The Saliva of Hemiptera

The Saliva of Hemiptera PETER W . MILES School of Natural Sciences. University of Zambia

Introduction . . . . . . . . . . . . . . . . Methods of Investigation . . . . . . . . . . . . Modes of Feeding . . . . . . . . . . . . . . Stylet-Sheath Feeding . . . . . . . . . . . . . A. Sampling the Surface . . . . . . . . . . . B. Secretion of the Flange . . . . . . . . . . . C. Formation of the Sheath . . . . . . . . . . D. Discharge of Watery Saliva . . . . . . . . . . . E. Ingestion . . . . . . . . . . . . . . . F . Withdrawal of the Stylets . . . . . . . . . . V . Lacerate-and-Flush Feeding . . . . . . . . . . . VI . Feeding by Carnivores . . . . . . . . . . . . . VII . Chemical Composition and Function of the Saliva . . . . A. Sheath Material . . . . . . . . . . . . . B. Watery Saliva . . . . . . . . . . . . . . VIII . Phytopathogenicity . . . . . . . . . . . . . . IX . Salivary Glands and Ducts . . . . . . . . . . . . A . Aphidoidea . . . . . . . . . . . . . . B . Jassomorpha . . . . . . . . . . . . . . C. Fulguromorpha . . . . . . . . . . . . . D. Other Auchenorrhyncha . . . . . . . . . . E. Heteroptera . . . . . . . . . . . . . . X. Origins of the Saliva . . . . . . . . . . . . . . A. Functions of the Accessory Gland . . . . . . . B . Functions of the Principal Gland . . . . . . . C. Sources of Oxidases . . . . . . . . . . . . D. Sources in the Homoptera . . . . . . . . . . E . Salivary Carbohydrate and Lipid . . . . . . . . XI . The Saliva as a Vehicle for Pathogens . . . . . . . . XI1. Evolution of Salivary Function in the Hemiptera: a Summary XI11. A Survey of Problems . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . I. I1. 111. IV .

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I . INTRODUCTION

Salivary function is especially interesting in Hemiptera because of the effects the saliva has on the living and surviving organisms on 183

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which many of these insects feed. Some of the exclusively phytophagous suborder Homoptera cause “phytotoxaemias” including galls in their food plants; some Homoptera and a few phytophagous Heteroptera transmit diseases to plants in their saliva and some of the blood-sucking Heteroptera similarly transmit diseases to their hosts. Even when Hemiptera transmit diseases apparently by contamination of their mouthparts, they do not function as mere “flying pins”; there is evidence that the saliva can modify the effectiveness of transmission. The saliva of Hemiptera is by no means a simple secretion: in addition to the usual salivary functions of moistening food and mixing it with hydrolytic enzymes before ingestion, the saliva of phytophagous species plays an important physiochemical role during the mechanical penetration of plant tissues by the piercing and sucking mouthparts and, in accomplishing this task, the saliva may vary in its chemical composition and physical consistency from one moment to the next. Moreover, deposits of solidifying components of the saliva of many species persist in the food plants, modifying the long term effects of feeding by the insects. At the same time, other salivary components dissolve solid materials during gustatory exploration of substrates and it seems highly probable that the saliva thus acts as a carrier of gustatory stimuli to sensilla remote from the “functional mouth” at the tip of the stylet-like mouthparts. Finally, there is evidence that the saliva may subserve an excretory function in some species, varying in composition in relation to the compositim of the haemolymph-indeed it would appear to provide the only means of excretion in those few, highly specialized Homoptera that lack an anus. In relation to the complex composition and varied functions of the saliva, the salivary glands of Hemiptera are themselves complex and in the suborder Homoptera may present a seemingly bewildering array of cell types that appear to secrete different products and to undergo individual cycles of secretory activity. Identification of the products of individual types of cells with externally recognizable salivary functions is difficult in the Homoptera, in which the main secretory parts of the salivary apparatus are without a distinct lumen; but in the Heteroptera the salivary glands take the form of sac-like lobes, and accumulations of the salivary secretions can be identified and even collected from the salivary glands of the larger species. The history of investigations of salivary functions in Hemiptera is largely one of independent studies: on the one hand of economically

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significant Homoptera, in which research on the saliva is beset with problems connected with the small size of the insect; and on the other of the larger Heteroptera which are easier to deal with but have evoked less interest because of their lesser economic importance. Only recently has a measure of similarity in salivary functions in the two taxa been recognized and explored. It is the intention of this article to bring together the various types of investigation on salivary functions in the Homoptera and Heteroptera, and to suggest profitable lines of future investigation based on analogous functions in different taxonomic groups. 11. METHODS OF INVESTIGATION

The effects of the feeding of phytophagous Hemiptera were first investigated by histological study of plants on which these insects had fed. Histochemical analyses were made of the tissues surrounding feeding punctures and physiological inferences drawn as long ago as Busgen (1 89 1); although the usefulness of such interpretations was limited by the uncertainty of whether observable effects were caused by the insects directly or were due to reactions on the part of the plant (Petri, 1909). A refinement in histological studies was the examination of tissues on which the insects had fed for a known length of time and in which the insects’ stylets remained in situ. This was achieved by rapid killing of the insects with drops of very hot wax (Smith, 1920), electric shocks or by snipping or shaving off the stylet bundle while the insect was still feeding (Mittler, 1957). Major advances in the determination of the nature of the salivary secretions of Hemiptera and of their possible roles during feeding became possible when it was found that the insects could be induced to feed on transparent media such as agar gels or solutions enclosed by membranes. McLean and Kinsey (1 967) brilliantly exploited the ability of aphids to probe through parafilm by passing a small electric current through the aphid and substrate and correlating changes in the current with observable feeding activities involving salivation and ingestion. By this means, McLean and Kinsey (1 968) were then able to determine the sequence of salivation and ingestion in opaque, natural substrates. Refinements and details of the method have been published by McLean and Weigt (1 968): electrical contact is made with the aphid by cementing a gold wire of 15.0 p thickness to the dorsum with a quick drying silver conducting paint. An example of the circuitry is outlined in Fig. 1. Patterns of change in the

. . Fig. 1. Block and schematic diagram of the various devices and components used in a system to record pea aphid salivation and ingestion. (From McLean and We&, 1968.) Rl-IOK; R2-150-500K; R3-99M; R4-47K; R5-16; R6-1M; R7-1M; R8-470K; R9-8; R10-1M; Rll-16; D1 to D4-1N1614; M-high impedance A-C voltmeter; T-Triad F-16X filament transformer; S1A,B-DPDT toggle switch; S2A,B-DPDT toggle switch.

Fig. 2. Strip chart recordings showing sequences of feeding activity by the pea aphid. To be read from right to left: base line at the bottom of chart Omv. S-salivation waveform associated with penetration of tissues by the stylets and the secretion of a stylet sheath; X-waveform associated with penetration of phloem sieve tube; t-probably a period of brief ingestion between unblocking of food canal by discharge of saliva; Y-waveform of unknown significance; I-prolonged ingestion. (From McLean and Kinsey, 1967.)

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conductance of the system can be recorded on a cathode ray oscilloscope, through a loud-speaker or on a chart recorder or any combination; although the chart recorder is probably the most useful as a means of recording data permanently (Fig. 2). A similar system has been used by Smith and Friend (1971) to record the feeding of the blood-sucking bug Rhodnius prolixus. These authors inserted a platinum electrode through the cuticle and fixed it in place with beeswax and colophony (2: 1). The insects were permitted to feed through a rubber membrane on artificial diets M ATP sodium salt. containing 0.15 M NaCl with or without Flows in the liquid were observed by adding washed frog erythrocytes. The authors used DC current rather than AC and added the refinement of television monitoring of the activity of the mouthparts (Fig. 3). Saliva has been obtained for chemical analysis by a number of means. Salivary deposits have been collected from solid surfaces or recovered from inert gels or membranes (Edwards, 1961; Miles, 1959b, 1965). Saliva has also been collected directly from the mouthparts (Anders, 196 1 ; Miles, 1967b; Strong, 1970), sometimes after the insects have been stimulated to salivate by placing them in a humid environment or by injecting them with solutions containing 10% pilocarpine (Miles and Slowiak, 1970). Saliva has also been collected by allowing the insects to probe inert absorbent materials. Feir and Beck (1961) collected the saliva of the milkweed bug (Lygaeidae) by allowing the insect to probe through the testa of a milkweed seed into powdered cellulose. Schaller ( 1968a) collected

0

P 47, recwder

Oscilloscope

recorder

Monitor

Fig. 3. System for simultaneously recording and displaying optical and electrical data from a feeding insect. (From Smith and Friend, 1971).

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the saliva of several hundred aphids at a time by placing them in successive batches on the undersurface of a piece of damp filter paper, illuminated from above with yellow-green light. The aphids were first deprived of food and water so that they no longer produced honeydew as a contaminant. The saliva was eventually eluted from the paper for analysis. Adams and McAllan (1958) analysed saliva for one particular enzyme by enclosing the insects directly over a spot of the substrate on a strip of filter paper. A membrane was interposed between the insects and substrate and the exploratory probes of the insects through e.g. parafilm caused their saliva to be directly applied to the substrate. Subsequent chromatography of the spot then revealed whether any enzymic hydrolysis of the substrate had occurred. In the larger Heteroptera particularly, recourse has been made to analysis of enzymic activity of the whole glands or of the contents of particular lobes (Miles, 1967a; Hori, 1968b). Chromatographic separation of components of the secretions of the glands can be conveniently carried out by using the whole glands themselves as the “spot” at the origin of the chromatogram (Miles, 1967b). Radioisotopes have been used both as a means of determining the extent of salivary deposition in substrates (Fig. 4)and as a method of tracing metabolites into the saliva and thus identifying salivary components. Kloft et al. (1968) reviewed the methods used for labelling the saliva of aphids by incorporating radioisotopes, 32P in particular, into host plants or “sachets” of artificial diet enclosed in parafilm. Onion leaves which had their cut stems placed in a solution containing one or two mCi/ml Na2 32P04became radioactive within three or more hours and aphids or Thysanoptera placed on the labelled leaves acquired sufficient radioactivity over 24 h to make their saliva traceable by autoradiography when the insects were transferred to non-radioactive hosts (Kloft and Ehrhardt 1959a, b; Kloft, 1960). Hennig (1 963) similarly placed the cut stalks of Vicia faba leaves in solution containing 16pCi/ml for 5 to 20 h to label the leaves in preparation for transfer of the label to Aphis fabae Scop. Injection of from 0.5 to 10 pCi of radioisotope-labelled metabolites directly into the haemolymph of Heteroptera has been used to trace the transport of materials from the haemolymph into the saliva and to demonstrate metabolic pathways leading to the occurrence of individual compounds or classes of compounds in the saliva (Miles, 1967b, 1969a). It became apparent that any compound injected into the haemolymph was likely to appear in the saliva

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Fig. 4. Autoradiograph: Distriibytion of saliva labelled with '*P 'discharged by Myzus ascolonicus in leaf of violet. (From Kloft, 1960).

within a few minutes unless it was first rapidly metabolized in the haemolymph; but by injecting a labelled metabolite already known to occur in the haemolymph, it was possible to demonstrate the normal presence in the saliva of those compounds of which the labelled metabolite was a precursor.

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111. MODES OF FEEDING

I. Scratch-and-suck Feeding

The Hemiptera, according to Goodchild (1 966), probably arose from insects that scratched the surfaces of growing plants, sucking out the cell contents (Fig. 5). Such an architypal scratch-and-suck mode of feeding is practised by the Thysanoptera, and probably also by the Tingidae within the Heteroptera. Little is known about the feeding of the tingids, however, except that these small insects feed on cells immediately below the epidermis, for the most part causing insignificant lesions. They are specialized Hemiptera nevertheless, and if their mode of feeding should indeed prove primitive, it is likely that it arose as a simplification of a more complex mode such as the stylet-sheath feeding described below. Even during the scratch-and-suck feeding of Thysanoptera, radioisotopic labelling of the saliva indicates that some of it is deposited in the cells on which the insects have fed (Kloft and Ehrhardt, 1959b) and species of both Thysanoptera and Tingidae are known to cause galls. Discharge of saliva into food sources is characteristic of the Hemiptera and it is convenient to distinguish between at least four major modes of feeding among the Hemiptera based partly on food source and partly on the function of the saliva.

2. Stylet-sheath feeding

All the Homoptera and the Heter0ptera:Pentatomorpha are able to discharge at least two different kinds of saliva; one that solidifies rapidly on ejection and another that always remains watery and that leaves only a trace of solid matter evaporation. During the normal course of feeding on growing plant tissues, the solidifying kind of saliva is ejected wherever the stylets penetrate and is moulded by the action of the stylets to form a tubular lining to the path taken by them, thus forming the so-called “stylet-sheath”. The watery saliva is also secreted before and during penetration of the substrate, as well as on withdrawal of the stylets and the functions of these secretions will be discussed in detail in Section VII.

3. Lacerate-and-gush feeding Some members of the Pentatomorpha, particularly the Lygaeidae and Pyrrhocoridae, feed on seeds. When they do so they secrete only a surface “flange” of sheath material, that is not continued as a stylet-sheath into the seed. Instead, the stylets and watery saliva are

Reduviidae Cimicidae etc. predatory forms

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subaquatic / Corixidae algophagous

Miriabe Tingidae return to plant-feeding carnivorous Geocorinae Asopinae Coreoidea \ / \ Amphibicorisae Cimicomorpha Pen tatornoidea - -- surface Saldidae skating litoral Pen tatornorpha habit stylet-sheath feeding on sap, shbre lacerate-and-flush on seeds or prey litter \? / ability to secrete a stylzsheath abhity to secrete a cornplite stylet sheath lost: mostly carnivorous retained: phytophagous

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Dipso corimorpha

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Jassomorpha Cicadomorpha

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\? litter-inhabiting OmniVOrOUS forms, lacerate-and-flush feeding evolved

other Sternorrhyncha /

Malpighian tubules reduced ;

Fulguromorpha

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posterior junctidn antehor junition of mid and hind gut of mid and hind gut stblet-sheath fehing Coleorrhyncha evolved; mesophyllfeeding, true Hemiptera

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scratch-and-suck pre-Hemipterans -THYSANOPTERA Fig. 5 . Evohtionary interrelationshipsbetween divisions of Hernipternbased on physiology of feeding. OVIodifi from Goodchild. 1966.)

