Application of Scanning Microscopy in the Study of Virus Transmission of Aphids

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Application of Scanning

Microscopy

in

the

Study of

Virus

Transmission of Aphids1

GUSTAAFA. DE ZOETEN

Department ofPlantPathology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 12 April 1968

The scanning electron microscopewasused in conjunction with the transmission

electronmicroscopetostudy stylet morphology and the role of stylets of thegreen

peach aphid in plant virus transmission. The morphology of thestylets generally agreed with earlier descriptions of these parts obtained with the transmission electron microscopy. A considerable variation seemed to exist in the extent and regularity of ridges observed in different individuals, especially among apterous adults andnymphs. Surface contamination of styletsaswellasof thebristleareaof

the labium could be detected. Thepossible role of these contaminantsinplant virus transmission is discussed. Working near the limit of resolution of the scanning

electronmicroscope, visualization oftwotypesofplant virus particleswasattempted.

Dippingaphid stylets in suspensions of purified virus did not leadto subsequent detection of these particles onthe stylets with the scanning electron microscope.

The scanning microscope is evaluated for its possibilities in the study of plant virus transmissionby insects.

Thestudyof plant virustransmissionbyaphids

and themechanismsinvolvedinthistransmission have attracted theattention ofmany researchers

during the last decades. Many experiments have

been performed and intricate hypotheses have been proposed regarding the mechanism

of

virus transmission by aphids. In light ofrecent

technical

developments

inelectron

microscopy,

a

reexamination of one of the earliest proposed

means ofplant virus transmission was thought tobewarranted.

In 1933, Hoggan (2) suggested that minute

amounts ofinfectious plant sap are withdrawn from infected plants as contaminants on the

stylets of feeding aphids. Upon resumption of

feeding,

these minute amounts are reinoculated.

Transmission in this fashion would be classified

asstylet-borne (3).

Earlier electron microscope studies of aphid stylet structure were done on Myzus persicae

(Sulz.) by H. A. van Hoof (Ph.D. Thesis, Inst. Plziektk., Onderz, Wageningen, 1958), who

described the anatomy and morphology of the

stylet tip regionandpostulateda mechanismfor stylet-borne aphid transmission.

With the scanning electron microscope, an attempt was made to detect plantor other

con-taminants on the aphid stylets and to evaluate 'Published with the approval of the Director of the WisconsinAgricultural Experiment Station.

the scanning electron microscope as a tool in theinvestigation ofthemorphology ofstructures

otherwise difficult to study. It was also

hoped

that virus particles could be resolved on the

aphids' stylets.

In the

scanning

electron

microscope,

a

rela-tively

low

accelerating

voltage,

10 to25

kv,

was used to produce amicrobeam that swept across

the

specimen.

Impingement

ofthe

scanning

beam

onthe specimen generated several kinds of

sec-ondary

emissions;

the secondary and back-scattered electrons from these emissions were

collected on adetector linked to an oscilloscope whose sweep was linked to the electron beam

sweep. For each point ofthespecimen hitbythe

electronbeam,a

point

wasrevealedonthescreen

of the oscilloscope.

MATERIALS AND METHODS

Greenpeach aphidswereculturedon noninfected radish undercontinuous lightatapproximately22C. First and second stage nymphs and apterous adults

wereused forscanning electronmicroscopy. Feeding aphidsweretouchedontheir backs withafine camel's hair brush. This caused cessation offeeding and

re-traction ofstyletsfrom the leaves. Immediately after retraction ofstylets from the leaves, the aphids were frozen in liquid nitrogen and subsequently freeze-dried. Carewastakentoprevent theaphids from ob-taining other contaminants on their stylets after feeding.

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labium was removed, whereas in others labium re-moval was followed by opening of the stylets; the latter treatment, performed by lowering a glass capillary filled with 70%, ethyl alcohol 3 over the stylets, resulted in the separation of the mandibular fromthemaxillary stylets.

The insects on the specimen tables were subse-quently subjected to a rotary shadow evaporation processin which palladium or mixtures of other metals were deposited on the specimens to a thickness of 200A. Aphids thus prepared were viewed in a Cam-bridge Stereoscan scanning electron microscope.

