Ann. appl. Biol. (1977)~ 85, 309-311 With I plate Printed in Great Brituin

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A technique for the location of planthopper feeding probes using labial imprints

BY HENRY WATERS Coconut Industry Board, P.O. Box 204, Kingston 10, Jamaica

(Accepted 17 September 1976)

S U M M A R Y

Labial imprints mark precisely a planthopper’s feeding probe. By staining a leaf surface with I % aniline blue in lactophenol, the labial imprints may be detected. This technique provides a simple method for the location of stylet tracks without having to kill feeding insects in situ. Using the leaf surface staining technique, the labial imprints of Haplaxius mudus van Duzee (Homoptera; Cixiidae), a possible vector of lethal yellowing of coconuts, were shown to be 70-80pm in diameter with a raised central crown. Such imprints are the mirror image of the distal labial surface.

During a study of possible planthopper vectors of lethal yellowing of coconuts, it was necessary to determine from leaf sections which tissues these insects most frequently probed. In other studies of homopteran feeding, sites have been located by killing insects in situ using toxic vapours or carbon dioxide anaesthesia (Botha, Evert & Walmsley, 1975), hot or cold fixatives (Wiegert, 1963), hot wax droplets (Smith, 1926) or even by crushing whilst feeding (Pollard, 1968). Planthoppers are readily disturbed while feeding and none of these methods proved suitable for preserving the exact location of the feeding site for subsequent sectioning. However, following preliminary observations of feeding planthoppers, a technique was developed using labial imprints, which gave the precise location of feeding probes without killing the insects in situ or requiring an unnecessary amount of sectioning.

The results reported here refer to Haplaxills crudus van Duzee (Homoptera; Cixiidae) feeding on pinnae of COCOS nucfera L. Experimental feeding was carried out either in the field using muslin bags over pinnae or in the laboratory using insects confined in test-tubes on pieces of coconut pinnae. After the insect left the feeding site, a droplet remained at the point where the tip of the rostrum had been applied to the leaf surface (Plate, fig. I). These droplets could be more readily seen through a dissecting microscope using incident illumination after staining the leaf surface. Suitable stains are I yo (w/v) aniline blue in lactophenol or 2 yo (w/v) aqueous acid fuschin or safranine, prepared according to Johansen (1940), staining for 3-60 s, followed by a gentle rinse with distilled water. Aniline blue stained most strongly and this was used for the present work on coconut pinnae.

The droplets stained by this simple technique were 70-Sopm in diameter with a more intensely stained raised central region 18-30 pm in diameter and an irregularly darkly stained margin (Plate, fig. 2). The pattern of the rostra1 apex was mirrored in

3 I* Short communication the stained droplets (cf. Plate, figs 2,3). The anterior sensoria and often the impressions of the sensory papillae could be distinguished (Plate, fig. 2). A more intensely stained, anteriorly-directed line from the central region to the droplet margin represented the labial cleft. The apical 5-25 pm of the stylet canal of the rostrum was reproduced as the darkly stained central region. Within this cast of the stylet canal, which was particularly obvious in sectioned material, the path taken by the stylets could usually be observed (Plate, fig. 4). Similar droplets after feeding by aphids have been described and have been referred to in a review by Pollard (1973) as labial imprints; this term is also used here for those formed by planthoppers.

The labial imprints, being an integral part of the stylet track, marks a feeding site precisely (Plate, fig. 4). It is reasonable to assume that the labial imprint consists of the Same salivary material as the stylet track itself, because of its obvious continuity and because it stains in the same way as the stylet sheath in sectioned material (Plate, fig.

The details of tbe rostral apex retained within the labial imprint reflect the stability of the gelled salivary material produced by feeding leafhoppers (Storey, 1939). Studies of the timing of imprint formation were beyond the scope of this work; but unless there is a backflow of saliva from the stylet sheath during feeding (and this Seems unlikely because of the more or less instantaneous gelling of saliva on exudation (Storey, 1939)), the imprint must be formed at the time of penetration. Whether the imprint serves any function or is formed passively during feeding, is not known. A search of the literature has not revealed a report of labial imprints made by any Homoptera other than aphids and although the labial imprints of aphids have been noted in sectioned material (Pollard, I973), their structure in surface view has not been described.