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v, h)

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used to lacerate and macerate a pocket of cells or the entire contents of the seed according to its size. The contents are finally flushed out with a copious flow of dilute saliva. Elsewhere among the Heteroptera, phytophagous forms are found within the Cimicomorpha: namely the Tingidae and many species of Miridae. These insects produce no stylet-sheath, yet they feed on the roots and shoots of growing plants. The tingids are mostly very small and probably feed on individual cells or small numbers of them near the epidermis, but the phytophagous mirids apparently use a lacerate-and-flush method of feeding on pockets of cells in a manner very much like that of the Pentatomorpha when they feed on seeds. Thus the derivation of lacerate-and-flush feeding on plant tissues by the Cimicomorpha is perhaps to be seen as a necessary consequence of their loss of ability to secrete a stylet-sheath.

4. Predation Many Heteroptera feed on small prey and they employ what is in effect lacerate-and-flush feeding. Indeed, some of the mirids among the Cimicomorpha are carnivorous as larvae and phytophagous as adults. In the Pentatomorpha too, cannibalism is practised and undoubtedly the seed-feeding forms employ much the same technique whether feeding on seeds or individuals of their own kind. The main difference between phytophagous lacerate-and-flush feeding and predation proper lies in the composition of the saliva; for the saliva of the predators causes rapid immobilization of their prey, while the phytophagous bugs can practise cannibalism only on incapacitated individuals or on eggs. Further, the true predators are not known to produce a stylet-sheath. 5. Blood-sucking Blood-sucking Hemiptera are found within the Cimicomorpha only. Despite the observation that the phytophagous Cimicomorpha secrete no sheath material, in at least one of the blood-sucking Reduvioidea, Rhodnius prolixus Stbl, traces of a solidifying secretion are deposited at the point on the surface of the skin about to be penetrated (Friend and Smith, 197 1). No stylet-sheath is formed, however. The mandibles force their way a short distance into the skin and the maxillae alone penetrate deeper, thrusting first one way and then another until a blood vessel is penetrated (Lavoipierre et al., 1959; Smith and Friend, 1971). Little or no histolysis of the tissues of the host occurs. Indeed, the success of feeding depends on avoidance of sensory reaction on the part of the host. Alp-9

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IV. STYLET-SHEATH FEEDING A . SAMPLING THE SURFACE

All phytophagous Hemiptera, Homoptera as well as Heteroptera, often touch a surface several times with the labium before settling down to feed. It has been suggested (Miles, 1959b) that during this initial period, gustatory stimuli are picked up by watery saliva that is ejected and almost immediately sucked back into contact with the gustatory receptors that form the epipharyngeal organ (Wensler and Filshie, 1969). Exploration of surfaces with the mouthparts by aphids may or may not be accompanied by actual penetration. Hennig ( 1963) observed that when the black bean aphid, Aphis fabae Scop. touched leaf surfaces several times with the labium, each touch could last between 5 and 60 s and that the epidermis was sometimes penetrated, sometimes not. Often sheath material was deposited to form an external flange (Fig. 6) and when the stylets penetrated the surface, more sheath material was sometimes secreted and formed a short canal through the surface. At other times, however, deposits of sheath material were almost imperceptible or non-existent.

I

200p d

Fig. 6. Flanges of sheath material. a: aphid probes into a leaf surface; a short sheath accompanies the probe into a mid-lamella. (After Hennig, 1963). b: aphid penetrating parafilm membrane and building a sheath in a solution-showing formation of the flange within the tip of the labium and the bead-like appearance of the sheath. (After Miles, 1968b). c: formation of flange by Rhodnius on rubber membrane. (After Friend and Smith, 1971). d: flange formed on testa of milkweed seed by Oncopeltus. (After Miles, 1967a). e: formation of flange external to labium by Dysdercus feeding on cotton seed. (After Saxena, 1963).

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Such observations make it important to distinguish between an actual penetration of a surface-a true probe-and a mere touching of the surface-a dab. Hennig ( 1963) was unable to obtain any evidence that during these dabs and probes, any radioactivity was taken up from plants labelled with 32P; on the other hand, his results indicate only that aphids had not taken up quantities of radioactivity large enough to be measured; it is also possible that the sucking back of watery saliva brought into very brief contact with the surface of a leaf plant labelled with 3? would not pick up the label at all. Miles (1 959a) observed that aphids with their stylets exposed and in contact with glass ejected and sucked back minute quantities of watery saliva quite distinct from the solidifying sheath material; while McLean and Kinsey ( 1964), during the electronic recording of feeding by aphids, also obtained evidence that some fluid was ejected and sucked back on impenetrable surfaces. Heteroptera will spend a considerable time dabbing surfaces that contain soluble food materials, while at the same time ejecting. and sucking back watery saliva (Miles, 1959b; Saxena, 1963; Bongers, 1969). B. SECRETION OF THE FLANGE

When Homoptera and Heteroptera : Pentatomorpha feed on plant tissues, typically a small amount of sheath material is first secreted on the surface and the stylets then push through the solidifying material and begin to penetrate the substrate. This external sheath material has been termed a “flange” (Nault and Gyrisco, 1966). Exceptions have been noted. Saxena (1963) stated that the pyrrhocorid Dysdercus koenigii ( F . ) produced a flange on the hard testa of a cotton seed or on the intact cuticle of stems and leaves, but no sheath material was secreted at the surface of slices of cottonseed kernel. Hori (1968a) claimed that when the pentatomid Eurydema rugosa Motschulsky fed on cabbage leaves, it did not always secrete a flange or any other sheath material when the stylets subsequently remained solely within the mesophyll; but the insect did produce both flange and sheath when the destination of the stylets was a phloem vessel. Hennig (1 963) indicated that short probes by Aphis fubae Scop. into epidermal cells were not necessarily accompanied by a flange or sheath. Saxena’s observation on Dysdercus can be interpreted as indicating that this insect secretes a sheath only when its stylets encounter a hard substrate during attempts at penetration. But there is no simple

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explanation for the observations of Hori and Hennig. They would seem to imply that, at the moment of penetration, the subsequent pattern of feeding is already determined-i.e. if no flange has been secreted, subsequent feeding will be on mesophyll cells (and not on vascular tissue) or only a short “exploratory” probe will be made (and the stylets will not penetrate further). Presumably such predetermination of stylet activity would have t o be due to the physiological state of the individual. One possibility is that subsequent feeding behaviour depends on the ability of the insect to produce sheath material when it first attempts penetration of the substrate. Nevertheless, McLean and Kinsey (1 969) found no loss of ability of an aphid to secrete sheath material with starvation and further investigation of this problem is clearly required. Despite the few reports to the contrary noted above, the usual course of events during feeding by Homoptera and Heteroptera: Pentatomorpha is the secretion of a drop of solidifying saliva on the surface at the point where penetration of the stylets will begin; and this now seems to be true even of a blood-sucking reduviid Rhodnius prolixus Stll (Friend and Smith, 1971) that until recently was thought unable to produce any secretion similar to the stylet sheath of the phytophagous bugs. The initial secretion of sheath material on to a surface results in a flange of more or less circular cross section surrounding the point of penetration. The flange extends either around the labium or into the labial groove (Fig. 6). C. FORMATION OF THE SHEATH

A flange is not necessarily continued as a stylet-sheath into the substrate below. When the seed-feeding lygaeids and pyrrhocorids penetrate a hard surface, the flange is continued only a short way beyond the surface (Miles, 1959a, 1967a; Saxena, 1963). When Aphis fubae probes the body of an epidermal cell, further sheath material is unlikely to be secreted; whereas if the stylets have struck an intercellular groove, secretion of a stylet sheath is more likely, whether or not the penetration continues further into the plant (Hennig, 1963). Mostly, however, when Homoptera and Heter0ptera:Pentatomorpha feed on the roots or shoots of plants, it is found that a flange of sheath material is secreted on the surface and is

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continuous with a stylet-sheath laid down wherever the tips of the stylets penetrate (Fig. 7). Precise details of the way in which a stylet-sheath is formed have been furnished by McLean and Kinsey (1965, 1968). The stylets characteristically move into tissues by a series of backward and forward movements. When the stylets are at the end of a backward sequence, a drop of sheath material is secreted; watery saliva is ejected into this, making it balloon out; the stylets then push forward through the material and pierce the end of it (McLean and Kinsey, 1968). At this stage, the substrate may be sampled by sucking. The stylets then move back again and sheath material is again secreted. In this way, each new drop of sheath material is secreted in contact with the last piece and becomes part of a coherent sheath. In a soft, uniform medium, the stylet-sheaths of aphids thus have a typically beaded structure (Fig. 6); but in materials of varied structure and texture, the sheath may flow into the space surrounding the stylets or be squeezed into a space so narrow that it almost disappears. When stylet-sheaths pass through solid materials (e.g. when an aphid’s stylets pass through unstretched parafilm) it would seem that the insect is sensitive to the backpressure caused by the medium and that less material is secreted. Even so, it is under these circumstances that some aphids begin to suck back some of the sheath material before it has completely solidified and it then remains as an indigestible deposit in the mid-gut. (Moericke and Mittler, 1965). Possibly the thinness of the sheath when it is secreted within narrow confines in relatively rigid structures accounts for some confusion on whether stylet sheaths are continuous within plant tissues or not (Miles, 1968b). D. DISCHARGE OF WATERY SALIVA

Some watery saliva is ejected by aphids as part of the process of formation of the sheath (McLean and Kinsey, 1965); and as the stylets push through each new drop of sheath material, this watery saliva is released. When the secretions of aphids have been labelled with 3?, diffusible components of the saliva can be traced for some distance beyond the stylet-sheath in, for instance, leaves (Fig. 4).In aphids and some other Hemiptera the diffusing secretion contains enzymes such as pectinase and cellulase (see Section VI1,B) that presumably aid penetration of the plant tissues. The phytophagous Hemiptera continually sample any substrate

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.-c

Fig. 7. The feeding of Aulacaspis tetalensis (Zhnt.), Coccidoidea, in sugar-cane stems. a-c: stylets and stylet-sheaths in cortical cells: . Williams, 1970.) c: pierced cell that has collapsed; d: barbed tip of stylet (scale = 5 0 ~ )(From

\o

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through which the stylets are passing by sucking back small amounts of liquid. Aphids periodically suck whatever fluids are present when the stylets have just emerged from the last drop of sheath material secreted; this has been indicated by the electronic methods of McLean and Kinsey (1967), the uptake of radioactivity from very heavily labelled plants (Garrett, personal communication) and from the sucking back of sheath material that has been unable to solidify when aphids probe unstretched parafilm (Moericke and Mittler, 1965). All Hemiptera would seem to react to an inability to suck fluids when they attempt to do so by alternately discharging small quantities of watery saliva and sucking back (Miles, 1959b; Saxena, 1963; McLean and Kinsey, 1967). In this way insoluble materials are brought into solution (Saxena, 1963) or viscous solutions diluted (Miles, 1959b). E. INGESTION

Once the stylets have penetrated a source of ingestible food (e.g. a phloem seive tube), no more saliva is ejected (Kloft, 1960) unless the food canal should subsequently become blocked. Rapid movements of the tips of the stylets then occur, and if this does not dislodge the

b

Dendrites

C

Fig. 8. Stylets of Myzus persicae. a: tip of left maxilla. b: cross section of maxillae. c: cross section of whole bundle. fc-food canal; md-mandible; mx-maxillae; sc-salivary canal. (After Forbes, 1969).

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particle, a small amount of sheath material is secreted (McLean and Kinsey, 1965). This last technique is effective because the functional opening of the salivary canal lies a short distance behind the functional opening of the food canal (Fig. 8). McLean and Kinsey (1967) ascribe a characteristic sequence of change of electrical resistance between aphid and substrate (the “X-waveform” Fig. 2) to the accumulation of callose slime around the tips of the stylets when they have been inserted into phloem sieve tubes and the clearing of the slime by short bursts of salivation. Those insects that feed on mesophyll tissue by the stylet-sheath method eject watery saliva continually (Kloft, 1960). Presumably they eject saliva into each cell on which they feed and suck out the contents before the secretion of the sheath is continued and the stylets move on to another cell. F. WITHDRAWAL OF THE STYLETS

The watery saliva has immediate effects on plant cells into which it diffuses, causing increased respiration and streaming of the protoplasm (Kloft, 1960); effects very likely caused primarily by an increase in the permeability of the cell membranes. Hemiptera may withdraw their stylets through the stylet-sheath completely, or they may draw back the tips of the stylets for a short distance before the stylet bundle is thrust through the side of the sheath to produce a branch in the sheath. Whenever the stylets retreat, even during the final withdrawal of the stylets, the surrounding cells again show transient increases in respiration, as though once more affected by the emission of saliva (Kloft, 1960). It has in fact been shown by Kinsey and McLean (1967) that when aphids withdraw their stylets voluntarily from plants, the end of the stylet sheath is sealed and the central canal filled up with secretion. It seems likely that any effects on the plant tissues that occur during withdrawal of the stylets are due to the saliva emitted at the moment withdrawal begins, as it is improbable that mechanical stimulation of the plants’ cells during withdrawal of the stylets would be significant and Kinsey and McLean (1967) have demonstrated that once the end of the sheath is sealed by the insect, dyes will not diffuse from the central canal to the exterior of the sheath. They conclude therefore that all ingestion by aphids is through the open end of the sheath and not from materials diffusing through its walls; at the same time it follows that saliva would be unlikely to diffuse outwards through the walls of the sheath.

