HISTOCHEMICAL ANALYSIS OF APHID SALIVA IN PLANT TISSUE

Anna Urbańska
Department of Biochemistry and Molecular Biology, University of Natural Sciences and Humanities in Siedlce, Poland

ABSTRACT

This paper concerns specific saliva of Aphididae injected into host-plant during feeding and commonly known as the salivary sheath. Fresh wheat leaves upon which grain aphid Sitobion avenae F. was feeding were hand sectioned. These cross sections were stained with acid fuchsin and examined for the histochemical localization and chemical character of the salivary sheaths by optical microscopy. The salivary secretion of a single aphid forms absolutely proteinaceus salivary sheath that is continuous and appreciable from upper or lower epidermis till sieve elements (sieve tubes). The majority of the salivary sheaths of S.avenae (approx. 88–83%) are located intercellularly through the intermediate tissues i.e. epidermis and mesophyll. Moreover, the salivary secretion is deposited into the epidermal and mesophyll cells and also vascular bundle, and moreover fills up the intercellular spaces. According to the histochemical examination of the salivary deposits of S.avenae its feeding path is established to be both intercellular and intracellular, and ends principally both in the phloem (approx. 63%) and in the mesophyll (approx. 33%), and additionally also in the xylem (approx. 4%). No obvious saliva diffusion into cells adjacent to the salivary sheaths of S.avenae has been found. It is concluded that the histochemical studies of the salivary secretions provide information both on probing mechanism and feeding source of S.avenae and also are useful for recognition of chemical resistance in wheat plants against Aphididae.

Key words: grain aphid, Sitobion avenae F., saliva, salivary sheath, salivary path, winter wheat, leaf tissue, probing mechanism, feeding source, plant-aphid interaction.

INTRODUCTION

Aphid feeds on plant using piercing and sucking mouth apparatus that is composed of four stylets. The internal maxillary stylets interlock and form the larger diameter (1–2µm) food canal and the smaller diameter (0.2–0.4µm) salivary canal. These stylets are surrounded and supported by the mandibular stylets. When a aphid inserts its stylets into the plant tissue then it secretes a liquid saliva through the salivary canal which opens on the tip of the stylet bundle. The aphid saliva hardens immediately as it is exuded and forms the salivary sheath, that is a solidified lining between the stylet bundle and the plant tissues. Presumably, the salivary sheath adheres to the plant tissues, but not to the surface of the stylet bundle. In consequence of that it remains in situ within the plant when the aphid withdraws its stylets. For this reason the salivary sheath is referred to as the feeding path or track, and also as the salivary path or track [13,14,16,18,19,20].

The salivary sheath of Aphididae has been studied for over a century (since 1891) [13,14,18]. Yet, rather speculations instead of detail knowledge have been formulated on the salivary sheath, specifically in the context of its function in aphid-plant interaction. A variety of subjects concerning the salivary sheath of Aphididae i.e. chemical composition, reactivity with plant compounds, distribution in plant cells and tissues are still not clear. In my opinion these aspects are important and useful in solving theoretical and practical problems e.g. feeding mechanisms, virus transmission and toxicogenesis of salivary secretions and also breeding of resistant plants to Aphididae etc.

This paper provides some data on histochemical localization and chemical nature of the salivary sheath of the grain aphid Sitobion avenae F. deposited into the winter wheat leaf, in particular: (I) staining of protein and visualization of the salivary sheath of a single aphid; (II) a variety of salivary 'paths' and 'directions' through the epidermis, mesophyll and vascular bundle; (III) extent of the aphid salivary secretion in the plant cell and tissue.

The examination was accomplished according to new technique by Brennan et al., published in 2001 and recommended for studying the saliva of homopteran insects [3].

MATERIAL AND METHODS

Plant tissue preparation. Seedlings of winter wheat that were densely colonized by S.avenae were collected and placed in plastic bags. Aphids fed on leaves for 1–2 weeks prior the collection. After collection, the seedlings were washed under running water to remove aphids prior to hand microtomy. The plant material (Fig. 1) was cut into sections approximately 15–35 µm thick. The fresh sections were floated into water in a Petri dish. Within 5–45 min after sectioning, the leaf sections were transferred to glass vials containing 70% ethanol for fixation and clearing. The ethanol was changed every 15 min. The sections were cleared after 3 changes of ethanol.

