 3–4 Å. A large number of mung bean varieties are found in India and a few of them are endowed with unusually good aroma. A project on the aroma molecules of mung bean by Brahmachary and Ghosh (unpub-

Cotyledon surface: a, Malda strain; b, Lalgola strain. CURRENT SCIENCE, VOL. 78, NO. 11, 10 JUNE 2000

SCIENTIFIC CORRESPONDENCE ACKNOWLEDGEMENTS. We thank the Director, IUC, Indore and Dr A. Dasgupta, Biophysics Department, Kalyani University for their kind co-operation. Received 22 December 1999; revised accepted 4 April 2000

T. GUHA †,* R. BHAR ‡ V. GANESAN # A. SEN † R. L. BRAHMACHARY ** USIC, Electron Microscope Centre, University College of Science, University of Calcutta, 92, Acharya Prafulla Chandra Road, Calcutta 700 009, India ‡ USIC, Jadavpur University, Calcutta 700 032, India # Inter University Consortium, University of Indore, Indore 452 001, India **21B, Moti Jheel, Calcutta 700 074, India *For correspondence (e-mail: [email protected]) †

Figure 2.

Seed coat surface, Lalgola strain.

lished) reports at least 6 different molecules. Together with the chemical studies an ultrastructural atlas of various strains could be prepared.

1. Binnig, G., Quate, C. F. and Gerber, C., Phys. Rev. Lett., 1986, 56, 930–933. 2. Bustamente, C. and Keller, D., Phys. Today, 1995, 48 , 32–38.

Heneicosane: An oviposition-attractant pheromone of larval origin in Aedes aegypti mosquito Oviposition aggregation pheromone can specifically influence many insect females to lay eggs in the same site resulting in more eggs deposition. The first unequivocal evidence for an oviposition pheromone occurrence in an insect vector mosquito was in Culex1. However, studies on the influence of eggs of conspecific and heterospecific larval stages on the site selection by various Aedes species have given conflicting results2,3. Surprisingly, in Anopheles mosquito the presence of conspecifics may actually be a deterrent4. Aedes aegypti prefers to oviposit on water containing the larvae of the same species5. This larval conditioned water (LCW) is found to be effective after removing the larvae by filtration and the attractant activity is retained for several weeks. Many groups earlier tried to iden-

tify the oviposition-attractant factors present in the LCW, but the extremely small amount released by the larvae thwarted its characterization3,6. Here we report the chemical primarily responsible for the oviposition activity of the LCW using gas chromatography coupled with mass spectrometry (GC/MS) followed by biological evaluation in the laboratory.

Table 1. Peak retention time (min)

For these studies, water used for rearing A. aegypti larvae only for twenty days continuously was taken after filtration as the LCW. We extracted this LCW with hexane and ether (HPLC grade) sequentially, combined the extracts, concentrated and analysed by GC/MS. Similarly control water was extracted for comparison (blank). GC/MS analyses were per-

Fragmentation pattern of the additional components in LCW Compounds identified

MW

17.25 17.63

Octadecane Isopropyl myristate

254 270

20.20 21.10 26.82

Heneicosane Docosane Nonacosane

296 310 408

CURRENT SCIENCE, VOL. 78, NO. 11, 10 JUNE 2000

Fragmentations 254 270 60 296 310 408

+

(M ), 57 (100), 71, 85, 99 (M+), 43 (100), 228, 102, (M+), 57 (100), 71, 85, 99 (M+), 57 (100), 71, 85, 99 (M+), 57 (100), 71, 85, 99 1295

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Extracts of LCW Retention time 20.20 min

Standard Heneicosane Retention time 20.20 min

Figure 1. Comparison of the mass spectra of heneicosane (standard) and one of the additional components present in the solvent extract of LCW of Aedes aegypti mosquito larvae.

formed on a HP 6890 gas chromatograph coupled to a 5973 quadrupole mass spectrometer using capillary column coated with 5% phenyl polydimethyl siloxane stationary phase (HP-5). The column temperature was programmed as follows: 50°C, 2 min, isothermal, then 10°C/min to 280°C ramp., 5 min isothermal (total 30 min run). Comparison between total ion chromatogram of the LCW and the control revealed that the LCW extract contained additional five peaks which could be of larval origin. The compounds were identified as heneicosane, docosane, nonacosane, octadecane and isopropyl myristate on the basis of comparison of their retention times and fragmentation patterns with the commercially available authentic samples (Table 1). The mass spectrum of heneicosane from the LCW and the authentic sample are shown in Figure 1 as a representative example. A GC flame ionization detector however could not identify these tiny amounts. In order to confirm the above identified compounds to be of larval origin, GC/MS analysis of the hexane extract of the cuticular components from the A. aegypti larvae was performed under identical conditions using selected ion monitoring procedure corresponding to the m/z values 254, 270, 296, 310 and 408. The retention times and mass spectra of the peaks were in agreement with the compounds obtained from the LCW extract as well as with the authentic samples. From 1296

this it is evident that the origin of heneicosane and other identified components must be from the larvae of A. aegypti in the LCW. We evaluated in the laboratory the oviposition attractancy essentially based on the method described by Allan and Kline3 with slight modification of the above five commercially available chemicals either individually at various concentrations or as a mixture. We varied the concentration of heneicosane (6.9, 69, 690; 50, 69, 90 ppm) in these experiments. This showed that heneicosane is the most promising oviposition attractant for A. aegypti females. The eggs laid per replicate (mean ± SE, n = 50) were 67.83 ± 10.79 in control water whereas eggs laid per replicate were 121.00 ± 16.26 (P < 0.01) in heneicosane-treated water at 69 ppm. Further comparison of egg laying was made with tap water, yeast water and heneicosane-treated water. While there was no statistical difference between the first two, there was 2-fold increase in heneicosanetreated water in comparison to the other two controls. However the eggs laid per replicate were comparatively less than in the LCW (eggs per replicate 179.00 ± 15.55, mean ± SE, n = 30) suggesting the possibility of the presence of some minor undetectable components. Oviposition attractants may have several benefits for the insect species. However, the oviposition behaviour of vectors7 is not yet fully studied. The oviposition-

attractant pheromone identified can be combined with novel vector control strategies (Shri Prakash et al., unpublished). 1. Osgood, C. E., J. Econ. Entomol., 1971, 64, 1038–1041. 2. Kalpage, K. S. P. and Brust, R. A., Environ. Entomol., 1973, 265, 729–730. 3. Allan, S. A. and Kline, D. L., J. Med. Entomol., 1998, 35, 943–947. 4. McCrae, A. W. R., Ann. Trop. Med. Parasitol., 1984, 78, 307–318. 5. Bentley, M. D. and Day, J. F., Annu. Rev. Entomol., 1989, 34, 401–421. 6. Soman, R. S. and Reuben, R., J. Med. Entomol., 1970, 7, 485–489. 7. McCall, P. J., Parasitol. Today, 1995, 11, 352–355. Received 4 February 2000; revised accepted 6 April 2000

M. J. M ENDKI K. GANESAN SHRI PRAKASH * M. V. S. SURYANARAYANA R. C. MALHOTRA K. M. RAO R. VAIDYANATHASWAMY Defence Research & Development Establishment, Jhansi Road, Gwalior 474 002, India *For correspondence (e-mail: [email protected])

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