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PETER W. MILES

These observations are a clear indication of the sealing properties of the sheath. Since the central canal of the sheath is moulded by the stylets themselves, the insect may be forced to fill the canal of the sheath with saliva during withdrawal of the stylets by the suction that would occur otherwise. What kind of saliva remains in the central canal of the sheath has not been determined with certainty. The photographic evidence produced by Kinsey and McLean (1 967) would seem to indicate that it is sheath material itself; although observations on lygaeids (Miles, 1959b) were that the central canal of the sheath is filled with watery saliva on voluntary withdrawal of the stylets. V. LACERATE-AND-FLUSH FEEDING

Lygaeids, pyrrhocorids and some pentatomids will feed either on seeds or on the growing tissues of plants. When feeding on phloem sap, the lygaeid Oncopeltus fusciutus (Dall.) secretes a complete stylet-sheath, but when feeding on seeds, it secretes a flange of sheath material which is continued as an abbreviated tubular structure within the seed (Miles, 1967a; Bongers, 1969), be it a milkweed seed on which the insect normally feeds or a peanut (Miles, 1959b). Saxena (1963) points out that Dysdercus secretes such a flange only when the surface covering the food material is non-porous and insoluble: the cut surface of a seed kernel does not induce the secretion of sheath material. Once the stylets of a seed-feeder are within the kernel, the feeding activity is very different from typical stylet-sheath feeding. The stylets are pushed first one way and then another for periods of up to two hours when Oncopeltus feeds on milkweed-seeds or Dysdercus on cotton seeds (Saxena, 1963). During this period, the whole contents of a small seed (Miles, 1969b) or a pocket of cells in a larger seed (Miles, 1959b; Saxena, 1963) will be rendered fluid and removed. How this is achieved may differ, however, with different species. Oncopeltus when feeding on a milkweed seed, reduces the solid contents to a thin juice which can be squirted out of the seed, the liquid being derived from the watery saliva. The insect finally sucks out this juice, leaving the testa practically empty. Bongers (1 969) calculated that an individual Oncopeltus injected some 1.14 mg of salivary solids into a milkweed seed during the process of digesting and flushing out its contents. How great a volume of saliva this

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203

represented was difficult to estimate, since Bongers found almost no solids at all in the watery saliva he was able to collect. This apparent anomaly is probably explicable in terms of the ability of Hemiptera to vary the dilution of the watery saliva they secrete (see Section VI1,B) Dysdercus appears to suck back its watery saliva immediately after it is discharged and thus significant quantities of watery saliva do not accumulate within the cotton seed kernels on which it feeds. External digestion of, for instance, starch grains too large to be sucked into the salivary food canal of the stylets can easily occur when Oncopeltus is feeding, but Saxena (1 963) concluded that this was impossible when Dysdercus fed and that disintegration of larger particles was due mainly to mechanical action aided, at the most, by physical solution in the watery saliva. Nevertheless, all seed feeders seem to require relatively large amounts of water to drink if they continue to feed exclusively on dry seeds and liquefaction of the contents of seeds, whether by physical solution and disruption or by enzymic action, requires the insect to secrete large amounts of watery saliva during feeding (Saxena, 1963; Bongers, 1969). The mirids and tingids of the Cimicomorpha secrete no sheath material in the sense of producing a coherent tubular structure; although early descriptions of feeding by mirids included accounts of “granular”, deeply staining material within histological sections through feeding punctures (Smith, 1926). In the light of recent reports of a secretion akin to sheath material being secreted by even blood-sucking reduviids (Friend and Smith, 197 l ) , the categorical statement that sheath material is not secreted by the Cimicomorpha (Miles, 1968b) must be revised. Mirids lacerate and flush out pockets of adjacent cells when they feed. They do not seem to be adapted to feed on dry seeds, and feed mostly on growing tissues and fruits. The general process of feeding is similar to that of lygaeids and pyrrhocorids, however, as indicated by the water-soaked patches of tissue that they produce, and the silvery appearance of the air pockets left behind when they have finished. VI. FEEDING BY CARNIVORES

Lacerate-and-flush feeding is clearly akin to feeding by predators; some species of mirids are carnivorous, as are some pentatomids and lygaeids and even the phytophagous Pentatomorpha are capable of

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cannibalism and suck eggs. It is interesting to note that when Oncopeltus feeds on the eggs or other individuals of its own species, it first secretes a flange of sheath material on the surface; thus seeds and prey are treated alike. The seed feeders, however, require an immobile food source and will attack only disabled individuals, while the true carnivores must rapidly immobilize their prey. The difference in the feeding process between the two forms thus resides in the chemical content of the saliva. Predatory (as opposed to blood-sucking) reduviids inflict painful bites on man. Some species spit their saliva for distances up to 30 cm as a defensive mechanism. As Edwards (1 96 1) showed, this spitting is without effect on the normal invertebrate prey of the insect, for the saliva is harmless when applied topically to arthropods or even when ingested by them. The spitting is apparently “aimed” at the sensitive mucous membranes of the eyes, nose and mouth of potential vertebrate enemies. Once the saliva is injected into an arthropod, however, it is highly toxic. The reduviid Platymerus rhadamanthus Gaerst immobilizes a large Periplanetu americana within two t o five s by injecting about 10 mg of saliva (Edwards, 1961). The saliva of predators in the Hydrocorisae and in other Geocorisae similarly rapidly immobilizes the prey. The offensive function of spitting by Platymerus probably explains the concentrated nature of this secretion, which Edwards used as a source for analysis of the saliva. In sharp contrast, the saliva ejected by the phytophagous forms outside their food is very dilute indeed (Strong and Kruitwagen, 1968; Bongers, 1969). Edwards (1961) compared the saliva of Platymerus to snake venom: both contain hyaluronidase, proteinase and phospholipase, although the activity of the salivary phospholipase of the insect was somewhat weaker than the phospholipase of snake venom. The hyaluronidase acted as a spreading agent, reducing the viscosity of some body fluids and attacking the intercellular matrix, thus speeding tissue lysis by the other enzymes. There was particularly rapid disruption of neural function, mainly due to lysis of the outer phospholipid membranes of nerves. The insect did not, however, contain ATP-ase or serotin, which are commonly present in snake venom, but which are presumably specifically adapted to vertebrate prey. By comparison, the saliva of the insects that suck vertebrate blood seems to contain no venom-indeed the habit depends for its effectiveness on the absence of immediate reaction on the part of the host (Table I). Little is known of the composition of the saliva of the blood-suckers, however.

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THE SALIVA OF HEMIPTERA

Table I Toxicity of Hemiptera salivary gland homogenates as indicated b y effect o n heart beat of Periplanetaa Donor species

Dilution factorb

Action

I. Predators Naucoris cimicoides Platymeris rhadamanthus R h in coris carm elita Reduvius personatus

40

Immediate cessation

40

Immediate cessation

11. Phytophagous bugs Pentatoma rufipes

1

Oncopeltus fasciatus

1 ,

Slight increase in rate: cessation after some minutes Slow decrease in amplitude

111. Blood-suckers

Rhodnius prolixus Triatoma protracta

i

10

No action

Data from Edwards (196 1). The unit is a pair of glands homogenized in 0.05 ml saline; larger numbers indicate relative increases in dilution. (I

VII. CHEMICAL COMPOSITION AND FUNCTION OF THE SALIVA A. SHEATH MATERIAL

The Homoptera and Heter0ptera:Pentatomorpha produce an oral secretion that originates from the salivary glands and that solidifies very rapidly after secretion. Such recent studies as have been done on this material, whether from aphids, pentatomids or lygaeids, indicate that it is mainly protein, but contains about 10% phospholipid and probably some conjugated carbohydrate (Miles, 1964b, 1967b). It is mainly hydrogen bonded, but is stabilized by disulphide linkages (Miles, 1967a, 1969a). Once formed it is impermeable (Kinsey and McLean, 1967). It is also inert, for when aphids ingest their own sheath material, they are unable to digest it (Moericke and Mittler, 1965, 1966). When first secreted, the precursors of the sheath material are admixed with soluble amino acids and these diffuse from

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the sheath during the second or less while it is solidifying (Miles, 1964b). Oxidizing enzymes are secreted with the sheath material, which in some species actually appears to be tanned by these enzymes (Miles, 1964b). The sheath can be formed in the absence of oxidizing enzymes however (Miles, 1967a), and these enzymes are probably also secreted by Hemiptera that secrete no sheath (Miles, 1964a). The precursors of the sheath material are secreted by specialized lobes of the salivary glands and are discharged more or less independently of the other products of the glands. The secretions of all parts of the glands are nevertheless freely miscible with one another and the sheath material shows no signs of beginning to solidify if for instance the contents of the glands are mixed together in vitro, as long as oxygen is rigorously excluded (Miles, 1959b, 1967a). Within the glands, the precursors of the sheath material are maintained in solution by reducing conditions, probably due to the presence of free sulphydryl groups (Miles, 1967a). The precursors are intimately mixed with amino acids that are presumed to prevent the formation of hydrogen bonds by maintaining a high dielectric constant (Miles, 1964b). Any other secretions of the salivary glands that are likely to be mixed with the sheath precursors before they are ejected are similarly reducing and contain free amino acids, although the analysis of freely soluble metabolites differs in different parts of the gland (Miles, 1967a, b). Once the precursors are secreted, diffusion of amino acids out of the mixture and the presence of free oxygen rapidly bring about solidification by hydrogen bonding and disulphide bonding respectively. The oxidizing enzymes present in the saliva undoubtedly assist in the oxidative part of the process of solidification, although they do not appear to be essential (Miles, 1967a). The work of Friend and Smith (1 97 1) indicates that the secretion of sheath material by Homoptera and Pentatomorpha is matched in Rhodnius prolixus, a blood-sucking bug within the Cimicomorpha which secretes a flange at the surfaces it penetrates; but nothing is yet known of its composition. The function of the stylet-sheath remains problematical, despite discussion by several authors (Mittler, 1954; Saxena, 1963; Miles, 1968b; Bongers, 1969). Mittler’s suggestion that it serves t o seal the stylets of Homoptera into cells (e.g. phloem sieve tubes), the contents of which are under turgor pressure, still seems sound when

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207

applied to sap-sucking aphids, particularly since recent studies have confirmed that these insects require a diet under pressure for their optimum growth (Wearing, 1968). The claim by Hori (1 968a) that the pentatomid Eurydema secretes a sheath when feeding on phloem sap, but not when feeding on mesophyll cells, would seem to be evidence that in the Pentatomorpha also, the sheath is an adaptation to feeding on pressure systems. But the foregoing does not explain why no sheath is produced by Dysdercus when it feeds on phloem sap (Saxena, 1963) or why Homoptera that feed only on mesophyll tissue do produce a stylet-sheat h. Saxena suggested that the flange secreted on to surfaces by Dysdercus enabled the insect to attach the labium firmly to the substrate without the danger of the mouthparts slipping sideways while the stylets were forced into the substrate. This explanation is supported by the cement-like nature of the sheath material and its ability to adhere to waxy surfaces of plants by virtue of its phospholipid content (Miles, 1968b), but such an explanation ignores the complete stylet-sheath that most phytophagous bugs secrete within plant tissues. Miles (1968b) contrasted the relatively small lesions made by stylet-sheath feeders of the Pentatomorpha with the large lesions produced by lacerate-and-flush feeders of comparable size in the Miridae. He suggested that if the stylet-sheath is seen as maximizing the efficiency of feeding on individual cells by preventing losses into intercellular spaces, then, in contrast, lacerate-and-flush feeders probably need to scavenge the contents of ruptured cells with large quantities of dilute saliva. Possibly the functional significance of the stylet-sheath can properly be appreciated only in an evolutionary context. Goodchild (1966) discussed the evolution of the physiology of the digestive system of Hemiptera in relation to sources of food. Although he did not refer to the secretion of sheath material, his ideas are relevant to it: as already noted in Section 11, he would consider the primitive mode of feeding in the Hemiptera to be stylet-sheath feeding (that arose from scratch-and-suck feeding, presumably when the insects had developed the ability to secrete a solidifying saliva). Secondarily, the Heteroptera became omnivorous. Even among the Homoptera, the secretion of a stylet-sheath may not occur automatically whenever the stylets are used (Hennig, 1963) and presumably some of the ancestral Heteroptera lost the ability to secrete a stylet-sheath

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almost entirely when they became predators, using the stylets and saliva as a lacerate-and-flush system. Goodchild assumed the mirids to be evolved from such predatory ancestors. If so, they retained the lacerate-and-flush system perforce because unable to secrete a stylet-sheath; indeed Goodchild specifically compares the feeding of the bryocorine mirids with that of the predatory Heteroptera. At the same time, it would appear that predators such as the reduviids include species that retain some of the ancestral ability to secrete sheath material, as the recent discovery of the salivary flange secreted by Rhodnius prolixus has demonstrated (Friend and Smith, 197 1 ). Within this evolutionary interpretation, the Heter0ptera:Pentatomorpha must be seen as Heteroptera that have retained the stylet-sheath feeding habit, but that nevertheless display some of the possible intermediate kinds of feeding between stylet-sheath and lacerate-and-flush. Thus the seed-feeders use both and also perhaps so do the pentatomids such as Eurydema which according t o Hori (1968a), uses the stylet-sheath method when it sucks phloem sap and the equivalent of a lacerate-and-flush technique when feeding on mesophyll. According to Saxena’s descriptions, Dysdercus has almost lost the use of a stylet-sheath, but retains the flange alone-perhaps because of its value in steadying the stylets during the initial penetration of a surface which, if a cotton seed, may be particularly hard to penetrate. An evolutionary interpretation in this way allows for the retention of ancestral habits and for changes in function that would be difficult to envisage in a strictly functional analysis. B. WATERY SALIVA

Some of the known components of the non-gelling secretions (watery saliva) of the salivary glands are summarized in Table 11. The watery saliva is a vehicle for hydrolysing (“digestive”) enzymes. Many of the Heteroptera can be induced t o secrete a watery liquid from the free tips of the stylets while these are outside food substrates; e.g. by manipulation of the labium, especially after subjecting the insects to a humid atmosphere or after injecting them with pilocarpine (Miles and Slowiak, 1970) or by amputation of the antennae (Clarke and Wilde, 1970a). Nevertheless, there is doubt whether this experimentally induced secretion properly represents the watery saliva secreted within substrates. Strong and Kruitwagen

Table I1

-$

Some recent studies of the soluble contents of the salivary secretions of Hemiptera

I

0

Compounds

Insects studied

Features noted

References

1. Enzymes Amylase

Oncopeltus fasciatus (Dall) (Lygaeidae) Heteroptera (various families) Lygus disponsi Linnavouri (Miridae)

Cellulase Oligosaccharases

Pectinpolyglacturonase

Empoasca fabae (Harris) (Jassoidea) Aphididae Oncopeltus fasciatus Dolycoris bascarum L. (Pentatomidae) Lygus disponsi Empoasca fabae Aphididae Auchenorrhyncha Heteroptera Lygus hesperus Knight (Miridae)

Occurrence

Feir and Beck (1 96 1)

