Studies on the salivary physiology of plant-bugs: Experimental induction of galls

ft. Insect Physiol., 1968, Vol. 14, pp. 97 to 106. Pergamon Press. Printed in Great Britain

STUDIES ON T H E SALIVARY PHYSIOLOGY OF PLANT-BUGS: EXPERIMENTAL I N D U C T I O N OF GALLS P. W. MILES Waite Agricultural Research Institute, University of Adelaide, South Australia

(Received 17 ffuly 1967) Abstract--The formation of nodules on the roots of vines grown in aqueous solutions is shown to be an inadequate test for hypotheses of cecidogenesis by Herniptera. 14C-labelledprecursors were used to demonstrate the production of the plant growth hormone, 3-indoleacetic acid, during salivation of a plant-bug; and a normally non-cecidogenic hug was made temporarily eeeidogenic by the injection into it of excess amounts of the metabolic precursors for IAA-production. It is suggested that IAA could be the universal cause of cecidogenesis by plant-bugs, and that the specificity of gall-morphology is explicable in terms of the insects' behaviour. INTRODUCTION

CECIDOGENESIS (the production of galls on plants) has long fascinated biologists, and many attempts at simulating the natural induction of galls have been made by the injection of plants with chemicals (ANNI~ND, 1927; KOSTOFF and KENDALL, 1933), or cecidogenic organisms or extracts of them (KosTOFF and KENDALL, 1933 ; MARTIN, 1938). The discovery in the mid-nineteen-thirties of the chemical nature of the growth substances of plants quickly led to comparisons between the effects of artificial applications of these substances and the galls produced by insects (LA RuE, 1935, 1937; MARTIN, 1938); and it was suggested either that cecidogenic insects themselves introduced growth substances (M~a~Tm, 1938; NYsreruu~IS, 1947) or that they indirectly caused the release of 'auxins' within the plant (ALLEN, 1951). In particular, claims have been made that 3-indoleacetic acid (IAA) is a cecidogenic or phytotoxic agent in the saliva of plant-bugs, and either that it can be synthesized in the salivary glands of the insects (DusPIvA, 1954) or that it may be transferred from the diet to the salivary glands (NuoR~WA, 1955, 1956). Interest in IAA as a possible cause of cecidogenesis by insects waned, however, with the publication of work by ANDF.~S(1958) on free amino acids as the cause of cecidogenesis by the grape phylloxera, Viteus vitifoliae Fitch. Anders not only found high concentrations of certain amino acids in the saliva of the insect, but he was also able to induce the formation of nodules on the roots of vine seedlings by growing them in a solution of just these amino acids--and not others. Nevertheless, in spite of the dramatic results obtained by Anders, the problem of the 7

97

98

P.W. MILES

chemical causation of insect-galls had not been solved. For SCHALLER (1960) reinvestigated the salivary amino acids of Viteus and found a different group of amino acids from that reported by Anders. Moreover, although KLOFT (1960) observed that amino acids had many physiological effects when injected into plantcells, these did not amount to a confirmation of cecidogenesis; and it is becoming increasingly apparent that the saliva of all the phytophagous Hemiptera contains free amino acids, whether the insects cause galls or not (KLOFT, 1960; NUORTEVA and LAURElVIA,1961; SCHALLER, 1963; MILES, 1967a, b). More recently, SCH~LLtm (1965) reopened the question of whether salivary IAA could be a cause of phytotoxicity or cecidogenesis. He found that IAA did indeed occur in the saliva of aphids, but he claimed that it was present in variable concentrations that could not be directly related to the effects of the insects' feeding. The present communication reports on the experimental induction of nodules on the roots of vines, and on an investigation of a possible metabolic pathway of the production of IAA in the saliva of plant-bugs (MILES, 1965 ; MILE8 and LLOYD, 1967). It is shown that the IAA content of the saliva can be increased experimentally, and the effects on the food-plant of the insect are described. Finally, the hypothesis that salivary IAA is responsible for cecidogenesis by plant-bugs is put forward in a modified form that takes into account the instinctive behaviour of the insect. MATERIALS AND METHODS Vine cuttings Canes selected from Vitis vinifera L. cv. 'Cabernet Sauvignon' were stored in plastic bags at 4°C until required. Propagation was by the method of MULLINS (1966). As soon as roots had begun to form, the cuttings were selected for uniformity and suspended in solutions at 20°C to determine the effect of the solutions on the subsequent formation of roots.