Staining reaction of salivary sheath in plant tissue. The cleared sections were transferred to glass vials with 70% ethanol and to each 10 drops of 70% ethanol, one drop of 0.2% acid fuchin in 95% ethanol and glacial acetic acid (1:1, v/v) was added. The sections were soaked in the stain for 10–30 min and they were transferred to water in a Petri dish for a few seconds. To increase the contrast of the plant tissues surrounding the salivary sheath, sections were counterstained with 1% aniline blue in 95% ethanol. A drop of the counterstain was placed directly on a section on the microscope slide and the excess stain was washed away with several drops of 95% ethanol after 3–10 sec. For permanent mounts, stained sections were transferred to microscope slides, dehydrated with a few drops of 100% ethanol for a few seconds, then mounted in Euparal. Stained sections were stored in a solution of 0.2% acid fuchsin in 70% ethanol (1:10, v/v). Excess stain was removed by soaking of the acid fuchsin stained sections in 70% ethanol.

Light microscopy microanalysis. The stained sections were transferred to microscope slides for examination with the Nikon Eclipse E 400. The acid fuchsin stained salivary sheaths/tracks were pink to red under light microscopy. Color light micrographs were produced from the stained sections.

RESULTS

Visualization of proteinaceus salivary sheath in plant tissue. Salivary sheath gives positive histochemical reaction for protein. Acid fuchsin (protein indicator) stains the salivary sheath of S.avenae uniformly throughout. In consequence of this staining reaction the pink salivary sheaths are clearly visible in the epidermis, mesophyll and vascular bundle using the light microscopy. Examples of the standard salivary sheaths S.avenae in the wheat leaf stained with acid fuchsin are presented on Fig. 2, 3, 4.

How salivary sheath is located in plant tissue. The salivary sheaths of S.avenae are present both in the adaxial and abaxial epidermis; in proportion approximately 83% and 17%, respectively. The majority of salivary 'tracks' (approx. 88%) is located via epidermis intercellularly, only 4% intracellularly, and 8% within the guard cells of a stoma (Table 1). Across the mesophyll the salivary 'paths' are in general intercellular (approx. 83%), and freguently branched. Some 17% of the salivary depositions of S.avenae 'passes' through mesophyll both inter-and intracellularly. Exclusively intra-cellular localization of the saliva along of the mesophyll cells has not been observed. Even 63% of the salivary 'tracks' of S.avenae ends in the phloem, approximately 33% in the parenchyma and only 4% in the xylem (Table 2, Fig. 2, 3, 4).

What is dimension of aphid saliva deposit within plant. Photomicrographs (Fig. 2, 3, 4) illustrate that S.avenae injects into the plant tissues considerable quantity of salivary secretion that is enough to form the salivary sheath extending via the epidermis, mesophyll, bundle sheath till phloem (sieve tubes) or xylem. Salivary sheaths with one or more branches have been formed. Yet, no obvious diffusion of saliva material towards cell walls and protoplasts adjacent to salivary sheath have been observed. In fact, the staining of the salivary protein by acid fuchsin is confined to the stylet 'track' (Fig. 2, 3). Typical salivary sheath appears to be tight and it sticks to cell walls and protoplasts all along, and its extent is not greater than 10 microns.

DISCUSSION

The present microphotographs demonstrate positive reaction of S.avenae saliva with acid fuchsin. Namely, the salivary sheaths are uniformly pink and clearly visible against the wheat leaf tissues. Acid fuchsin is commonly used as histochemical stain that indicates protein [3,6,18]. Then, the aphid salivary sheath is predominantly peroteinaceous.