Origin in glands Activation by C1and NO3 Characteristics

Miles (1 967a) Hori (1969) Hori (1970a, d)

Variability in quantity Role in digestion Occurrence

Hori (1 970b) Hori (1 970c) Berlin and Hibbs (1 963)

Occurrence Heat stability Occurrence . Occurrence

Adams and Drew (1963a) Adams and Drew (1963b) Feir and Beck (1 96 1 ) Nuorteva and Laurema ( 1 96 1b)

Absence Occurrence Variable occurrence

Hori (1 970c) Berlin and Hibbs (1 963) Adams and McAllan (1958)

Absence Occurrence in Miridae only Role as spreader in phytotoxicity

Laurema and Nuorteva ( 1 96 1) Laurema and Nuorteva (1961) Strong (1 970)

N

0 \D

Table 11-cont. Compounds

Insects studied

Features noted

References

1. Enzymes Hyaluronidase Proteinase

Platymeris rhadamanthus Gaerst. (Reduviidae) Platymeris rhadamanthus Oncopeltus fasciatus (Lygaeidae) Empoasca fabae (Jassoidea) Dolycoris baccarum L. (Pentatomidae) Lygus disponsi (Miridae) Aphididae

EsteraseILipase

Oncopeltus fasciatus (Lygaeidae)

Phospholipase

Platymeris rhadamanthus (Reduviidae) Pyrrhocoris apterus L. (Pyrrhocoridae) Pyrrhocoris apterus L. (Pyrrhocoridae) Hemiptera

Acid phosphatase Phosphorylase Phenolase/ peroxidase

Pentatomorpha (Pentatomidae and Lygaeidae)

Edwards (1 96 1)

Role as spreader in toxicity to prey Role in toxicity Occurrence

Edwards (1 96 1) Feir and Beck (1961)

Occurrence

Berlin and Hibbs (1963)

Variability in quantity Variability in quantity

Nuorteva and Laurema (196 1b)

General properties Occurrence Occurrence

Hori (1970d) Schaller (1 968a) Feir and Beck (1 96 1)

Origins in glands Role as neurotoxin

Miles (1967a) Edwards (1 96 1)

Occurrence

Kloft (1 960)

Occurrence

Kloft (1960)

Presence in glands of phytophagous and predatory species

Miles (1 964a)

Occurrence

Hori (1970b)

Miles (1964a) and Miles and Slowiak (1 970)

Oncopeltus fasciatus (Lygaeidae) Aphididae

Origin in glands

Miles (1 967a)

Occurrence

General

Discussion of role

Miles (1965) and Miles and Slowiak (1970) Miles (1 969b)

2. Metabolites Amino acids

Phenylalanine/ Tyrosine/DOPA Cysteine and derivatives

Aphididae

Occurrence and phytotoxicity

Kloft (1960); Anders (1961) and Schaller (1 968b)

Occurrence and transfer to saliva Role in experimental induction of galls

Miles (1964a, 1967a, b)

Pentatomorpha (Pentatomidae and Lygaeidae) Pentatomorpha (Pentatomidae and Lygaeidae) Aphids

Occurrence

Schaller (1968a)

Eumecopus punctiventris Stil (Pentat omidae)

Occurrence and m etab o h m

Miles ( 1 969a)

Miles (1964b, 1968a)

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PETER W. MILES

(1968) have shown that the secretion discharged from the free tips of stylets of Lygus hesperus Knight outside plant tissue lacks detectable quantities of the enzymes the insects discharge within feeding punctures. The lygaeid Oncopeltus is known to secrete esterase into substrates (Feir and Beck, 1961) and the enzyme can be found in its salivary glands-yet Miles (1967a) was unable t o detect it in saliva collected directly from the tips of the stylets. It would thus seem that the watery saliva of phytophagous Hemiptera is of varying composition. When secreted within plants it contains hydrolysing enzymes, but it is subject t o varying degrees of dilution and when discharged outside plants may contain little but water. Studies based on the watery saliva collected from the free tips of the mouthparts are thus of limited use in determining the composition of the saliva; yet it is indeed difficult to collect the watery saliva secreted within plants. Saxena (1 963) managed to collect drops of saliva discharged by Dysdercus while it was feeding on thin slices of cotton seed kernel. The best attempts so far t o analyse watery saliva as actually discharged into plants have been made by allowing aphids t o probe into damp filter paper (Schaller, 1963); by caging Homoptera over filter paper covered with plastic membrane or parafilm (Adams and McAllan, 1958) or by allowing larger insects to probe through the surface of a normal food material into a pocket of powdered cellulose (Feir and Beck, 1961). The results of such experiments are still open to some doubt. One of the functions of the very dilute saliva discharged by Hemiptera is probably the bringing back of soluble materials to the gustatory sensilla of the epipharyngeal organ; thus in the absence of the gustatory stimuli normally associated with food-for instance in filter paper or powdered cellulose-the watery saliva discharged might still lack the normal concentration of salivary enzymes secreted into food. The closest approach to determining salivary enzymes under the natural conditions of their secretion is probably that of Adams and McAllan (1 958) who have caged insects over filter paper impregnated with the pure substrates for the enzyme. By determining the breakdown or otherwise of these substances chromatographically, they were the first t o demonstrate the secretion of pectin polygalacturonase by Hemiptera. Other studies of this enzyme are summarized in Table 111. Salivary polygalacturonases are assumed to aid the disruption of the midlamellae of the cell walls of plants rather

THE SALIVA OF HEMIPTERA

213

Table 111 Occurrence of pectinase in the saliva of Hemiptera ~

1. Present Aphididaea Aphis abbreviata Patch Dactynotus sp. Macrosiphoniella millefolii (DeGeer) Myzus persicae (Sulz) Rhopalosiphum rhois (Monell) Aphis fabae Scop. Aphis sedi Kltb. Aphis spireacola Patch Eriosoma americanum (Riley) Eriosoma lanigerum (Hausm.) Phorodon humili (Schrank) Aulacorthum solani Cinara spp. Periphyllus negundinis (Thomas) Pterocomma spp. Schiz olach n is pin i-radiata (Davidson) Thomasiniellula populicola (Thomas) Ceruraphis viburnicola (Gill)

In both apterae and alatae In both apterae and alatae In both apterae and alatae In both apterae and alatae In both apterae and alatae In apterae, not always in alatae In apterae, not always in alatae In apterae, not always in alatae In apterae, not always in alatae In apterae, not always in alatae In apterae, not always in alatae Known from apterae only Known from apterae only Known from apterae only Known from apterae only Known from apterae only Known from apterae only Known only from immature alatae

Auchenorrhyncha (Cicadellidae)" Dalbulus maidis (Del and Wohl)

Demonstrated in adults

Heteroptera (Miridae)b ~~~

~

Adelphocoris seticornis (F) Poeciloscytus unifasciatus (F) Miris dolabratus (L) Stenodema calcaratum (Fall.) Capsus ater (L) Lygus pratensis (L) Lygus hesperus KnightC

~

~~

In males and females In males and females In males and females In males and females Demonstrated in female only Demonstrated in female only In saliva discharged in plants, but not in saliva discharged outside plants

214

PETER W. MILES

Table 111-cont. Heteroptera (Lygaeidae)' Demonstrated in adults

Liocoris lineolaris (Beauv.)

2. Absent Aphididae:' Aphis cardui L. Ceruraphis eriophori (Wlk.) Melaphis rhois (Fitch) Myzus cerasi (F) Prociphilus tessellata (Fitch)

Psyllidae:' Unidentified spp. Auchenorrhyncha:b Various spp. in Fulguroidea (5), Cercopoidea (2) and Jassoidea (10). Heteroptera:b ~

Various spp. in Lygaeidae ( l ) , Coreidae (2) and Pentatomidae (4) a

In saliva secreted into filter paper (Adams and McAllan, 1958). In salivary glands (Laurema and Nuorteva, 1961). (Strong, 1970).

than to function primarily in the digestion of a major nutrient (Adams and McAllan, 1958; Strong, 1970). Many workers have contented themselves with analysis of whole gland homogenates or (from the larger Heteroptera) the secretion expressed from the sac-like lobes of the principal gland. The main generalizations that have emerged from such studies can be summarized as follows:

(1) The saliva of phytophagous Hemiptera most often contains amylase. Other carbohydrases are sometimes present, but Hori ( 1970c) states that Lygus disponsi Linnavouri secretes a salivary amylase and no enzymes that hydrolyse oligosaccharides; all further digestion of carbohydrates occurring in the midgut. (2) Those insects that suck phloem sap as their principal food

THE SALIVA OF HEMIPTERA

215

are said not t o have hydrolytic enzymes other than carbohydrases in their saliva, although among these enzymes may be counted the pectin polygalacturonases secreted mainly by aphids and mirids (Adams and McAllan, 1958; Laurema and Nuorteva, 1961). (3) Those insects that feed on mesophyll tissues or seeds however, are likely to have both proteinases and esterases (or lipase) in their saliva (Nuorteva, 1958; Feir and Beck, 196 I ). Other salivary enzymes have also been found. Kloft (1960) showed that the mesophyll-feeder Pyrrhocoris upterus L. secreted acid phosphatase and a phorphorylase among the other enzymes in its saliva (whereas these enzymes were not secreted by sap-sucking aphids). Miles (1968b) reported that the saliva of all the Hemiptera excepting those that suck vertebrate blood secrete a salivary polyphenol oxidase (PPO). The latter generalization is perhaps too sweeping, however, for the Jopeicidae in the Cimicomorpha are predatory bugs that apparently have no .detectable oxidase in the glands (Davis and Usinger, 1970). In addition t o PPO, Miles and Slowiak ( 1970) report that the insects also secrete peroxidase and despite the possible confusion of the two enzymes (van Loon, 1971), the discovery of insects that secrete the one without the other would seem to corroborate the finding. The function of the oxidases is discussed in the concluding section; so far no function for them has been convincingly demonstrated. The enzymic content of the saliva of any particular species cannot be assumed t o be constant. There is considerable evidence for the variation in content of proteinase especially. Nuorteva ( 1 956b) found that in the mirid Miris dolubrutus L. salivary proteinases occurred in the juveniles and in only female adults and Nuorteva and Laurema ( 196 1b) found that the amount of proteinase in the salivary glands of the pentatomid Dolycoris buccururn (L) increased when the amount of protein in the diet increased. Similarly Hori (1970b) found that not only did both amylase and proteinase content of the salivary glands of the mirid Lygus disponsi vary with the physiological state of the insect, but that the proteinase content was variable even between the two glands of a single individual and that there was no correlation between the amount of proteinase in the glands and the less variable amount of amylase present. Similarly, Adams and McAllan (1 958) found that whether detectable pectinase occurred in the saliva of aphids depended on the species of plant on which they were feeding, or whether they were apterous or alate; sexual differences were also noted in the latter (Table 111).

216

PETER W. MILES

The saliva of Hemiptera is noted for its content of amino acids and, as indicated in SectionVI1,A the presence of these may be essential in those species that secrete a stylet-sheath. In the grape phylloxera, Viteus vitifolii Fitch, Anders (1 96 1) and Schaller (1 960) have shown that especially large quantities of amino acids are secreted, no doubt because this insect is without an anus and must excrete via the salivary glands. A distinctive feature of the saliva of Viteus is that it contains an unusually high content of the basic amino acids asparagine and glutamine, which presumably represent the end points of nitrogenous excretion in the insect. In other species of aphid, however, there are only trace quantities of the dibasic amino acids, although aspartic acid and glutamic acid may be present in significant amounts (Table IV). Lately, the work of Miles (1967b) with radioisotopes has shown that any soluble metabolite introduced into the haemolymph will appear also in the watery saliva. Miles worked mainly with the watery saliva discharged from the free tips of the mouthparts and this presumably represented direct excretion of water from the haemolymph along with traces of any metabolites present in it. Nevertheless, analysis of the contents of the whole glands (Miles, 1967b, 1969a) suggests that some metabolites are selectively sequestered from the haemolymph by the salivary glands. Thus a large part of labelled cysteine injected into a pentatomid subsequently appeared in the salivary glands and secretions; Hagley (1 969) noted a disproportionately large amount of phenylalanine in the salivary glands of the cercopid Aeneolamia compared with the composition of the haemolymph. It is therefore safe to conclude that all the soluble metabolites that occur in the haemolymph, be they salts, amino acids, sugars or the precursors of lipids (e.g. glycerol), will occur in the salivary glands and will be secreted (or excreted) in the very dilute secretion that comes mainly from the accessory gland (see below); but some metabolites are specifically sequestered in the principal glands. Of particular interest in relation to phytopathogenicity is the possible occurrence of the plant hormone p-indolyl acetic acid (IAA) in the saliva. Duspiva (1954) and Miles (1968a) claimed to have demonstrated the in vitro production of IAA from tryptophan by homogenates of the salivary glands of an aphid and heteropteron respectively. Nuorteva ( 1962) claimed evidence for the transfer of IAA and gibberellic acid from diet to saliva of the auchenorrhychan Calligyponu pellucidu. On the other hand, IAA is a common

THE SALIVA OF HEMIPTERA

217

endpoint of indole metabolism in animals, and the recent work of Schaller (1 965, 1968a) has demonstrated conclusively that the saliva of some aphids contains IAA: whether in sufficient quantity to account for their phytopathogenicity will be discussed in the next section. A possible means of demonstrating the origin of a salivary secretion is provided if the pH of the secretion can be determined. Thus the pH of the sheath material is slightly on the acid side of neutral (Saxena, 1963; Miles, 1965, 1968b); and the pH of the watery saliva is alkaline, with pH values of up to 9 (Miles, 1965, 1968a). On the other hand, the contents of the principal salivary gland never have as high a pH as this, and thus the more strongly alkaline the watery saliva, the more it is likely to be the secretion of the accessory gland (see Section X) and the less it comes from the principal gland and thus contains the digestive enzymes and other compounds secreted by that gland. VIII. PHYTOPATHOGENICITY

Large numbers of some species of aphids or leafhoppers can abound on plants and apart from some stunting, or contamination of the foliage with honeydew, little effect on the growth pattern of the plant can be observed. Yet individual mirids of some species produce large lesions or such violent necrosis at the points where they feed that the fruit will absciss prematurely or grow with characteristic deformations (Carter, 1962). Although lacerate-and-flush feeding necessarily results in necrosis, the lesions produced by cocoa capsids on young stems may become surrounded by a zone of increased growth, such that in time the lesion becomes occluded (Carter, 1962). Stylet-sheath feeding is usually accompanied by less immediate damage, and is more likely to cause longer-term disturbances in growth. Colonies of the woolly aphis, Erisoma lunigerum (Hausm.) produce intumescences on the part of the stem or root on which they feed. The grape phylloxera, Viteus vitifolii Fitch causes similar swellings on roots; by depressing growth at the point where it feeds and stimulating it further away, this insect causes characteristic pocket galls on the vine leaves (Fig. 9). Similarly, many (but not all) sedentary psyllids and coccids produce galls. Among the scratch-and-suck feeders also, some tingids (and Thysanoptera) cause pocket galls in which they live colonially.