Sunflower seedlings Seeds of Helianthus annuus L. cv. 'Grey Stripe' were germinated in soil in a glass-house. At the cotyledon + 2 leaf stage, the seedlings were selected for uniformity of growth and condition of cotyledons, and they were enclosed in lamp-glasses covered at the top with 'mosquito netting'. Insects were then introduced, ten to a plant. For every experiment, all treatments (of the insects) and a growth-control (a plant without insects) were replicated five times and the plants were randomized on the bench. The insects Adults of Elasmolomus sordidus (F.) (Heteroptera: Lygaeidae) were obtained from a culture maintained at between 25 and 35°C on peanuts and water. Injections were done with an Agla syringe (Burroughs, Welcome) as described by MILES (1967b).

I N D U C T I O N OF G A L L S

99

Chromatography and autoradiography Thin-layer chromatography and autoradiography of the chromatograms was done as described by Mm~s (1967b).

Histochemistry Staining of nucleic acids was done by the method of DE BOER and SAm~AKERas outlined in PEARSE(1960). RESULTS

Induction of nodules on vine rootlets In an initial experiment, the vine cuttings were placed in aqueous solutions of the amino acids that ANDEaS (1958) claimed caused the development of rootnodules, namely tryptophan, histidine, lysine, and a mixture of these three amino acids together with valine and glutamic acid. The histidine and lysine were used as the monohydrochloride, and all amino acids were present as 0"025 per cent w/v of the L-isomer. Every treatment was replicated ten times. After 3 weeks, typical growth of the plants and their roots was as shown in Fig. 1. Thereafter, the parts of cuttings immersed in the mixture of amino acids became increasingly covered with moulds and bacterial slime, and the experiment was discontinued. It was apparent, however, that no nodules had formed on any of the roots that had developed, and that the order of vigour of growth (by solutions) was (i) tryptophan, (ii) histidine, (iii) lysine, and (iv) the mixture, with little or no growth in the last. Cuttings grown in water were similar in appearance to those grown in the solution of tryptophan. ANDEaS (1958) made no mention of the pH of his solutions of amino acids, but tryptophan in solution will undergo spontaneous oxidative deamination to IAA at a pH above 7 and in the presence of air. Because of the possible importance of pH, therefore, the experiment was repeated using solutions that were buffered by the presence of 0.02 M potassium dihydrogen phosphate, adjusted with KOH (5%) to pH 8.0. Cuttings were also grown in the buffer alone. Three weeks later, there had been very little growth of roots in any of the buffered solutions of amino acid, but the growth in the solution of buffer alone was as shown in Fig. 2. Nodules had indeed been produced--but not in solutions of amino acids. The results illustrated in Fig. 2 may open up an interesting problem in plant physiology, but they did not seem to have any direct relevance to the production of galls by insects; for it is most unlikely that potassium or phosphate as such are cecidogenic, or that the pH alone of the small volumes of saliva that plant-bugs inject into plants would have such profound effects. Experiments on the effect of solutions on the growth in them of vine-roots were therefore discontinued.

S3mthesis of IAA by the salivary apparatus of Elasmolomus It has been established that amino acids present in the haemolymph of plantbugs also occur in the saliva (MILES, 1967b) and almost certainly one of these amino

100

P . W . MILm

acids is tryptophan (MILES, 1964b, 1967b). Dihydroxyphenylalanine (DOPA) is probably also present in the glands (MILES, 1964b, 1967b) and a polyphenol oxidase is certainly present in another part of the apparatus, namely the accessory gland (MILm, 1964a). From the work of GOXDONand PALEG(1961) it is possible, therefore, that reactions leading to the production of IAA (Fig. 3) could take place at the moment of salivation when all the salivary secretions come together; and this is especially so because the watery saliva (i.e. that part of the secretions that remains liquid after discharge) has a pH ~>8-0 (MILES, 1965) that would favour the reaction. In the principal salivary gland

TRYPTOPHA~

In the accessory salivary gland

DOPA Polyphenol oxidase

+½Os In the saliva

L

TRYPTOPHAN

DOPA-QUINONE -> 3-1NDOLEAC'ErlCACID

FIG. 3. Hypothetical reactions leading to the production of IAA in the saliva of a plant-bug.