The stylets of some aphids were prepared for transmission microscopy (H. A. van Hoof, Ph.D. Thesis, Inst. Plziektk, Onderz., Wageningen, 1958) in order to compare these results with those obtained with thescanning electron microscope.

Dilute solutions of purified tobacco mosaic virus (TMV) and Southern bean mosaic virus (SBMV)

were prepared and sprayed on specimen tables in order to establish the feasibility of visualizing these particles in the scanning electron microscope. Stain-less-steel and standard aluminum tables were used to

assess back scatter properties of these specimen holders with regard to their specimen.

Anumber ofaphids were also prepared for scanning microscopy after their stylets had been dipped in the purified virus solution mentioned above.

RESULTS

The structures on those aphid stylets (Fig. 1)

observed by scanning electron microscopy are

existing at the stylet tips were very pronounced and could bestudiedathigh magnification (Fig. 1, 3-5). Proximally, the mandibular stylets seemed to be rather smooth (Fig. 1 and 2), at least no pronounced, regularly arranged struc-tures could be observed. Irregularities were observed, however, especially along the suture of mandibular and maxillary stylets (Fig. 2, parts 2and 3). The rectangular nature of the maxillary stylets at the site where the stylets emerge from the labium could be seen (Fig. 3, part 4). The sharp ridges at the end of the maxillary stylets described by van Hoof couldnotberesolved by

scanning electron microscopy. They were ob-served in some cases, however, by employing

transmission microscopy.

Ingeneral,thepointed tip of one of the mandib-ular stylets was found to extend beyond the other mandibular stylet (Fig. 1, parts 3 and 4 Fig. 2, part 1; Fig. 3, part 1), in accordance with the alternate probe theory for mandibular

stylets. It was observed that ridging is not

con-sistent from individual to individual and that variation in the extent of the ridges is more

pronounced with the apterous and alate adults. In the photographs presented here, it can be seen that a fairly extensive amount of outside

contamination exists on the mandibular stylets (Fig. 2, parts 1-3). This contamination seems to

FIG. 1. (1)Mouth partsof Myzuspersicae (Sulz). Note thejointarea(J) ofthelabiuim (L), thebristle hairs (B) atthe endofthelabium, and the mandibularstylets(MS). Black and whitearrowsindicate mandibularstylet apposedtoopposite stylet. X 800. (2) Close-up of the mandibularstylets (MS) and the endofthe labium(L).

Notethe smoothness of the surface ofthemandibularstylets wheretheyemergefromthe labium. Thesutureof

themandibular and maxillary styletscan be seenzconstituting unevenness alongtheside ofthestylets. X 1,700. (3) Tipof the mandibular stylets (MS). Ridging (R) ofthe tipisevident onbothmandibularstylets. Theoverlap ofone stylet on theother is acommonphenomenon. X 17,000. (4) Face viewofone ofthe mandibularstylets (MS). Theridging (R) isextensiveand theridgesarefairlydeep,butnoevidenceof barbingcanbeseenin

scan-nzingmicroscopy. X 20,000. (5) Transmission electron micrograph ofamandibular stylet (MS). Note canal (C, whitearrows).R, ridging.Someindication of barbingmaybeobserved. X 15,000.

FIG. 2. (1) Tips of the mandibular stylets (MS) carrying outside contaminants (CO) believed to be saliva.

X 6,000. (2) Tipsofthe mandibularstylets (MS) carrying outside contaminants (CO) believed to be ofplant

origin. B,bristle hairs; L, labium. X 1,740. (3) Tipof labium with emerging stylets. Contamination (CO) on

stylets (MS),as wellas onthelabium (L),isevident(whitearrows). Sutureofmandibular andmaxillary stylets

canbe seen(blackand whitearrows). B, bristlehairs. (4) Transmissionelectronmicrograph ofapartially purified

preparationof Southern beanmosaic virus(SBMV). Compare withFig. 3,parts 5and 6. X 100,000.