Table IV Analyses of aphid saliva for some metabolites Viteus Eriosoma vitifolii lanigerum

Serine Alanine Aspartic acid Glutamic acid Glycine “Underasparagine” Leucine Valine Asparagine Glutamine Arginine Lysine Histidine Cysteine/Cystine Tyrosine PAlanine Threonine a-Aminobutyric acid Methionine

++ ++ ++ + + + + f

+++

++ + +

Aphis pomi

+

+++

+

f

+++

i

-? -

f f

+

+

Sappaphis Myzus Hyalopterus Crypto- Aphis myz.us smnbuci mali cerasi pruni rib is Amino acids

Myzus ascalonicus

Megoura viciae

f f f f

?

++ f f f

-

i i

-

-

f f

*3M %

Phenolics Naringenin Phloridzin? Quercitrin Quercetin Chlorogenic acid? Rutin? Hyperin? Robinetinaglycone? Protocatechuic acid? Ferrulic acid? pCumaric acid?

+ +

?

f

-

+ +

?

f

+

+ ++ + + f +

f f

+ f +

+

-

+

f

-

?

?

-

-

? ?

f

Indole acetic acid

*

*

+

Proteinase

+

?

-

?

-

-

-

+

-

-

-

-

Results for some unidentified amino acids and phenolics not included: Number of ++ indicates strength of reaction, * indicates trace only, and ? an uncertain result. Results for Viteus vitifolii represent the pooled results for seven biotypes of the species. (From Schiiller, 1968a).

4

E

Bt: <

E

s

M

p

>

220

PETER W. MILES

a. Dreyfusia spp. Increase in cell protein but not cell growth (from Kloft, 1960).

b. Eriosma lanigerum Simple surface gall.

a

d. Wteus vitifolii At root tip of vine causes swelling surrounding feeding puncture (from Anders,1961).

e. Viteus vitifoii on foliage causes occluded gall and with daughters o calonial gall forms (adapted from AndersJ961).

a c. Cocoa capsids Cause occluded necrosis on young shoots by stimulating peripheral growth.

Fig. 9. Formation of galls: a: “physiological gall”. b: surface gall; c: occluded necrosis; d: root gall; e: pocket gall on leaf.

Such localized effects of feeding must be differentiated from systemic stunting or deformation that are caused by viruses or by mycoplasmas (Raine and Forbes, 1969; Whitcomb and Davis, 1970). Localized effects, whether or not they take the form of necrotic lesions or galls, can usually be shown to be independent of any disease organism and to be due to the toxic effects of the saliva discharged by the insects into the plants during feeding. The identity of the salivary toxins responsible has elicited much interest. In recent work on the causal agents of galls, a major controversy has been whether phytopathogenicity resides in free amino acids present in the saliva of plant bugs (Anders, 1961 ; Kloft, 1960) or in the IAA that is either secreted in the saliva (Schaller, 1968a) or may be formed from tryptophan by oxidative deamination brought about by salivary oxidases (Miles, 1968a, b). Recently an explanation of the formation of lesions by mirids has been advanced as a separate hypothesis; namely that pectin polygalacturonase in the saliva causes a rapid spread of other salivary enzymes into the tissues of plants during the lacerate-and-flush type of feeding of these insects (Strong, 1970). Kloft (1 960) showed that the amino acids secreted by aphids in their saliva caused increases in the permeability of plant cells and

THE SALIVA O F HEMIPTERA

22 1

probably thereby increased respiration, transpiration and protoplasmic streaming. Insects that suck phloem sap release their saliva only while penetrating to the phloem or at the moment of withdrawal of the stylets and the pathological effects of an insect such as the onion aphid, Myzus ascalonicus Donc. are thus not pronounced. In contrast, the Dreyfusi spp. (Adelgidae) that multiply on silver fir, feed on the parenchyma and secrete saliva continually; the net result of their feeding is similarly progressive, resulting in the accumulation of proteins in the bark just under the points of feeding of the insects. Such an accumulation of nutrient materials without any obvious hypertrophy of the plant tissues Kloft called a “physiological gall” and ascribed it principally to a stimulation of protein synthesis brought about by the amino acids discharged by the insect in its saliva. Anders in a series of papers ( 1960a, by 196 1) described his analysis of the saliva of larvae of phylloxera that were producing galls on vines. As has been discussed in Section VI1,B the unusually high concentrations of amino acids that this species has in its saliva have been considered excretory in nature. Anders claimed t o have identified three amino acids in particularly high concentrations namely lysine, histidine and tryptophan, with significant quantities also of glutamic acid and valine. When germinated in solutions of these amino acids (but not others), the roots of grape seedlings produced subapical swellings that Anders compared to the subapical galls produced by phylloxera feeding just behind the root cap (Fig. 9d). On the other hand, similar results have been described when vine roots are grown in potassium phosphate solution (Miles, 1968a) and perhaps solutions of other compounds that affect the permeability of the cells in the meristematic subapical region of the rootlet would cause similar growth disturbances. Careful analysis of the saliva of phylloxera and several other species of aphids with varying degrees of phytopathogenicity led Schaller ( 1 968a) to conclude that in fact the amino acids named by Anders were not typical of the saliva of phylloxera, in which he found the principal amino acids to be glutamine, serine, alanine and aspartic acid. The saliva analysed by Schaller was collected from dampened filter paper and there is thus the problem of whether the saliva discharged in what were presumably exploratory probes was of the normal strength and composition secreted by the insects when they feed on plants.

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PETER W. MILES

Nevertheless, that amino acids were found at all gives a good indication of those that feature most prominently in the salivary secretions of the various species. Schaller also found IAA and several phenolic compounds in the saliva of aphids. The average quantities of compounds collected from an individual aphid during the 6 to 8 h when it was permitted to probe the filter paper were up to a maximum of 2 x 10-Fg each of the major amino acids present in the saliva. Eriosomu Zunigerum secreted a similar quantity of IAA in its saliva and somewhat less IAA was retrieved from the saliva of Viteus vitifolii and of Myzus cerusi (Table IV). Schaller concluded that although the analysis of the saliva of aphids showed each to have a specific composition qualitatively and quantitatively, certain generalizations emerged. The greater the ability to cause hypertropy of tissues in their host plants, the greater the overall concentration of amino acids, the greater the concentration of IAA and the lower the concentration of phenolics. He concluded that IAA must work together with free amino acids to produce galls while the phenolic compounds, by producing quinones that reacted with amino acids and destroyed IAA, reduced the likelihood of gall formation. In a series of brilliant experiments, Schaller (1968b) tested his conclusions by introducing solutions that simulated the saliva of aphids into the petioles of developing vine leaves. To do this he filled hollow glass microneedles with the solution and allowed the needles to remain permanently in place while the leaf grew (Fig. 10). He found he could imitate the galls of phylloxera with remarkable felicity and the nearer the solution approached the actual composition of phylloxera saliva with respect to amino acids and IAA, the more “perfect” the gall produced. Strong ( 1970), meanwhile, has investigated the destructive lesions produced by Lygus hesperus Knight. He concluded that the saliva contained no IAA and lacked any physiological system by which IAA could be synthesized by the saliva released within plants. He ascribed the large lesions produced by individual bugs to the salivary pectin polygalacturonase which, by greatly increasing the effective spread of the digestive enzymes in the saliva during the lacerate-andflush feeding practised by this and other mirids, caused a much larger lesion than would be produced by a sap-sucking insect of comparable size. Presumably the effects of the feeding of the mirids that attack cocoa must involve more than the destructive processes in the feeding of Lygus, since Strong’s explanation does not dispose of the

THE SALIVA OF HEMIPTERA

223

Fig. 10. Formation of “histoid” gall on petiole of vine leaf by a mixture of amino acids and IAA simulating the saliva of the grape phylloxera, V . vitifolii. (After Schiller, 1968b).

observation that hypertrophy of cells can occur at the edge of the lesions produced by the “cocoa capsids” (Carter, 1962). In fact, Scott (1 970) showed that the feeding both of L. hesperus and of L. elisus Van Duzee on either carrot seeds or beans, while depressing initial growth of the seedlings after germination, actually increased their eventual growth compared with plants grown from seeds on which the insects had not fed. A third approach to the phytotoxicity of the saliva of Hemiptera is the proposal that the salivary phenols and phenolases of Hemiptera directly interfere with the growth-controlling mechanisms of meristematic cells. As Gordon and Paleg (1 96 1) demonstrated in vitro, quinones produced by a system such as dihydroxy phenylalanine (DOPA) plus polyphenol oxidase (PPO) will transform tryptophan by oxidative deamination to IAA. On this basis, Miles (1969b) proposed that an increase in IAA content of cells surrounding the feeding punctures of Hemiptera would be likely. The PPO would seem to be provided in the saliva of all the phytophagous forms; phenylalanine is transformed in the salivary glands to DOPA and the tryptophan, although present in small amounts only, would

224

PETER

W. MILES

nevertheless occur in the haemolymph and therefore saliva as the free metabolite. Miles thus considered all phytophagous Hemiptera as possessing the potential for forming galls on their food plants and he considered they would do so provided the precursors of the IAA-producing reaction were present in sufficient concentration and the insect injected enough of its saliva in a place where the plant’s cells were still sufficiently meristematic. As a demonstration of this hypothesis, he made a non-cecidogenic lygaeid temporarily produce galls on cotyledons of a sunflower seedling by implanting into the bug flakes of agar containing phenylalanine and tryptophan (Miles, 1968a). Scott (1970) similarly suggested that the potential of the saliva to form IAA was a likely explanation for the enhanced growth of plants grown from seeds that had been attacked by Lygus spp. A number of problems arise from the hypothesis that salivary phenols and phenolases cause increases in IAA concentrations in the tissues of plants. Phenolases have also been implicated in the breakdown of IAA (Pilet et al., 1970); Schaller (1 968a) correlated an increase in salivary phenolics with decreases in both IAA-content and phytopathogenicity of the saliva. Moreover, as Schaller ( 1968b) has pointed out, the total amounts of known metabolites injected by the insects into plant tissues are small compared with the amounts already present within the tissues themselves. Indeed the amino acids in the saliva of a sap-sucking aphid represent merely a small proportion of the amino acids obtained by the insect from the plant’s own phloem sieve tubes and the galls induced by phylloxera are much richer in IAA than the saliva injected into them by the insects. The question thus arises whether the observations so far made on possible gall-inducing substances have in fact been observations of secondary effects: the amino acids and IAA in the saliva of cecidogenic species being derived from the metabolites already present in rich supply in the galls, rather than the galls being due to the substances introduced in the saliva of the aphids. Nuorteva’s (1962) work on the transfer of dietary amino acids t o the saliva of Calligypona and the evidence that even enzymes are readily transported unaltered from haemolymph to the saliva of plant bugs (Miles and Slowiak, 1970) would add weight to such an interpretation. Schaller’s successful demonstration that the injection of amino acids and IAA does cause galls must tip the balance in favour of the explanation that cecidogenesis indeed lies in the presence of these

THE SALIVA OF HEMIPTERA

225

compounds in the saliva of the insects, even though the apparent anomaly that they are already present in higher quantities in the plant remains. A definitive explanation of cecidogenesis is likely to be concerned with the precise location and movement of physiologically active substances, rather than overall concentrations. A sap-sucking insect in effect removes amino acids from sieve tubes and subsequently reinjects them elsewhere in the plant. The hypertrophy that occurs at the edges of the lesions of cocoa-capsids must be due either to a gradient in concentration of substances that cause necrosis at higher concentration and increased growth at lower or to differential rates of diffusion of necrosis-causing metabolites and growth-promoting ones. And it is possible to reconcile the in vitro production of IAA by a phenol-phenolase system, with demonstrations that the same system causes the destruction of IAA, by assuming that the end point of the reaction is dependent on a precise relation between the concentrations of the reactants. IX. SALIVARY GLANDS AND DUCTS

The salivary glands of Hemiptera are labial glands, lying mainly in the anterior region of the thorax, alongside and partly above the gut. Typically, there is on each side a principal gland and an accessory gland. The duct of the accessory gland unites with the duct of the principal gland near where the latter duct leaves the principal gland (Fig. 1 l), and the principal ducts unite to form a common salivary duct that discharges into the salivary pump. From the pump there is a short meatus leading to the salivary canal, the posterior of the canals formed by the opposition of the maxillae (Fig. 8). There is usually a high degree of uniformity of structure in the glands of members of the same family and a degree of likeness is also apparent within higher taxa. At the same time, considerable differences exist between the glands of such taxa as the Sternorrhyncha and Auchenorrhyncha in the Homoptera and between the superfamilies of the Auchenorrhyncha. In contrast, the glands of all the Heteroptera show a remarkable similarity of anatomical pattern, seemingly derived from the relatively simple pattern within the Coleorrhyncha, a small unspecialized group once regarded as heteropteran, but now usually placed in a separate division of the Homoptera.

226

PETER W. MILES

Fig. 1 1. Salivary glands of Hemiodoecus veitchi Hawker (Co1eorrhyncha:Peloridiidae). (From Prendergast, 1962). A nerve (not shown) also innervates the principal gland.