The hypothesis represented by Fig. 3 would be strongly supported if, for instance, the injection into the haemolymph of l~C-labeUed phenylalanine (which may be assumed to be a normal constituent of the haemolymph) gave rise to labelled DOPA in the salivary glands; and if l~C-labelled tryptophan gave rise to labelled IAA in the saliva. Attempts to establish that these transformations do indeed occur in Elasmolomus are described below. Injections of 0.5/~c of DL-3-phenyl(alanine-l-14C) in 3/A of solution were made and, 1 hr later, the glands were dissected out and chromatographed on silica gel G using methylethylketone-propionic acid-water ( 3 : 1 : 1 ) as solvents, and the chromatogram was autoradiographed. Apart from radioactive material at the origin and some streaking, three distinct radioactive spots were found, corresponding in R t to phenylalanine, tyrosine, and DOPA. In a second experiment, 0.5/~c of DL-tryptophan (methylene-14C) was injected into the insects, along with 2 #g unlabelled phenylalanine. The latter compound was added to ensure that the precursor for DOPA would not be limiting in any reaction leading to the formation of IAA. The collection of saliva from Elannolomus proved unsatisfactory both in relation to the quantity collected and to its content of solutes. The dilute nature of such collected saliva and the possibility that it has no obvious relation to the saliva secreted during the insect's natural feeding process have been discussed in a previous paper (MILES, 1967b); hence the fact that little radioactivity could be recovered from it an hour or more after the injection was not accepted as negative evidence for the relationships indicated in Fig. 3. Analysis of saliva recovered from food plants, even had this proved practicable, would have been equivocal

0-1

FIG. 4. Photographs of 'galls' that developed within 2 weeks around the feeding punctures of Elasmolomus sordidus when the insects fed on sunflower cotyledons within 24 hr of the implantation into the body cavity of the insect of an agar flake containing 3/zg each of tryptophan and phenylalanine. A, base of a cotyledon with several of the intumescences, showing their natural pigmentation. B and C, photomicrographs of the intumescences showing the external 'collar' of sheath material (arrowed) protruding from the centre, and the additional darkening of the galls produced by treating the preparations with gallocyanin to stain nucleic acids. All scales in ram.

FIG. 1. Vine cuttings showing roots developed after 3 weeks in unbuffered solutions of: A, tryptophan; B, histidine; C, lysine; and D, a mixture of tryptophan, histidine, lysine, glutamic acid, and valine. All compounds were present as 0"025 per cent w/v.

FIG. 2. Development of roots on vine cuttings immersed for 3 weeks in 0"02 M potassium phosphate buffer, p H 8.0, showing subapical nodules.

t •

*

.

°

t* "~

.

FIG. 5. A, cross-sections of a feeding puncture made by one of the insects within 24 hr of the implantation into the body cavity of a flake of agar containing 3/zg each of tryptophan and phenylalanine, showing the sheath material (arrowed), and the enlarged parenchyrna that makes up the bulk of the 'gall' (reversed print of photomicrograph by dark-ground illumination). B, part of the preparation shown in A stained with gallocyanin and photographed by normal light-ground illumination--nucleic acids have stained darkly. C, cross-section of a feeding puncture made by one of the treated insects 3 days after implantation, showing the relatively small amount of disturbance to the leaf tissue normally caused by Elasmolomus (photomicrograph by normal transmitted light after staining with safranin and light green; the darkly stained sheath material is arrowed). All preparations were fixed 2 weeks after the feeding puncture was made. Scales are in ram.

