2021358a0Nature202493919640627135813590028-0836196410.1038/2021358a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v202/n4939issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue2021358a0A Technique for Electronically Recording Aphid Feeding and Salivation
AU  - McLEAN, D. L.
AU  - KINSEY, M. G.Department of Entomology, University of California, Davis.APHID feeding and salivation has long intrigued entomologists. The knowledge of when and where these activities occur in the plant may contribute to the solution of the problems of plant virus acquisition and inoculation by aphids as well as by other insects. At present, no satisfactory techniques exist to detail these activities. In our search for a method to explain these phenomena, we developed a technique to record electronically some phases of aphid feeding and salivation within a plant or other electrically conductive substrates.Approximately 2-0-V, 60-cycle a.c. were introduced into the substrate, and this voltage passed through the slightly resistant substrate to ground. With the more conductive substrates, a resistance was placed at the ground end. The source of the voltage was a 6 VAC filament transformer (Stancor P-6466, Chicago Standard Transformer Corp., Chicago, Illinois). The transformer secondary was connected to a 10,000-ohm linear potentiometer so that the amount of voltage could be regulated.
A rectangular feeding cage measuring | in. wide x 5/16 in. deep was used to confine the aphids on the substrate. This cage was constructed out of J in. 'Plexiglass'. A gold-plated tungsten grid (approximately 900 holes/in.2) was glued to the bottom of this cage. The bottom part of the grid was insulated with 'Glyptal'. The cage was placed on a plant leaf or other substrates and held fast with a woman's hair clip. The grid was connected to a 25-W monaural amplifier (Heath -kit model AA-18I, Bent on Harbor, Michigan). Connected in parallel to the 16 ohm output of the amplifier \vere a cathode ray oscilloscope (Heath-kit model 10-10), a chart recorder (Esterline-Angus model AW, 0-100 mV, Indianapolis, Indiana), and a speaker. All instruments used in the testing were grounded to an 8-ft. copper pipe driven into the soil (Fig. 1).
The pea aphid, Acyrthosiphon pisum (Harris), was used for most testing. However, the green peach aphid,
in. long x Myzus persicae (Sulzer), and the lettuce seed stem aphid, Acyrthosiphon scariolae Nevsky, were also observed. The empty stylets of the 3 species were found to be electrically non-conductive, and the tarsi of the 3 species were very low in electrical conductivity. We had hoped that the tarsi would provide electrical contact from the aphid to the grid. In order to make this contact, it was necessary to attach a No. 38 tinned copper wire to the dorsum of the aphid and to bend the wire in such a way that it would contact the grid. At the point of contact with the grid, the wire was bent so that, when the aphid was probing, approximately 1 cm of wire laid flat on the grid. The wire did not markedly disturb the aphids, but it did slightly impede locomotion. The wire was attached to the aphid with a rapid drying silver conducting paint ('Silver Print', Walsco Electronics, Los Angeles 18, California). Although no long-term observations havo been made, this paint does not appear to harm the aphids.
When the aphid probes into the substrate and the stylets fill with saliva or substrate liquid, an electrical circuit is completed between the substrate voltage, the aphid, the grid, and the amplifier. Changes in voltage are observed on the oscilloscope and recorded on the chart. The hypothesis was that the peak and depression activity on the chart was correlated with liquid volume increases or decreases through the stylets, that is, the more liquid the aphid ingests or salivates, the higher the voltage recorded. In order to test this hypothesis, a technique was designed to record liquid volume passing at different rates through a glass capillary tube. A 3-mm glass tube was drawn out so that a capillary tube with an inside diameter of 98a resulted. One end of this tube was placed in a small chamber containing tap water. The chamber was so constructed as to permit the introduction of voltage. A N"o. 38 wire was affixed to the inside of the tube at the opposite end, and this wire was connected to the amplifier. When the tube was placed in the wrater, capillarity caused the water to flow through the tube making contact with the wire. When this occurred, the pen on the recorder moved upward and remained at a constant level. When mouth suction was applied at the upper end of the tube, the pen rose higher indicating increase in voltage, and when the suction was released, the pen would drop to the capillary-level. Increasing suction caused the pen to rise further. When water was blown through the tube, an increase in voltage was also evident and increases in pressure also caused an increase in voltage. These results would indicate that the proposed hypothesis was valid.