FIG. 3. (1) Distal viewof mandibularstylets (MS). Tipofthe upperstylet isbent downover thelowerpart

(black and white arrows). Ridges (R) onz thelowerstyletare verypronounced. Notedepth of focus. X 42,000. (2)Scanningelectron micrographofthe tipofapairofmaxillarystylets(MX). Note sutureofmaxillarystylets (black and white arrows)aswellassurfacestructure(whitearrows).MS, mandibularstylets. X21,000. (3) Trans-mission electronmicrograph ofa partofamaxillarystylet (MX). No evidence ofbarbingwas regularly found. Note that some of the barbs (black arrows) point towards the base, others towards the tip. Twocanalscan be resolved (whitearrows). X 28,000. (4) Stylet bundle opened bytreatmentwith

70%,0

ethyl alcohol. Although the maxillarystylet (MX)isroundedatthe end(incrosssection),it isrectangularorellipsoidatthebase(blackarrow, andwhitearrows).Fuzzyappearanceofstylettipswastheresultofheatinstabilityof tip.MS,mandibularstylets.

X 1,900.(5) Southern bean mosaic virus (SBMV)photographed on an aluminum specimen holder. Note that image wasobliteratedby backscatterfrom specimen table. X 400,000. (6) Partially purified tobacco mosaic

virus(TMV).Notefibrillarimpression oftheimageas aresultofthedefinite morphology ofTMV. A stainless-steelspecimenholderwasuised. X48,000.

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FIG. 2

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ofplant debris can be found outside, on the end of thelabium contained within the ring of bristle hairs or on these hairs (Fig. 2, part 3).

The contaminants were usually of twodifferent types (Fig. 2, parts 1 and 2), namely plantdebris and droplets of some freeze-dried fluid, pre-sumably saliva, since it was observed that in-sects handled as described above continue to salivate for a short time after being removed from the leaves. Virus directly deposited on the specimen tables could be visualized (Fig. 3,

parts 5 and 6), but the images did not compare favorably with those of the transmission micro-scope (Fig. 2, part 4). Virus could not be found, however, in its particulate form on stylets that had been immersed in concentrated virus solu-tions.

The mandibular stylets were fairlystable under a 25-kv beam. However, the unsupported, pro-jecting distal ends of the maxillary stylets that were sticking up unsupported soon started to move under electron bombardment (Fig. 3, parts 2-4).

Dis(-ussioN

There was considerable variation in the

mor-phology of the stylet tips in the different stages of development of the aphids.

However,

no

systematic studyofanaphid populationwasdone to carry these observations beyond the point of reasonable doubt. In the apterous adults

es-pecially, the extent of ridging of the mandibular

stylets varied considerably. Very often, one of the mandibular stylets had very few ridges. The difference in ridging among aphids in different stages of development may in part explain

discrepancies in efficiency of virus transmission observed in aphidsat different stagesof

develop-ment. Differences in probing habits may

con-stitute another reason for differences in

trans-mission frequencies.

Mandibular stylets were always found to be closely apposed at their distal ends, and no aperture was ever seen at these ends. In general, the observations confirmed those of van Hoof with regard to structure, and proved the useful-ness of the scanning microscope for direct ob-servationsof insectmorphology.

A comparison of ridges on the mandibular and maxillary stylets, using the scanning and transmission electron microscopes, showed that the barbed appearance of some of the ridges may bearesult ofelectron transparency of parts of these ridges in transmission microscopy, in

were of peripheral interest in this study. Also, exact measurements of the long protruding structures are of rather doubtful value because ofthe projected nature ofthe three-dimensional image in scanningelectron microscopy.

Figure 2, parts 2and 3, seems to show beyond doubt that contaminants of plant origin, and therefore plant virus, can be carried on the out-side of the mandibular stylets. After a fasting period, the contaminants carried on the bristle hairs may cause recontamination of stylets en-gaged in probing. No claim can be made that thisis the only possible way of infection, because intrastylet contamination is a distinct possibility as a sourceofinoculum.