INDUCTION OF GALLS

101

because of the possibility that labelled compounds injected by the insect would be metabolized by the plant. T h e only alternative seemed to be homogenization of those parts of the salivary glands thought to provide the necessary reactants in the saliva, and the following procedure was adopted. Thirty insects were injected, and approximately 1.5 hr after the first injection, the whole salivary apparatus, including the accessory glands, was dissected out and accumulated in a glass homogenizer kept at 35°C. T h e glands were homogenized as they were collected, and the collection was completed within 1 hr. The accumulated brei was then chromatographed immediately, even though the last glands had only just been added. This was done so that there would be a minimum chance for bacterial contamination to affect the results. T h e pH of the brei was found, using pH papers (Merck, May & Baker 'ROTA', and Johnson's of Hendon), to be 6.5+0.1. Chromatography was on silica gel G using methyl acetateisopropanol-25~/o conc. NH4OH in water (45 : 35 : 20). Autoradiography of the chromatogram revealed that only two radioactive compounds were present beyond the origin, one corresponding in position to tryptophan and the other (present in very much smaller quantity) to IAA. This result was remarkable considering that the low pH of the brei would not have favoured the reaction, which is presumed to proceed normally at the pH of the watery saliva of >/8"0. Autoradiographs of chromatograms of the unhomogenized salivary glands showed no radioactive spot other than tryptophan beyond the origin, and solutions of the labelled tryptophan in phosphate buffer, pH 6"5, showed no evidence of the formation of IAA within 1 hr at 35°C.

Experimental induction of galls In spite of the success of the experimental production of IAA from labelled tryptophan in the homogenized salivary glands of Elasmolomus, such experiments are always open to the objection that conditions in a homogenate are too abnormal for rigorous conclusions relating to natural processes to be drawn from them. Nevertheless, several possibilities arise if the result obtained is indeed indicative of a natural process. It is already known that injections of an amino acid in excess into the haemolymph of plant-bugs results in its appearance in excess in the salivary glands and saliva as well (MILES, 1967b). Thus, it should follow that injection of tryptophan into Elasmolomus should give rise to an increase in the amount of this amino acid in the saliva and, as long as neither DOPA nor the salivary polyphenol oxidase is limiting, an increase in the production of salivary IAA. The polyphenol oxidase at least seems to be present in the saliva of lygaeids in large amounts (MILES, 1964a-1967a) and injection of phenylalanine along with tryptophan would ensure that the precursor for DOPA was not limiting. Hence, if salivary IAA is truly responsible for cecidogenesis by plant-bugs, insects injected with tryptophan and phenylalanine should show enhanced cecidogenic ability. Elasmolomusdoes not naturally produce galls, either on its natural food plant, the peanut (Arachis hypogaea L.) or on other plants on which it will feed, such as bean

102

P.W. MILES

(Viciafaba L.) or sunflower (Helianthus annuus L.). The theoretical implications of the use of such an insect to demonstrate a ceeidogenic process will be discussed later: that it can be made temporarily cecidogenic was established as follows. In these experiments, the insects used were adults that were still soft after ecdysis, but had developed nearly all their colour. Dry tryptophan and phenylalanine were mixed with liquid 2% agar (cooled to 50°C) at the rate of 100 mg of each amino acid to every 1 g of agar sol. The mixture was spread thinly and allowed to dry, and small flakes weighing about 6/zg were then inserted into the insects through an abdominal intersegmental membrane. Subsequent dissection of such insects and treatment of the flakes with the same reagents that were used to reveal the compounds on chromatograms showed that the flakes had lost most but not quite all their charge of amino acids within 24 hr. (This observation was made by comparing the implanted flakes with 'control' flakes of agar, also implanted in the insects but not originally containing arnino acids.) During the time of implantation, the flakes became invested with a melanized material, but the melanization was not due to the amino acids originally present in the flake, for flakes of agar alone were indistinguishable in appearance 24 hr after implantation from those that had contained tryptophan and phenylalanine. Immediately after implantation of the agar flake, the insects were released on sunflower seedlings. Over half the insects that were implanted with agar/amino acid flakes died within the first 24 hr, and no more than 1 in 10 were alive at the end of 3 days. More of the insects that were implanted with agar alone survived, but over half of these were dead by the third day. Few of the untreated insects died during this period. After 3 days, all insects and lamp-glasses were removed from the plants. It was observed at the end of 2 weeks that the plants exposed to the insects treated with the amino acids had begun to display (i) swelling and splitting of the stem at the first node, (ii) stem bent at this point, and (iii) various degrees of epinasty of the leaves (MIL~ and LLOYD, 1967). Although these results seemed to be associated with the feeding of the treated insects, which had been observed feeding at the first node, the exact sequence of events that led to the results was uncertain. In several repetitions of the experiments, occasional plants that had been exposed to insects implanted with agar alone, or even to untreated insects, showed the same symptoms. It appeared that the epinasty and the splitting of the stem at the first node were probably due to an initial bending of the stem at this point; so that there was doubt whether the insects were causing the symptoms by merely weakening the stem or by causing hormonal disturbances of growth. Further observation led to the discovery that a far more significant localized effect was produced by the treated insects when they fed on the cotyledons (Fig. 4). The point of feeding by such insects is clearly visible afterwards because of the 'collar' of sheath material that the insects leave externally on any firm substrate on which they feed (MILEs, 1959). Around some of those made by insects implanted with tryptophan and phenylalanine, there appeared small, usually pigmented, intumescences; and insects implanted with agar alone and untreated insects were never found to produce these intumescences.