A. APHIDOIDEA

The salivary glands, although apparently simple, have been shown by Weidemann (1968, 1970) to be composed of a number of different kinds of cells (Fig. 12) that show very different cycles of activity and produce visibly different kinds of secretion (Moericke and Wohlfarth-Bottermann, 1960a). At least nine histological types of cell occur in the principal gland apart from the canal cells. Weidemann arranged them into four functional groups, at least three of which produce components of the saliva. Using the electron microscope, Moericke and Wohlfarth-Bottermann ( 1960b) have shown that the secretory products accumulate in vesicles that are extruded from the cell border, and the membrane surrounding the vesicle then breaks down. The principal salivary glands of Aphididae exhibit a partial bilateral division into two symmetrical halves. Down each half runs a stout walled central canal, formed intracellularly by the “canal cells” through which it runs. In the principal glands there is a region of at least seven kinds of “Main Cells”, and at its proximal end, the region of main cells fits into a region of “Cover Cells” rather as an acorn fits into its cup. Both main and cover cells are secretory and small canalulae ramify among the cover cells from the central canals.

227

THE SALIVA O F HEMIPTERA

gland

b

Fig. 12. Salivary glands ofMyzus persicae. a: principal gland; left side showing cell types and numbers (on each side), right side showing nuclei; canal cells and their nuclei not shown; cross-hatching indicates histochemical grouping: A and F are rich in cysteine; B, C, and I in hyaluronic acid; D and E in carbohydrate. (After Weidemann; 1968, 1970). b: principal and accessory gland showing ducts. c: schematic diagram of the structure of the salivary ducts, showing the canal wall surrounded by intercellular spaces and the interdigitating processes of the canal cells which form the corona sinuosa (Faltenkranz). (Redrawn from Moericke and Wohlfarth-Bottermann, 1960b).

228

PETER W. MILES

According to Moericke and Wohlfarth-Bottermann ( 1 960b) the cells of the principal gland discharge vesicles containing their secretory products into the intercellular spaces that surround and indent the canal cells. The surface area of the cells of the central canal is increased by numerous radiating folds, forming the corona sinuosa (Moericke and Wohlfarth-Bottermann, 1 960b) and this is particularly well developed in the region of the cover cells. The secretion of the main cells appears to be relatively electron transparent, while in the region of the cover cells the contents of the canal become electron dense. It is not certain, however, whether this can be ascribed solely to the secretory products of the cover cells or to changes occurring to the products within the canal, possibly mediated by activity of the canal cells themselves. The accessory gland of Aphids is usually a small structure with a few cells producing a very electron transparent secretion. Its canal is continuous with numerous intracellular canalulae that ramify into the secretory cells-an arrangement consistent with the rapid discharge of a dilute secretion. In the phylloxera, Viteus vitifolii, as Rilling (1 966) has shown, the accessory gland is enormously increased in size and is much larger than the principal gland (Fig. 13). The cells of the accessory gland form a syncytium and appear to be greatly enlarged canal cells. Surrounding the intracellular accessory canal is a system of radial, laminar vacuoles and surrounding these again vesicular vacuoles that occupy most of the bulk of the cells. Rilling relates this development of the accessory gland to the gall-forming propensities of the insects, but it is more probable that the hypertrophy of the accessory gland is related primarily to its assumption of the main excretory role in the insects, which not only lack Malpighian tubes, in common with the other Aphidoidea, but also have no anus and must therefore excrete, if at all, through their salivary glands. Viteus show other interesting phenomena. The partial division of the principal glands of aphids into bilaterally symmetrical halves has apparently become complete in Viteus for the principal gland is now completely bilobed, although each lobe has a histologically similar structure. Each is composed of four “giant cells” and two “eosinophilous cells”, and a further group of cells that Rilling homologizes with cover cells, although they are arranged centrally in a “hilus”. The giant cells, the eosinophilous cells, and the cover cells all show secretory activity. In addition there are the presumably

THE SALIVA O F HEMIPTERA

229

Fig. 13. Salivary glands of Viteus vitifolii (Phylloxeridae), semidiagrammatic. a: bilobed principal gland and multilobular (semisyncytial) accessory gland. b: one lobe of the principal gland, showing the four “giant” cells, the branched, double nuclei and the hilus cells. c: cross section through one lobe of the principal gland, principal duct and part of the accessory gland. I to 1V-giant cells; e-one of the two eosinophilous cells; hilus cells in c cross hatched. (Adapted from Rilling, 1966).

non-secreting canal cells. The secretory cells are typically binucleate, and the nuclei of the giant cells are greatly enlarged and branched structures. €3.

JASSOMORPHA

In the superfamilies Jassoidea and Cercopoidea, the various kinds of secretory cells of the principal salivary glands are grouped

230

PETER W. MILES

separately into distinct lobes or regions, while the accessory gland takes on a more or less elongated, vesicular appearance. In Empoasca fabae (Hausm.), the four separate lobes of the principal gland remain a solid mass of cells (Fig. 14a), although between the cells at the centre of the lobe there is an intercellular space, sometimes extending as indentations (“intracellular canniculae”) into the

Fig. 14. Salivary glands of Jassoidea, semidiagrammatic. a: Empoasca fabae (Harris) (Typhlocybidae). (After Berlin and Hibbs, 1963). b: Erythroneura limbatu Matsumura (Cicadellidae). (After Sogawa, 1965). c: Nephotetrix cincticeps Uhler (Deltocephalidae). (After Sogawa, 1965).

secretory cells (Fig. 15). This space communicates at the origin of the lobe with a chitin-lined duct. The accessory gland is typically elongated with at least one portion having a distinct lumen and looking similar in structure to a Malpighian tube. The glandular cells of the salivary apparatus are typically binucleate and the nuclei of the large secretory cells may be ramified (Berlin and Hibbs, 1963). In other Jassoidea, the cells in each lobe tend to remain separated

THE SALIVA OF HEMIPTERA

23 1

Fig. 15. Lumen of glands. a: Xenophyes cuscus Bergroth (Coleorrhyncha), with canniculae only. (After Prendergast, 1962). b: Empwscu f a h e (Harris) (Jassoidea), with confluent intracellular vesicles. (After Berlin and Hibbs, 1963). c: Notonectu glaucu L. (Hydrocorisae), with true but poorly developed lumen approximating the condition in Empouscu. (After Baptist, 1941).

from one another (Fig. 14b) and in the principal glands of some families, Sogawa (1965) described up to seven kinds of secretory cells, arranged not into distinct lobes, but as rosettes of cells (Fig. 14c). He nevertheless distinguished two groups of these cells: the principal salivary duct branches just as it enters the gland into two “ducteoles”; about the end of the one (“posterior”) branch, four kinds of cells form a concentric rosette, while two other kinds of cells are arranged in radial layers along the “anterior” branch. The anterior ducteole remains short in some species, but becomes more elongate in others and may end in a terminal appendage formed from a cluster of small cells of yet another type. The accessory gland of the Jassoidea is typically elongated and may be differentiated into a tubular distal (“tail”) region and a more

232

PETER W. MILES

bulbous proximal (“head” or “cephalic”) region in which the lumen becomes less evident. Sogawa believes the bulbous region t o be secretory or resorptive, possibly increasing the analogy between the accessory gland and an excretory organ. Very little information is available about the glands of the Cercopoidea. Nuorteva ( 1956a) provided illustrations of some glands (Fig. 16), indicating that there occur in them compact groups of cells

Fig. 16. Salivary glands of Cercopidae. a: Aphrophora alni (Fall.) b: Philaenus spumarius (L.) (After Nuorteva, 1956a).

corresponding to Sogawa’s “anterior” lobe, and a “crown” of sometimes tubular acini corresponding to the rosettes of the posterior lobe of some Jassoidea. C . FULGUROMORPHA

The principal gland in the Delphacidae is composed of a large number of multicellular acini, each with its separate ducteole (Fig. 17). It differs from the gland of Empoasca (Fig. 14) chiefly in the larger number of acini and in the absence of a tubular accessory gland. Sogawa (1 965) describes nine kinds of acini, one of which he designates an accessory gland, although it is reniform and not always well separated from the other acini. Balasubramanian and Davies (1968) also distinguish nine commonly occurring acini in the family Delphacidae and a tenth type occurring in some species. Miles ( 1 964a) similarly described ten lobes in the glands of a flatid. Neither Balasubramanian and Davies nor Miles distinguished an accessory gland. Sogawa lists the numbers of cells in each type of acinus as

233

THE SALIVA OF HEMIPTERA

d

I

loop

Fig. 17. Salivary gland of Laodelphax striatellus (Fu1guromorpha:Delphacidae). a to h-distinguishable acini of the principal gland; Ag-accessory gland. (After Sogawa, 1965).

varying between two and eight, while Balasubramanian and Davis list two kinds of acinus as unicellular. The latter authors point out that all cells are binucleate; a feature they appear to share with the Jassomorpha. The ducts in the Fulguromorpha are similar to those of the Cicadellidae in the Jassomorpha: intracellular spaces in the cells of the acini finally join up with ducteoles that have their own characteristic small cells with compact ovoid nuclei. The ducteoles join up with one another and eventually a common salivary duct is formed on each side of the body; these join to form a very short median duct just before this enters the salivary syringe. D.OTHERAUCHENORRHYNCHA

Very little work has been done on the other groups within the Homoptera. The Cicadomorpha are described by Nuorteva ( 1956a) as having bilobed principal glands and a tubular accessory gland, but this account was based on early studies and, as Balasubramanian and Davies (1 968) point out, many past generalizations may need careful revision as more information on the comparative morphology of the salivary glands of Homoptera begins to accumulate. The salivary glands of the small family Peloridiidae, now placed in the taxon Coleorrhyncha separate from the other Hornoptera, are of especial interest. These glands bear a superficial resemblance to the glands of many of the relatively unspeciaIized, carnivorous

234

THE SALIVA OF HEMIPTERA

Heteroptera. Balasubramanian and Davies object that this similarity is more apparent than real, quoting as the major difference between the glands of the heteroptera and Homoptera the presence or absence of a lumen. The glands of the Peloridiidae have no distinct lumen and therefore are typically Homopteran , according to these authors. But the distinction is not necessarily valid, for the salivary glands of many of the Heteroptera Cryptocerata, as Baptist (1941) has shown, have no more lumen than those of the Cicadellidae (Fig. 15). E. HETEROPTERA

Most of the Heteroptera have principal salivary glands that are clearly differentiated into an anterior and a posterior lobe, each of which has a distinct lumen. In most, also, the accessory gland is vesicular. In the Cimicomorpha the accessory gland is a thin-walled bladder, whereas in the Pentatomorpha, the terminal vesicle is completely lost, but the cells at the distal end of the elongated accessory duct take on a swollen, glandular appearance. The nerve supply in the lygaeid Oncopeltus (Bronskill et al., 1958; Miles, 1960b) is two-fold, one nerve supplying the anterior lobe and the other, which travels alongside the principal salivary duct, supplying the posterior lobe and accessory gland and duct (Fig. 18). Although Baptist ( 1 941) illustrated only a single nerve supplying the gland of any one species of heteropteran, that nerve might variously appear to be the homologue of either of those described in Oncopeltus. It is therefore possible that the double nerve supply is typical throughout. According to Miles, this arrangement provides for the discharges either of the anterior and related lobes of the principal gland or of the posterior lobe and accessory gland, thus accounting for the independent discharge of the two kinds of saliva secreted by the Pentatomorpha. In the Cimicomorpha, blood-sucking forms tend to lose the division between the anterior and posterior lobes of the principal gland (Rhodnius) or lose the anterior lobe entirely as in Cimex. Whereas, in the Pentatomorpha, only the Pentatomidae retain the simple bilobed condition; in all other families, the glands are multilobed. Baptist (1941) claimed that such proliferation of lobes was due to subdivisions of the posterior lobe, but studies of innervation and of physico-chemical composition of the secretions could indicate that, on the contrary, the subdivisions are of the

PETER W. MILES

235

Fig. 18. Salivary glands of Heteroptera. a: a predator, Notonecta glauca L. (Hydrocorisae) b: a blood-sucker, Rhodnius prolixus Stal (Reduviidae). c: a blood-sucker, Cimex lectularius L. (Cimicidae). d: a phytophagous lacerateand-flush feeder, Lygus pratensis L. (Miridae). e: a stylet-sheath feeder, Pentatoma rufipes L. (Pentatomidae). f a stylet-sheath feeder, Oncopeltus fusciatus (Dall.) (Lygaeidae). Ag-accessory gland; anterior-anterior lobe; lateral-lateral lobe; n, ,na -nerves; posterior-posterior lobe. (a to e from Baptist, 1941; f from Miles, 1960b). All drawings approximately to scale.

anterior lobe and are concerned with the secretion of sheath material (Miles, 1967a). If, as Goodchild (1 966) has suggested, all Heteroptera are to be derived from phytophagous Hemiptera through omnivorous forms in which the number of lobes was reduced or primitively small as in the aquatic Heteroptera, then the return to a phytophagous condition in the Pentatomorpha has apparently required a more complex division of labour in the salivary secretions and a convergent elaboration of the salivary glands in the Homoptera on the one hand and Heteroptera : Pentatomorpha on the other.

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THE SALIVA OF HEMIPTERA

X. ORIGINS OF THE SALIVA

Tracing the origins of the salivary components within the salivary glands is easiest in the Heteroptera because the great majority have glands that are morphologically simple and each lobe of the gland has its own lumen in which the secretory products of the lobe collect. Little is known of the contents of the salivary glands of Heteroptera other than the Geocorisae, however. Work has been done mainly on the glands of Pentatomorpha; but amongst these, only the Pentatomidae retain a simple two-lobed principal gland and in the other families the problem of identifying the origins of any specific salivary secretion has proved more difficult because of secondary multiplication of the lobes.

A. FUNCTION OF THE ACCESSORY GLAND As a result of the work of Goodchild (1966) it can be accepted that, throughout the Hemiptera, the function of the accessory gland is the production of the copious flows of very dilute secretion that the insects employ when they “taste” surfaces with which the stylets come into contact, as a means of flushing out food sources or as a means of excreting excess water. In all Hemiptera, with only the possible exception of the Fulguromorpha, that part of the salivary apparatus identifiable as the accessory gland is either vesicular or tubular. When the accessory gland is a compact organ, it often has greatly enlarged intracellular vesicles or extensive canniculae; i.e. ways of increasing the surface area of the functional ducts. In the Fulguromorpha, Sogawa ( 1965) identified the lobe in which the duct had a distinctive “corona sinuosa” as the accessory gland. Where it has been specifically identified, the secretion of the accessory gland is alkaline (Miles, 1968b) and presumably the accessory gland thus functions as the first stage of a typical diuretic organ, producing a dilute ultrafiltrate of the circulatory fluid by the active secretion of cations. In a complete excretory organ, there would follow a part that served to resorb useful metabolites such as amino acids and sugars, while exchanging the cations for hydroxonium ions, thus “acidifying” the original secretion. Clearly, however, in the Heteroptera at least, such a resorptive region of the accessory gland does not occur (or is at best only partially effective).