Miles [11,12] first published results of successful physiological studies on protein presence in the salivary secretions of Aphididae, other Homoptera and also Heteroptera. Next, Bauman and Bauman [2] reported on main three proteins in the saliva of Schizaphis graminum. In 2008 I 'revealed' the protein occurrence in the salivary secretions of S.avenae injected into the agarose (1.25%) – sucrose (30%) gels by classical biochemical stain, the Coomassie Brillant Blue R-250 [22]. Chemicals found by microchemical tests to occur in aphid salivary sheath include: calcium pectate, callose, lipoproteins, phospholipids, tannin, amino acids i.e. dihydroxyphenylalanine (DOPA), cysteine, cystine, and also phenolics, and even some conjugated carbohydrates [13,14,16]. Yet, most authors have considered the salivary sheath to be mainly proteinaceous [2,3,4,5,9,13,14,16,22]. Miles [15,16] described that it is composed of catechol oxidase and a substrate for this enzyme, probably DOPA. According to my study two oxidoreductases i.e. polyphenol oxidase and peroxidase seem to be primary proteins/macromolecules in the aphid salivary sheath [21,22,23,24]. Some authors discussed the suggestion that the salivary secretions may contain enzymatic proteins that break down cell walls e.g. pectinases and cellulases, or digest plant food material e.g. amylases, proteases, phosphatases [5,9,10,13,14,15,16,18]. In my opinion they are rather wrong.

In general, S.avenae deposits saliva on upper epidermis of wheat leaf but occasional deposition on lower surface also occurs. The reason for this may be difference in physical and chemical nature between upper and lower cuticle e.g. composition and content of wax, cellulose, cutin and even pectin. 'Entry' of S.avenae saliva into wheat leaf is mostly intercellular (approx. 87%), rarely intracellular and through a stoma. Next, the 'salivary paths' are located between cell walls and also across mesophyll cells. Salivary deposits of the cereal aphids are predominantly intercellular through epidermis, whereas both intercellular and intracellular via mesophyll. Examples are Rhopalosiphu padi L. on wheat [17], Macrosiphum (Sitobion) avenae F. on Avena sativa L. and Schizaphis graminum on barley [10,18]. Although for R. maidis on Zea mays L. leaf intracellular 'entry' was recorded only [18].

The saliva injection by S.avenae into wheat leaf is continued, from epidermis surface till phloem cells (sieve tubes). Moreover, the salivary deposits tightly adhere to the 'passed cells' both to their cell walls and protoplasts. Typical salivary path/track of S.avenae found in the wheat tissues is rather thick, under 10 µm diameter. No obvious diffusion of the aphid saliva is visible in plant sections illustrated. Then, saliva of S.avenae affects rather 'subtle' host-plant tissue. It taps but not 'destroys' individual cells completely.

'Transference' of aphid saliva into host-plant is debatable. In some cases, saliva diffuses for a distance of 30 – 40 µm on both sides of the stylets e.g. S.graminum on wheat and Therioaphis maculate on alfalfa [1,7]. Similarly, salivary secretions of S.avenae diffuse within the agarose gel (1.25%) till 60µm [21]. On the other hand, Aphis fabae on Vicia faba deposits saliva inside pierced cell wall alone [8].

According to the results presented in this paper S.avenae saliva appears within vascular bundle. The phloem is favored tissue of S.avenae and the sieve tubes are the food source. Over 60% of the feeding tracks reaches phloem. Contrary to the phloem 4% of the salivary tracks ends in the xylem alone. Aphids are rather not known as xylem feeders [18, 20]. In quite a few cases (approx. 33%), the feeding tracks of S.avenae terminate in mesophyll. In general, a few species of Aphididae can feed on the mesophyll cells [1,7,18].

As for S.avenae, the salivary tracks may show branches ending in mesophyll. Moreau and Loon [17] presented similar results for R.padi feeding on Triticum sp. In fact, branched salivary tracks are generally common around the mechanical tissues [20], whereas their occurrence in mesophyll may evidence that aphid stylets 'err' following phloem.

The characteristics of S.avenae saliva in wheat tissues seems to be valuable and useful for researchers that are interested in chemicals responsible for plant resistance to Aphididae. The technique with acid fuchsin appears to be excellent for fast microscopic visualization of aphid saliva proteins both in fresh plant material and artificial diets. It may be also applicable to a broad range of biochemical studies concerning aphid – plant interactions.

CONCLUSIONS

1. Protein is predominant chemical compound of aphid saliva.

2. S.avenae deposits saliva mainly on adaxial epidermis of wheat leaf.

3. Saliva of S.avenae  is located essentially between plant cells.

4. Occurrence of aphid saliva within mesophyll cells is occasional.

5. Saliva of S.avenae is not diffused into cells adjacent to aphid feeding path.

6. Presence of aphid saliva is easily visible in vascular bundle.

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Accepted for print: 30.11.2010

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