INDUCTION OF GALLS

103

In a further experiment, some insects were implanted with (i) agar flakes alone, (ii) flakes containing about 3 ~g tryptophan, (iii) flakes containing about 3 ~g phenylalanine, (iv) flakes containing about 3/~g each of both amino acids; and (v) other insects were left untreated. Insects representing all five 'treatments' were caged on the seedlings and transferred daily to new plants for the next 3 days, after which the insects were removed from the experiment. The cotyledons of all seedlings used in the experiment were inspected every day. Within a week, the intumescences such as those illustrated in Fig. 4 had become noticeable at all the feeding sites made during the first day by insects treated with tryptophan and phenylalanine, and the growths had probably developed to their maximum size within 2 weeks. They were found particularly on the petioles and edges of the cotyledons but also on all parts of the upper and lower surfaces. None were found on the main stems of the seedlings, however. After 3 weeks, the cotyledons were removed and fixed in a mixture of ethanol, glycerol, 'formalin', acetic acid, and water (34 : 5 : 4 : 2 : 5). Some were subsequently treated entire with gallocyanin to stain nucleic acids, and some were sectioned first in paraffin wax. Feeding sites that had been made on the first day by insects implanted with flakes of agar containing tryptophan or phenylalanine separately were sometimes surrounded by small intumescences with varying degrees of pigmentation: there was no obvious independent relation between which of the two amino acids had been introduced and the amount of swelling or pigmentation produced. Occasional sites at which bugs had fed on the second day were also surrounded by intumescences if the insects had been implanted with a flake containing either or both of the amino acids. On the other hand, the feeding sites of insects injected with agar alone, and those of untreated insects, were never observed to develop an intumescence. Histological preparations (Fig. 5) showed that the growth was due mainly to hypertrophy of the cells surrounding the 'stylet sheath', and the enlarged cells stained with gallocyanin, indicating the presence of unusual amounts of ribonucleic acid within them. There was also evidence of increased meristematic activity in the cambium of the vascular bundles immediately below the feeding puncture, and it was apparently due to this that some larger intumescenees extended through the cotyledon and caused an outgrowth of normal sized cells from the other surface (Fig. 5A). DISCUSSION One of the problems that any hypothesis of cecidogenesis has to overcome is that of the specificity of gall morphology. Yet the chemical hypotheses that have been advanced so far have seemingly been applicable to all species. Thus amino acids appear to be universally present in the saliva of plant-bugs, and it now seems probable that IAA too is universally present, even if in minute quantities. The problem of why some species produce a gall and others do not is not necessarily a separate problem, for 'no gall' can, perhaps, be considered as just one of the possible types of gall that the phytophagous Hemiptera are capable of producing.