PETER W . MILES

237

B. FUNCTIONS OF THE PRINCIPAL GLAND

In the two-lobed gland of pentatomids, the contents of the anterior lobe rapidly gel on exposure to gaseous or dissolved air or to hypotonic liquids, whereas the contents of the posterior lobe do not. The anterior lobe has thus been identified as the origin of the sheath material. The contents of the anterior lobe gel particularly rapidly in water, in which the free amino acids that are presumed to keep the solution of sheath precursors (Section VI1,A) stable, can rapidly diffuse from the secretion (Miles, 1964b); whereas under paraffin oil, the contents remain fluid. Nevertheless, paraffin oil readily dissolves oxygen and a skin of solidified material forms at the interface of the secretion and the oil. The cells of the anterior lobe in pentatomids do not show uniform chemical activity, however. The cells that contribute most of the sulphydryl groups of the sheath precursors are located at the anterior end of the lobe. In lygaeids, both anterior and lateral lobe of the three-lobed principal gland produce sheath precursors, but the secretion of the lateral lobe contains less sulphydryl groups than does that of the anterior lobe. Thus Miles (1 967a) concluded that the two lobes (anterior and lateral) of lygaeids represent a secondarily bifid anterior lobe (of pentatomids) with a division of function between the two; the lateral lobe providing a protein that readily forms hydrogen bonds and that provides the bulk of the protein (or lipoprotein) of the sheath, while the anterior lobe provides the sulphydryl groups that give the sheath chemical stability and initiate particularly rapid gelling in oxidizing conditions. In the Pyrrhocoridae, the principal gland is four-lobed and the contents of all the lobes except the posterior can form irreversible gels; thus Miles (1960b, 1967a) considered that in the Pentatomorpha as a whole, any more than two distinct lobes (not counting digitations) in the principal gland represented subdivisions of the anterior lobe. This point of view is at variance with that of Baptist (1941) who believed any subdivision to belong morphologically to the posterior lobe. But so little is known of the functions of the various lobes in the multilobed glands of some families of Pentatomorpha, that any conclusion must be considered conjectural at the present time. The posterior lobe of the salivary glands of Heter0ptera:Pentatomorpha secretes salivary enzymes. Miles (1 967a, 1968b) stated that the digestive enzymes occur only in the posterior lobe of the lygaeid

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PETER W. MILES

Oncopeltus, but other authors claim the occurrence of such enzymes in other lobes of the salivary glands of Heteroptera (Baptist, 1941; Bronskill et al., 1958; Salkeld, 1960). In Heteroptera that secrete no sheath, the division of function between anterior and posterior lobes is almost unknown. Edwards (1961) found that in the carnivorous bug Plutymerus, extracts of both anterior and posterior lobes were proteolytic and zootoxic, whereas the contents of the accessory gland were neither. Hori (1968b) stated that the main source of salivary amylase in the mirid Lygus disponsi was the posterior lobe and that only traces of activity occurred in the anterior lobe and accessory gland; but it has also been pointed out (Miles, 1960b) that very careful separation of the lobes and removal of contaminants from the haemolymph is necessary to pinpoint the origin especially of amylase in view of its rapid activity and the sensitivity of tests for it. In the blood-sucking bugs, the distinction between the lobes in the principal gland tends to disappear and Cimex lacks an anterior lobe entirely. This secondary reduction of structure is presumably ascribable to a reduction in the number of functions performed by the saliva and comparative investigation of salivary function in the Cimicomorpha, phytophagous, predatory and blood-sucking, should be most rewarding, especially in the light of the discovery by Friend and Smith (197 1) of the secretion of traces of a sheath-like material by Rhodnius. C. SOURCES OF OXIDASES

In most Hemiptera, some part of the salivary glands secrete polyphenol oxidase (Miles, 1965) and perhaps peroxidase as well (Miles and Slowiak, 1970). In the Homoptera it is cells of the principal glands and in Heteroptera it is the cells of the accessory gland or its ducts and often those of the principal salivary duct also that secrete polyphenol oxidase. Within the phytophagous Hemiptera, these secretions could be expected to aid the oxidative process accompanying sheath formation; but experiments in which the oxidase of the accessory gland was effectively isolated from the feeding mechanism failed to prevent the normal formation of a stylet sheath within plant tissues (Miles, 1967a). At the same time, carnivorous bugs which are presumed to secrete no stylet-sheath also secrete a polyphenol oxidase in the accessory glands or ducts (Miles, 1964a).

THE SALIVA O F HEMIPTERA

239

It is noteworthy that the cells reacting with DOPA in the Heteroptera are those surrounding the ducts, all of which have a chitinous intima: the oxidase could thus be part of the normal cuticle-producing mechanism of epidermal cells, except in those insects in which there is definite evidence of secretion of the enzyme in the saliva itself. Locke ( 1969) has recently shown that peroxidase too is involved in the oxidative processes carried out by the epidermal cells when they produce cuticle, and peroxidase is the latest enzyme to join the list of those secreted in the saliva of Hemiptera (Miles and Slowiak, 1970). One puzzling feature of the secretion of salivary oxidases by Hemiptera is that the enzymes are secreted in high concentration along with the sheath of aphids (Miles, 1965) and Pentatomorpha, and they can be found in high concentration in secretions remaining within the lumen of the sheath (Miles, 1967a); yet in the Pentatomorpha these enzymes are secreted by the accessory gland and this produces so dilute a secretion that .it is often impossible to detect any solutes in it. Possibly it is always secreted by the cells of some parts of the accessory gland or duct (Miles, 1964a) and is subject to varying degrees of dilution by water discharged by other parts. D. SOURCES IN THE HOMOPTERA

In the Homoptera, discovery of the origin of salivary components is considerably more difficult than in the Heteroptera, and recourse has mainly been had to histochemistry. It is not always easy to differentiate between the secretory products and the synthetic processes that give rise to them, however, and although prosecretory granules are separately identifiable by electron microscopy in the cells of the salivary glands of Homoptera (Moericke and WohlfarthBottermann, 1960a) the composition of the secretion cannot be determined by this means. Comparison between the work of different authors on the histochemistry of the salivary glands of Homoptera is also complicated by their use of different dyes and reagents. Another difficulty, pointed out by Weidemann (1970) is that in Myzus persicae the cells do not react consistently with reagents-a likely result in view of the cycles of activity which the cells undergo (Moericke and Wohlfarth-Bottermann, 1960a). Miles (1964a) published a comparison of the cells that produce

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PETER W. MILES

PPO in the salivary glands of Homoptera; Sogawa ( 1 965, 1967) made a histochemical comparison of the glands of delphacids (Fulguromorpha) and deltophacids (Jassomorpha) and Weidemann (1 970) made a histochemical study of the glands of the aphid Myzus persicae. Since the sheath material secreted by Hornoptera, like that secreted by Heteroptera, most probably contains sulphydryl groups (Miles, 1965), attempts have been made to trace the origins of the sheath material by looking for a major source of the sulphydryl compounds, for example cysteine/cystine in the glands. Results have tended to depend very much on the particular reagent chosen, however. Perhaps the most specific test for sulphydryl groups is the nitroprusside reaction, but it is insensitive and unsuitable for small sections; a highly sensitive method, said to be specific, is with DDD (dihydroxy dinaphthyldisulphide), but this test gave no clearly positive results with the salivary glands of leaf hoppers (Sogawa, 1967). Other methods, found more likely to react positively, were of less certain chemical protocol-e.g. the alkaline tetrazolium reaction (Sogawa, 1967) and performic acid-alcian blue (Weidemann, 1970). Finally, quite apart from the occurrence nt sulphydryl groups in the sheath, any other secretion with which the sheath precursors are likely to come into contact within the conducting system of the glands are also likely to contain reducing groups (Miles, 1967a). Other properties of sheath material that may be of use in identifying its origins in the glands of Homoptera are its content of phospholipid and its periodic acid-Schiff (PAS) reaction. Using tests for lipids and the PAS-reaction, Sogawa homologized some lobes of the salivary glands of the jassomorph N. cincticeps and the fulguromorph L. striatellus (Figs 14, 17). These cells gave somewhat different combinations of reactions, but Sogawa concluded that two or three sets of cells were responsible for the secretion of the sheath precursors out of the six kinds of cells in the principal glands of N. cincticeps and out of eight kinds in the glands of L. striatellus. The strongest homologies according to Sogawa are between the IV cells of N. cinticeps and the A cells of L. striatellus (main secretion of protein); the I11 cells and the H and G cells (main secretion of lipids) and the V cells and the Ag cells (secretion of conjugated carbohydrate). E. SALIVARY CARBOHYDRATE AND LIPID

One histochemical peculiarity of sheath material and of some cells in the salivary glands may be of use in future studies of its glandular

THE SALIVA OF HEMIPTERA

24 1

origins. The PAS-reaction is usually given by carbohydrates, but also by some lipids: these sources can be differentiated by either treating with an acetylating agent, which blocks the groups in carbohydrates that are PAS-positive or by extraction with solvents that remove the source of the reaction when it is due to lipids. One or both of these pre-treatments should prevent the PAS-reaction, thus indicating the identity of the PAS-positive source. Sheath material (Miles, 1960a) and certain cells of the Homoptera (Sogawa, 1967) and Heteroptera (Miles, 1960b) remain PAS-positive despite prior blocking of carbohydrates or extraction of lipids. Sogawa (1 967) suggested this was due to the presence of both sources of reactivity in a conjugated form. His hypothesis received some support from experiments with injected radioactive glucose and glycerol (Miles, 1967b), both of which are incorporated into the sheath precursors of the pentatomid Eumecopus, the glycerol presumably being metabolized into the 10% or so lipid moiety. According to Hori (1968b), the mirid Lygus also has a strongly PAS-reacting secretion in the anterior lobe (compared with the posterior) of the principal gland. This insect produces no sheath material and the anterior lobe secretes relatively little protein. In the Pentatomorpha, by comparison, the anterior lobe, which does secrete sheath precursors, produces a secretion that is strongly PAS-positive and also contains a large amount of protein. These results, taken with the finding that the blood-sucking reduviid, Rhodnius produces a salivary material akin to sheath material, may mean that in all Heteroptera the anterior lobe retains an ability to secrete protein conjugated with carbohydrate and lipid, although in the forms that secrete no sheath the amount of protein secreted is reduced. Even in the Pentatomorpha, there is apparently a tendency for the most anterior part of the anterior lobe to lose its ability to secrete a large bulk of protein (Miles, 1967b), this function being taken over by another part of the glands or perhaps by the hind region of the anterior lobe, which becomes separated off as a distinct lateral lobe. XI. THE SALIVA AS A VEHICLE FOR PATHOGENS

The pentatomid Acrosternum hilare (Say) acquires and transmits the fungus Nematospora coryli Peglion when it feeds on lima or soy beans or the berries of dogwood, Cornus drummondi Meyer; and Dysdercus transmits N. gossypii to cotton bolls similarly (Clarke and Wilde, 1970a, b). The spores contaminate the stylets and salivary syringe and infectivity is lost during moulting. Clarke and Wilde AIP-

11

242

PETER W. MILES

(1970a) were unable to recover the fungus from watery saliva secreted outside plants (the insect was induced to salivate by amputation of its antennae). Even so, the authors did not discard salivary transmission as the most likely and they had not entertained the possibility that this secretion might have been atypical of those normally secreted within plant tissues (see Section VI1,B)-perhaps a more viscous secretion, such as the sheath material, is normally required to dislodge the contaminating spores. By far the most significant instances of transmission of plant pathogens by Hemiptera are the virus diseases carried by Homoptera. The “non-persistent” viruses are transmitted immediately after they have been acquired by the insects and the latter very rapidly lose their infectivity; the “persistent” viruses are transmitted only after a period of hours to weeks, the latent period, and usually the insect then continues to be infectious until it dies. The non-persistent viruses are not ingested before transmission and they are somehow contaminants of the stylets or food canal of the feeding mechanism; whereas the persistent viruses are ingested, transferred first to the haemolymph and thence to the saliva. The non-persistent viruses are typically transmitted by aphids and are acquired from and transmitted to the subepidermal tissues of plants. The persistent viruses are typically transmitted by the jassomorph Homoptera and are acquired from and transmitted to the vascular tissues of the plant. A number of authors have suggested that the saliva is by no means an inert carrier of the viral particles and may either inactivate them or modify the plant cells in such a way that they become more or less susceptible (Sylvester, 1962; Miles, 1968b). Of most recent interest in the field of salivary transmission of plant diseases is the finding of mycoplasma-like bodies (Fig. 19) in the salivary syringe and ducts of the jassomorph leafhopper Mucrosteles fascifrons (Raine and Forbes, 197 1) and in its ejected saliva (Raine and Forbes, 1969), and the evidence that such bodies are normally associated with individuals carrying the Aster Yellows disease of China Aster and annual Chrysanthemum (Davis and Whitcomb, 1970). Moreover, both the disease and the organisms within the insect are alike attacked by certain antibiotics, according to Whitcomb and Davis (1970), who nevertheless warn that some non-infective individuals also carried the mycoplasma-like bodies and that an absolute link of organism with disease had thus not been established beyond all possible doubt.