No contamination resembling that on the mandibular stylets was seen on the maxillary

stylets. However, the maxillary stylets very seldom protruded from the mandibular stylets. Thus, stylets that had been wiped clean of gross contamination upon retraction were mainly ob-served. On the basisofstylet morphology alone, no explanation can be given for specificity of virus transmission by aphids However, it can be seen in Fig. 2, part 1, that salivary contami-nantsdo occur on aphid stylets. Thus, individual virus resistance to drying and inhibiting sub-stances inplant sap or insectsaliva isa probable explanation for specificity, as has already been proposed by Day and Irzykiewicz (1).

Although in some instances virtIs was applied

to the aphid stylets, in no case could particles be resolved on these mouth parts. Several pos-sible reasons can be mentioned: (i) particles did not adhere to stylets in purified form; (ii) par-ticles were buried in the evaporated metal; (iii) not enough metal was deposited on the stylets to increase contrast of these virus particles to visualize them; (iv) the work was done close to the limit of resolution of the scanning micro-scope, 150-200A.

The first assumption does not seem to be sup-ported by data from research concerned with transmission ofplantviruses afterthe aphids fed on purified preparations of these viruses (5). To compensate for the possibility that puri-fied virus preparations in water may not adhere tostyletsurfaces,somebovine serumalbumin was addedtothesepreparationsbefore dippingstylets

inthesesolutions. Again, no virus could be found on thestylets.

As can be seenfrom Fig. 3, part 5, resolutionof single particles of SBMV was completely ob-literated because of serious back scattering problems encountered with thealuminumn

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men holders. The outline of SBMV aggregates could be resolved and identified only because of the typical angularity of the aggregates which were familiar from transmrnission microscopy (Fig. 2, part 4). With highly polished stainless-steel specimen tables, the images improved suffi-cientlytoenable identificationof particles such as TMV, which are morphologically distinct from other cellular particles (Fig. 3, part 6).

There is good reason to believe that eventually singlevirus particles can be resolvedby scanning microscopy. However, unless "spherical" virus particles form paracrystalline arrays when con-taminating stylets, it will be difficult to dis-tinguish them from otherplant debris. From Fig. 3, part6, itcanbeseenthat,evenwhenembedded in other debris, viruses of distinct morphology can be identified.

Heat instability of the mouth parts, especially

the tips of the maxillary stylets, was a problem since it caused movement of stylets which made

photography more difficult. Although heat generation here is only a fraction ofthat in the trans-.nission electron microscope, as reported by Pease (4), it seems that it is serious on tiny

unsupported surfaces and where heat conduction maybeaproblem.

The present investigationemphasizes thevalue of scanning microscopy in the study of insect morphology and indicates that valuable infor-mation may begatheredinthisway fortaxonomic

purposes. However, to make scanning micros-copya valuable tool in the study of insect virus relationship, specimen handling techniques must be improved and higher resolution capabilities than are possible with present-day scanning microscopes are required.

ACKNOWLEDGMENTS

This investigation was supported by Institutional Grant no. I.N.-35from The American Cancer Society and by Hatchproject 1494.

I thank F. Rossi of the Engis Equipment Co., Chicago, IlL., for his valuable help with the scanning microscope. The technical help of G. Gaard and F. B. Diez is acknowledged.

LITERATURE CITED

1. Day, M. F., and H. trzykiewicz. 1954. On the mechanism of transmissions of non-persistent

phytopathogenic viruses by aphids. Australian

J. Biol. Sci. 7:251-273.

Hoggan, 1. A. 1933. Some factors involved in aphid transmission of the cucumber mosaic virus to tobacco. J. Agr. Res. 47:689-704.

3. Kennedy, J. S., M. F. Day, and V. F. Eastop. 1962. A conspectus of aphids as vectors of plant

viruses. Commonwealth Institute of Entomol-ogy, London.

4. P'ease, R. F. W., T. L. Hayes, A. S. Camp, and N. Mv. Amer. 1966. Electron microscopy of living insects. Science 155:1185-1186.

5. Pirone, T. P.,and E. Megahed. 1966.Aphid

trans-missibility of some purified viruses and viral

RNA's. Virology30:631-637.

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