104

P.W. MILES

A general hypothesis that seeks to explain the causation of galls in terms of a single chemical compound can nevertheless be made to explain specificities in gallstructure if the hypothesis takes into account two modifying factors. These are (i) the type and stage of development of the plant-tissue which is attacked, and (ii) the behaviour of the insect once feeding has begun. It seems likely that some types of gall depend for their full development on the stage of differentiation of the plant tissues when they first come under the influence of the cecidogenic stimulus. At the same time, it has been observed in this laboratory that if certain types of psyllid are killed in their immature galls, whether by systemic insecticides or by heat or by mechanical injury, the galls cease to develop. It follows that the continued development of the gall is under the continuous stimulation and hence control of the insect. For not only are the cecidogenic sucking insects all sedentary, and therefore able to exercise control of cecidogenesis over a prolonged period, but they also possess the power, through their flexible mouth-parts, of influencing different parts of the same plant, even though the body of the insect may remain sessile. In this sense, the stylet-bundle can be thought of as providing the functional mouth of the insect with powers of locomotion that are retained to the end of the insect's life. Certainly, it is easy to account for the fact that an insect such as Elasmolornus is not naturally cecidogenic, even though it has been demonstrated here that the insect possesses the potential for cecidogenesis. The insect attacks mature cells and it influences these for little more than half an hour at a time. Under these circumstances, the amount of IAA in the saliva produces little or no effect. Nevertheless, we have seen that, if the quantity of salivary IAA dispensed in the plant is increased artificially, it may reach a threshold of concentration at which its diffusion into the cells surrounding the feeding puncture is sufficient to give rise to a local intumescence. If one could imagine such an insect settling down to feed in one place only and for a month or more, during which time it dispensed IAA to cells surrounding it in an instinctively controlled pattern, the result would surely be an elaborate and specifically shaped gall. SCH~a.LER (1965) was unable to relate IAA-content of the saliva of aphids to the effects they produced on their food-plants; but quantity alone may not be of over-riding importance if development of a gall depends on prolonged stimulation of just the right points within the plant-tissues, and if summation of the stimulus can also occur over a long period. T h e relevance of experimental condition is also important when assessing results such as those of Sch~iller: saliva collected directly from the mouth-parts of a Hemipteron may have no simple relation to the saliva dispensed within food (MILES,1967b); and feeding activity in synthetic substrates appears to be different from that in natural ones (MCLEAN and KINSEY, 1965). Thus it is always possible that the saliva collected by Sch~iller on filter paper was not identical in composition to the saliva injected by the insects into their food plants. What is of fundamental significance is that IAA/~ injected by the insects and that it can be demonstrated to produce gall-like structures experimentally. As NUOaTEVA(1962) first demonstrated, insects with enhanced amounts of IAA

INDUCTION OF GALLS

105

in their saliva can cause an increase in the growth of their food plants. If this results in an increase in the a m o u n t of food available to the insect, then salivary IAA would have an adaptive value; and it is easy to derive from the widespread or general occurrence of IAA in the saliva of plant-bugs, firstly, the ability of some sedentary or colonial forms to produce relatively undifferentiated intumescences-such as those made by the woolly aphid, Eriosoma lanigerum (Hausm.)--and, secondly, the formation by entirely sessile insects of a more elaborate structure able to provide not only an enhanced food source, but varying degrees of physical protection as well. It has been suggested above that IAA production in the saliva of plant-bugs is dependent on the salivary polyphenol oxidase. Nevertheless, there is no reason to believe that this is the sole raison d'dtre of the enzyme; for it occurs in a variety of carnivorous as well as phytophagous insects (MILES, 1964a). T h e r61e of the enzyme in the production of I A A and its possible rble in the formation of the salivary 'stylet sheath' (MILES, 1964b-1967a) should probably be considered as secondary, therefore, to some underlying primary function, whatever this might be.