Fig. 1. A block and schematic diagram showing the circuit and equipment used to record aphid feeding and salivation
A feeding chamber was constructed so that the aphid's stylets could be observed within a liquid media. The feeding chamber was in 2 parts: a cage with a gold grid similar to the clip-on type, and a well to contain the liquid. Both units were constructed out of 1/16 in. 'Plexiglass'. A 'Parafilm' membrane was stretched over the bottom surface of the cage. The cage was bolted to the section containing the well. The liquid to be used was forced through a hole in the rear of the well. Liquids used included a 20 per cent sucrose solution, filtered plant sap with sucrose added, and 3 artificial diets1'4'5. Voltage was then introduced into the liquid. The observation chamber was designed so that the entire length of stylets and sheath could be observed under a magnification of times 150.
All three species of aphids secreted visible salivary sheaths in all liquids during each probe. Initially, it appeared that production of sheath was related to changes in voltage recorded on the chart. However, after further observations, it became apparent that the salivary sheath was of low electrical conductivity and the pen would move upward only slightly. In most instances, however, strong increases in voltage occurred during sheath production, but obviously not related to the secretion of sheath material. It appeared as though another material was being secreted or that the liquid substrate wras being ingested.
To eliminate the liquid substrate as a variable, further tests were conducted using gold leaf as the substrate. A rectangular piece of gold leaf of the type used for lettering signs was affixed to a small 'Plexiglass' sheet. The size of the leaf was slightly larger than the clip-on cage. The gold leaf was covered by a sheet of stretched 'Parafilm' and the feeding cage clipped over it. Voltage was introduced into the gold leaf substrate. Twenty-five A. pisum were allowed 3 probes each and each recording was noted. Thirty-nine probes showed the slight flat rise attributed to sheath production. The remaining 36 probes were accompanied by higher voltage showing higher peaks on the chart. These results indicate that the aphid secreted another material and possibly a watery saliva similar to that observed by Miles2-3 for Aphis craccivora Koch and for Oncopeltus fasciatus (Dallas). This material may be the phytotoxin secreted by some aphids. However, the possibility that ingestion took place when the aphid probed into the liquid cannot as yet be ruled out.
We have not been completely successful in distinguishing between the liquid or watery secretion and ingestion either by visual observation or differences in recorded curves. When the aphids probed into crushed and filtered Viciafaba L. stems, certain rhythmic curves were recorded. Nearly all individuals tested with these media reproduced these curves. When separate aphids probed F. faba plants, similar curves were produced. Observation of stylet activity within the feeding chamber showed that the rhythmic curves being recorded were correlated with pumping of the stylets within the sheath. The peaks and depressions of the curves were related to the position of the stylets as they moved up and down within the sheath. When the stylets were down, the recorded voltage was high, and when the stylets were up, the voltage was lower. This rhythm may be related to ingestion (cibarial pump activity), secretion of a liquid saliva (salivary pump activity), or the combination of both. Of the 75 probes on gold leaf, 8 were accompanied by a similar rhythm, but they were shorter in duration and not quite as symmetrical.
When the other liquids were used, the rhythm curves were rare and the aphids usually withdrew after a shorter period of probing. If it becomes possible to relate each recorded curve to either ingestion or salivation, it can then be used to determine the acceptability or rejection of any media, and perhaps the volume of fluid ingested during any aphid probe. Provided the substrate is electrically conductive, the exact length of any aphid probe can be recorded from actual stylet contact with the substrate to the probe termination. This recorded information could then be used to establish aphid acceptability of any substrate.
We thank Drs. R. Craig and E. S. Sylvester, Department of Entomology and Parasitology, University of California, Berkeley, California, for their advice. We also thank Mr. D, K. Johnson, of Jennings Radio, Salinas, California, Mr. P. D. Williams, of Eitel-McCullough, Inc., San Carlos, California, and Mr. C. Johnson, of Watsonville, California, for supplying us with some of the equipment and materials used.Auclair, , J. L., and Cartier, , J. J., Science, 142, 1068 (1963).ISIMiles, , P. W., Nature, 183, 756 (1959).ISIMiles, , P. W., J. Inst. Physiol., 3, 243 (1959).Mittler, , T. E., and Dadd, , R. H., Nature, 195, 404 (1962).ISIBragdon, , J. C., and Mittler, , T. E., Nature, 198, 209 (1963).ISI