THE SALIVA O F HEMIPTERA

243 m

$

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244

PETER W. MILES

Observations on identifiable infective organisms in the salivary glands are of especial interest in the light of the report by Ossiannilsson ( 196 1) and Moericke and Wohlfarth-Bottermann (1960a, b) that the wall of the gut and the basement membrane of the salivary glands respectively have no pores in them large enough to allow viral particles (e.g. 32 x 300 mp in size) to move through. Moericke (1961) found large viral bodies up to 50 x 400 mp in the cells of the salivary glands of Myzus persicue infected with potato leaf-roll disease; he reasoned that the infective unit must be very much smaller and was probably molecular nucleic acid. The mycoplasma-like bodies described by Raine and Forbes in the salivary glands and ducts of Mucrosteles were up to 400 mp across. Even larger particles, up to 800 mp across, have been described in the cells of the glands themselves by Hirumi and Maramarosch (1 969); Raine and Forbes (1 97 1) concluded that these must produce small intermediate bodies that leave the cells and develop into the organisms found in the saliva. On the other hand, Moericke and Wolfarth-Bottermann ( 1960a) describe the discharge of secretory granules up to 260 mp across from the cells of the salivary glands of Myzus persicae by a membrane flow mechanism-i.e. a prosecretory granule bulges out from the secretory cell into a canal cell, a part of the cell membrane surrounding the granule detaches itself from the parent membrane and the granule is eventually liberated when the membrane dissolves. Just how large a particle can pass all the way through the glands is now known, however. Slama (personal communication) found that the concentration of enzymes in the saliva of the phyrrocorid Pyrrhocon's apterus was much the same as their concentration in the haemolymph and the possibility must therefore be considered that the enzymes merely found their way into the saliva by transport from the haemolymph. In the only known check of this possibility (Miles and Slowiak, 1970), peroxidase, with a molecular weight of 40,000, was shown to pass into the saliva of a pentatomid after injection into the haemolymph, but this is a relatively small molecule for a protein. XII. EVOLUTION OF SALIVARY FUNCTION IN THE HEMIPTERA: A SUMMARY

The salivary glands of Hemiptera can be thought of as having two kinds of function:

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245

(1) those of excretory organs due to their origin as the segmental nephridia of the labial segment and (2) those of epidermal cells due to their ectodermal origin. The excretory function resides mainly in the accessory gland and this is seen very clearly indeed in the Phylloxeridae. Whereas, in the related aphids, the main excretion occurs into the midgut and the accessory gland is small, in Viteus there is no anus and the accessory gland is enormously increased in size. Characteristically, the cells concerned with salivary excretion contain either large intracellular vacuoles or an elaborate system of intracellular canals as in the Aphidoidea or much of the accessory gland takes on the characteristics of a Malpighian tube, as in the Jassoidea and the He terop tera. Epidermal cells, as Locke (1 970) has shown, can carry out a series of functions simultaneously : they secrete the carbohydrate and protein of the cuticle, pass on the lipid elements that are eventually incorporated in the exocuticle and epicuticle, secrete the polyphenol oxidase and peroxidase involved in the oxidative bonding that stabilizes the cuticle, at the same time they secrete the hydrolysing enzymes that digest the untanned parts of the old cuticle and they assimilate the products. The cells of the principal salivary gland and ducts of the salivary glands of Hemiptera similarly secrete the precursors of the sheath material, as well as oxidizing enzymes and hydrolysing enzymes, although there would seem to be a considerable separation of specialized functions between the various parts of the glands. It is noteworthy that the ducts of the salivary glands have chitinous linings. Miles (1 960a) drew attention t o the histochemical and histological similarity of newly secreted sheath material and newly secreted cockroach cuticle. It was found later that gelling of the sheath material involved the formation of disulphide linkages, at a time when it was widely reported that the cuticle of insects contained no sulphydryl amino acids. Recently, however, this generalization has been shown to be incorrect (Hackmann and Goldberg, 197 1) and perhaps in the cuticle, as in sheath material, the oxidative bonding of sulphydryl groups plays a part in the gelling and stabilization of the precursor proteins. If all the Hemiptera are to be derived from forms similar to the Peloridiidae (Coleorrhyncha), then the salivary glands of the Heteroptera are the more primitive in shape and form within the Hemiptera as a whole. In both Homoptera and Heteroptera the

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PETER W. MILES

accessory gland has retained a diuretic function, although there is doubt whether a true accessory gland persists in the Fulguromorpha (Balasubramanian and Davies, 1968). In the Heteroptera, the accessory gland assumes a definitive form as a chitin-lined duct surrounded towards its distal end by more or less glandular cells. In all but the Pentatomorpha, the accessory gland has a thin walled vesicle at its distal extremity. This vesicle, far from being a reservoir for secretory products (Baptist, 1941), appears to be a bladder that collects a dilute ultrafiltrate of the haemolymph. It is probably an adaptation to predatory and other lacerate-and-flush feeding, in which a copious flow of water is finally required to flush out the food source. In the Pentatomorpha, although lacerate-and-flush can be practised by the seed-feeders, the absence of a vesicle on the accessory gland is possibly associated with a reduced need for large quantities of watery secretion by insects adapted to the more conservative stylet-sheath feeding. Within all the Hemiptera, there is a clear division of labour between the different lobes or types of lobe of the principal gland. Even in the Peloridiidae, the poorly developed principal gland is composed of an anterior and posterior lobe that have different staining reactions (Prendergast, 1962). In the Heteroptera, the posterior lobe is probably the main source of the hydrolysing enzymes of the saliva. All the secretions of the principal gland are relatively concentrated and it is likely that the posterior lobe in particular produces a secretion that in normal function requires dilution by the secretion of the accessory gland. Although the anterior lobe in the Heter0ptera:Pentatomorpha produces components of the sheath material, its primary function throughout the Heteroptera may be the production of mucoid substances and conjugated lipids that both lubricate the stylets and are sufficiently viscous to dislodge particles at their tips; for, as McLean and Kinsey (1965) have shown, this is one of the functions of the sheath material of aphids. At the very beginning of the evolution of the Hemiptera, some species must have begun to utilize the solidification of this viscous secretion, brought about perhaps by the release into the saliva of oxidases secreted by the ectodermal cells of the ducts, perhaps by the increased content of sulphydryl groups within the protein moiety of the secretion, perhaps by a combination of both. However evolved, this “sheath material” may originally have been used to fix the stylets at one point on a hard or slippery surface

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during its initial penetration (Saxena, 1963): but for all those species that fed on phloem sap, the evolution of stylet-sheath feeding has clearly become the most unique and characteristic of their salivary functions. It also seems likely that evolution of the production of sheath material has tended to result in a further division of labour and proliferation of morphological divisions in those parts of the glands responsible for its secretion; a convergent tendency in both Homo p tera and Heteroptera :Penta tomorpha despite their p hy letic separation. XIII. A SURVEY OF PROBLEMS

Of the problems concerned with salivary function in the Hemiptera that remain t o be solved, perhaps one of the most intriguing is the function of the salivary oxidases. Little is known about their secretion by Hemiptera other than that they can be readily demonstrated in the stylet-sheaths of aphids and Pentatomorpha in concentrations that must be physiologically active. Miles (1969b) has attempted to rationalize the occurrence of oxidase in the salivary glands and saliva of Hemiptera by suggesting that the enzymes when first secreted may have subserved a detoxifying function. This suggestion was based on the facts that:

(1) many of the toxic and repellant substances produced by insects are based on phenolic compounds (their production no doubt also being derived from the abilities of ectodermal cells to secrete substances that tan the cuticle) and (2) the reaction of plants t o wounding also involves the secretion of toxic phenols. Thus for insects that feed either on plants or other arthropods, the possession of an antidote against toxic phenols and quinones, by oxidizing them all the way to insoluble polymers, would be an advantage. It would seem t o be possible t o test this hypothesis by experimental inactivation of the salivary phenolase system, either by surgery on the salivary gland (Miles, 1967a) or by the use of reducing compounds in artificial diets. It could then be determined whether the insects’ sensitivity t o toxins was increased. A related problem is the occurrence and function of salivary peroxidases and their interaction with the phenol/phenolase system. A further line of investigation of a possible detoxicant function of

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PETER W. MILES

the saliva of phytophagous Hemiptera is indicated by a recent investigation of the degree of resistance of various tissues from apple trees to attack by the woolly aphis, Eriosoma Zanigerum (Sen Gupta and Miles, unpublished). These insects will begin to colonize a variety of seedlings germinated on wet filter paper, but survive on only apple as the seedlings grow. In the field and greenhouse, a relation existed between how readily the insect attacked different varieties of apple-or different parts of any one tree-and the chemical composition of the tissues. High amino acid content predisposed tissues to attack, but phenolics appeared t o protect them; thus the ratio of phenolics t o amino acids provided a rough means of prediction of the resistance of any particular tissue t o attack (Table V). Of further interest was the discovery that the galls produced by the insects were more readily colonizable than surrounding tissues. A number of contingent questions are raised by these observations and as yet remain unanswered. Is host specificity due to the presence of specific phagostimulants (e.g. in apple and not in other plants) or absence of repellents (in young as opposed to older seedlings)? Is Table V The susceptibility of tissues of the apple to attack by E. lanigerum in relation to their chemical composition Susceptibility ratin&

a-amino Nb

phenolics (8)"

@/N

14.5f0.04 lO.lf0.04 8.5f0.12

115 105 59

2 1 .OfO.1 1 16.3f0.11 13.2f0.33

43 20 12

Shoot

+

tip base gall

++ +++

0.126f0.002 0.096f0.002 0.143fO.007 Root

+++ ++++

8mm 4mm gall

*U+

0.493f0.02 0.806f0.02 1.069f0.05

0 Based on measurement of rate of development of colonies, expressed semiquantitatively because of incomplete equivalence of, for instance, physical conditions. b mg N/g wet weight: colorimetric determination with ninhydrin against a D-alanine standard, standard deviation. mg/g wet weight: colorimetric determination with Folin and Denis reagent against a catechin standard, standard deviation.

*

*

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249

ability to feed on apple due to the possession by the insect of a specific detoxicant for repellent (e.g. phenolic) substances present in apple? Is the reduced toxicity of galls a direct result of salivary action or an indirect result of the stimulation of cells to grow at a rate greater than they can accumulate phenolics? The problem of cecidogenesis itself is still far from closed. Despite the elegant way in which Schaller (1 968b) has shown that the amino acids and IAA injected by some aphids cause galls in plant tissues-how the IAA comes to be present is an interesting problem in insect physiology, while the roles of the salivary IAA and the specific amino acids said to determine gall morphogenesis represent an even more challenging problem in plant physiology. The possible functions of the stylet-sheath and the salivary oxidases in moderating the effect of feeding on the plants also requires investigation. It has been suggested that the sheath, besides increasing the efficiency of feeding by the phytophagous Hemiptera, may also reduce those interactions between insect and plant that cause necrosis in the latter (Miles, 1969b) with an obvious advantage to both. According to Hille Ris Lambers (personal communication), however, the aphid Aulucorthum soluni is a stylet-sheath feeder that causes necrosis as a result of a single feed and this possible exception should be especially worthy of investigation. The problem of the role of the phenolic compounds as distinct from the phenolases in the saliva of phytophagous insects also require elucidation. Schaller ( 1 968a) has demonstrated that phenolic compounds occur in the saliva of aphids and, at the same time, has correlated their presence with a lack of pathogenicity of the insect. Although some of these phenolics could well have been transferred directly from the diet, Miles (1964a) demonstrated that one of the phenolics present in the saliva of the phytophagous Heteroptera is DOPA and that it is the end product of a specific metabolic pathway in the salivary glands. Thus any investigation of the salivary oxidases should take into account the complete phenol/phenolase system and its eventual products. The functions of the salivary glands and saliva of Heteroptera other than the Pentatomorpha are still almost unknown. The division of labour between the various lobes of their salivary glands remains undetermined; the way in which Rhodnius produces its flange of sheath material and in what way the loss of the anterior lobe has affected the salivary physiology of the Cimicids remain further intriguing problems.

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W. MILES

How viral or mycoplasmal particles find their way seemingly from the gut to the saliva of Homoptera and details of the transmission of rickettsia1 diseases by reduviids are further aspects of the salivary physiology of Hemiptera that remain undetermined. Indeed, the degree to which salivary composition is normally due to transport of whole molecules including proteins from the haemolymph to the saliva is largely unknown. Finally, the control of salivary discharge and compositionwhether entirely by neural pathways or partly subject to hormonal influences-remains apparently unexplored. Yet this knowledge will be necessary to integrate what is known of salivary function into the broader, but still unique, subject of the physiology of feeding of these highly specialized and economically important insects. REFERENCES Adams, J. B. and Drew, M. E. (1 963a). A cellulose-hydrolysing factor in aphids. Can. J. Zool. 41, 1205-1212. Adams, J . B. and Drew, M. E. (1 963b). The effects of heating on the hydrolytic activity of aphid extracts on soluble cellulose substrates. Can. J. 2001.41. Adams, J. B. and McAllan, J. W. (1958). Pectinase in certain insects. Can. J. ZOO^, 36, 305-308. Anders, F. (1960a). Untersuchungen uber das cecidogene Prinzip der Reblaus ( Viteus vitifolii Shimer) I. Untersuchungen an der Reblausgalle. Biol. Zbl. 79,47-58. Anders, F. ( 1960b). Untersuchungen iiber das cecidogene Prinzip der Reblaus ( Viteus vitifolii Shimer) 11. Biologische Untersuchungen uber das galleninduzierende Sekret der Reblaus. Biol. Zbl. 79,679-700. Anders, F. (1961). Untersuchungen Uber das cecidogene Prinzip der Reblaus ( Viteus vitifolii Shimer) 111. Biochemische Untersuchungen iiber das galleninduzierende Agenus. Biol. Zbl. 80, 199-233. Balasubramanian, A. and Davies, R. G. (1 968). The histology of the labial glands of some Delphacidae (Hemiptera:Homoptera). Trans. R . ent. SOC.Lond. 120, 239-251. Baptist, B. A. (1 941). The morphology and physiology of the salivary glands of Hemiptera-Heteroptera. Quart. J. micr. Sci. 82, 9 1-139. Berlin, L. C. and Hibbs, E. J. (1963). Digestive system morphology and salivary enzymes of the potato leafhopper, Empousca fabae (Harris). Iowa Acud. Sci. 7 0 , 527-540. Bongers, J. ( 1969). Saugverhalten und Nahrungsaufnahme von Oncopeltus fusciatus Dallas (Heteroptera, Lygaeidae). Oecologia, Berl. 3 , 374-389. Bronskill, J. F., Salkeld, E. H. and Friend, W. G . (1 958). Anatomy, histology, and secretions of salivary glands of the large milkweed bug Oncopeltus fasciatus (Dallas) (Hemiptera:Lygaeidae). Can. J. Zool. 36,961-968. BUsgen, M. (1891). Der Honigtau Biologische Studien an Pflanzen und Pflanzenlausen. Z . Naturwiss. 2 5 , 340-428. Carter, W. (1962). “Insects in Relation to Plant Disease”. Interscience

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