REFERENCES

ALLEN T. C. (1951) Deformities caused by insects. In Plant Growth Substances (Ed. by SKOOG F.), pp. 411-415. University Press, Wisconsin. ANDERS F. (1958) Aminosiiuren als gallenerregende Stoffe der Reblaus [Viteus (Phylloxera) vitifolii Shimer]. Experientia 14, 62--63. Ar~ P. N. (1927) Tumours in kale. Science, N . Y . 65, 553-554. DusPxvA F. (1954) Weitere Untersuchungen tiber stoffwechselphysiologische Beziehungen zwisehen Rhynehoten und ihren Wirtspflanzen. Mitt. biol. ZentAnst. Berl. 80, 155-162. GORDON S. A. and P ~ G L. G. (1961) Formation of auxin from tryptophan through action of polyphenols. Plant Physiol. 36, 838-845. t~oF'r W. (1960) Weehselwirkungen zwischen pflanzensaugenden Insekten und den yon ihnen besogenen Pflanzengeweben. Z. angew. Ent. 45, 337-381 ; 46, 42-70. KosTo~ D. and KENDALLJ. (1933) Studies on plant tumours and polyploidy produced by bacteria and other agents. Arch. Mikrobiol. 4, 487-508. LA Rtr~ C. D. (1935) The role of auxin in the development of inturnescenees on poplar leaves; in the production of cell outgrowth in the tunnels of leaf miners ; in the leaf-fall of Coleus. Am. ft. Bot. 22, 908. LA RuE C. D. (1937) The part played by auxin in the formation of internal intumescences in the tunnels of leaf miners. Bull. Torrey Club 64, 97-102. McLEAN D. L. and KINSmr M. G. (1965) Identification of electrically recorded curve patterns associated with aphid salivation and ingestion. Nature, Lond. 205, 1130-1131. MARTIN J. P. (1938) Stem galls of sugar cane induced with an insect extract. Hawaff Plant. Rec. 42, 129-134. MXLES P. W. (1959) The salivary secretions of a plant-sucking bug, Oncopeltus fasciatus (Dall.) (Heteroptera: Lygaeidae)--I. The types of secretion and their r61es during feeding..7. Insect Physiol. 3, 243-255. MILES P. W. (1964a) Studies on the salivary physiology of plant-bugs: oxidase activity in the salivary apparatus and saliva, ft. Insect Physiol. 10, 121-129. MXLF.SP. W. (1964b) Studies on the salivary physiology of plant-bugs: the chemistry of formation of the sheath material, ft. Insect Physiol. 10, 147-160.

106

P . W . MILES

MILES P. W. (1965) Studies on the salivary physiology of plant-bugs: the salivary secretions of aphids. J. Insect Physiol. 11, 1261-1268. MILES P. W. (1967a) The physiological division of labour in the salivary glands of Oncopeltus fasciatus (Dall.) (Heteroptera: Lygaeidae). Aust.f. biol. Sci. 10, 785-797. MILES P. W. (1967b) Studies on the salivary physiology of plant-bugs: transport from haemolyrnph to saliva, f . Insect Physiol. 13, 1787-1801. MILES P. W. and LLOYDJ. (1967) Synthesis of a plant-hormone by the salivary apparatus of plant-sucking Hemiptera. Nature, Lond. 213, 801-802. MULLINS M. G. (1966) Test-plants for investigations of the physiology of fruiting in Vitis vinifera L. Nature, Lond. 209, 419-420. NUORa~VAP. (1955) On the nature of the plant injuring salivary toxins of insects. Ann. ent. fenn. 21, 33-38. NUORTIVCAP. (1956) Studies on the effect of salivary secretions of some Heteroptera and Homoptera on plant growth. Ann. ent. fenn. 22, 108-117. Nuoaan~VA P. (1962) Studies on the causes of the phytopathogenicity of Calligypona pellucida (F.) (Horn., Araeopidae). Ann. Zool. Soc. 'Vanamo' 23, 1-57. NUORa~VA P. and LAtrREMA S. (1961) The effect of diet on the amino acids in the haemolymph and salivary glands of Heteroptera. Ann. ent. fenn. 27, 57-65. NYsrm~aas F. (1947) Zooctcidies et substances de eroissance. C. R. Soc. Biol., Paris 141, 1218-1219. t~AaSE A. G. E. (1960) Histochemistry: Theoretical and Applied. Churchill, London. SCn/a.~R G. (1960) Untersuchungen fiber der Aminos~iuregehalt des Speicheldrfisensekretes der Reblaus [Viteus (Phylloxera) vitifolii Shimer], Homoptera. Entomologia exp. appL 3, 128-136. SclJ/a.um G. (1963) Papierchromatographisehe Analyse der Aminosiiuren und Amiden des Speiehels und Honigtaues yon 10 Aphidenarten re_itunterschiedlicher Phytopathogenitiit. Zool. fib. (Physiol.) 70, 399--406. SCHgL~R G. (1965) Untersuchungen tiber den ~-Indolylessigsiiure-Gehalt des Speichels yon Aphidenarten mit untersehiedlicher Phytopathogenit~t. Zool. fib. (PhysioL) 71, 385-392.