The labial imprints of planthoppers and aphids probably differ because of the marked differences between the flattened rostral apices of planthoppers (Plate, fig. 5) and the pointed proboscis apices of aphids (Adams & Fyfe, 1970). The method described by Hagley & Blackman (1966) of surface staining to locate approximately the feeding probes of the sugarcane froghopper relied on staining excreta and not a labial imprint. In the studies of planthopper feeding, excreta and labial imprints were visible on coconut pinnae before staining, but after staining only the labial imprints were stained irrespective of the dye used.

Although the particular features of the labial imprint of H . crudus are described here, adults of the Fulgoroidea, Antillixius sp., Colpoptwa elevans Walker, Omolicana sp. and PseTzoJEata brevis van Duzee leave labial imprints after feeding on coconut pinnae. In addition both nymphs and adults of Proana hilaris van Duzee (Homoptera; Cicadidae) leave labial imprints on roots and pinnae respectively during feeding on coconut. For all these insects, the labial imprint was similar to that of H . crudus although differing in details which reflect differences in their rostral apices.

This technique, because it marks a probing event, may be of wider application since observations show that, for coconut pinnae, labial imprints remain detectable for at least 72 h after feeding, thus making it possible to determine probing frequency without recourse to time-consuming sectioning.

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This work was carried out as part of the Lethal Yellowing of Coconuts Research Scheme supported by the United Kingdom Ministry of Overseas Development and the Government of Jamaica.

R E F E R E N C E S

ADAMS, J. B. & FYFE, F. W. (1970). Stereoscan views of some aphid mouthparts. Canadian Journal of Zoology 48, 1033-1034.

BOTHA, C. E. J., EVERT, R. F. & WALMSLEY, R. D. (1975). Studies on Gomphocarpm physo- carpus: further evidence of preferential feeding by the aphid Aphis nerii on the internal phloem. Protoplasma 84, 345-356.

HAGLEY, E. A. C. & BLACKMAN, J. A. (1966). Site of feeding of the sugarcane froghopper, Aeneolamia varia sacchavina (Homoptera : Cercopidae). Annals of the Entomological Society of America 59, 1289-1291.

JOHANSEN, D. A. (1940). Plant Microtechnique. 1st ed. New York: McGraw Hill. POLLARD, D. G. (1968). Stylet penetration and feeding damage of Eupteryx melissae Curtis

(Hemiptera, Cicadellidae) on sage. Bulletin of Entomological Research 58, 5 5-71. POLLARD, D. G. (1973). Plant penetrations by feeding aphids (Hemiptera, Aphidoidae) : a

review. Bulletin of Entomological Research 62, 63 1-14. SMITH, K. M. (1926). A comparative study of the feeding methods of certain Hemiptera and

of the resulting effects upon the plant tissue, with special reference to the potato plant. Annals of Applied Biology 13, 109-139.

STOREY, H. H. (1939). Investigations of the mechanism of the transmission of plant viruses by insect vectors. 111. The insects saliva. Proceedings of the Royal Society (B): 127, 526-543.

WIEGERT, R. G. (1963). The ingestion of xylem sap by meadow spittle bugs, Philaenus spu- marius (L.), American Midland Naturalist 71, 422-428.

E X P L A N A T I O N O F P L A T E

All figures refer to Haplaxius mudus van Duzee. Fig. I . An adult insect feeding on the abaxial pinna surface of COCOS nucifera. Arrow marks the rostral apex applied to the pinna surface. Several leafhairs are also shown (H). Bar represents

Fig, 2. The labial imprint on the adaxial surface of a coconut pinna by transmitted light. The central densely stained region (CR), the impressions of the anterior sensoria (AS), the sensory papillae (SP) the labial cleft (LC) and the irregular margin (M) are shown. Bar represents 10 pm. Fig. 3. The rostral apex by incident illumination showing the stylet canal (SC), the anterior sensoria (AS) and the labial cleft (LC). Bar represents 10 pm. Fig. 4. Transverse section of a labial imprint on the abaxial surface of a coconut pinna showing the raised central region (CR), the path taken by the stylet (SP) and the margin (M). Bar represents 10 pm. Fig. 5. Cleared specimen showing the mouth parts in lateral view. The flattened apex of the rostrum is evident (RA) as are the labrum (LB), stylets (ST), labium (LA) and stylet canal (SC). Bar represents 100 pm.

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