The structural sheath protein of aphids is required for phloem feeding

Aphids produce two types of saliva that mediate their interactions with plants. Watery saliva is secreted during cell penetration and ingestion, whereas gel saliva is secreted during stylet movement through the apoplast where it forms a sheath around the stylet to facilitate penetration and seal puncture sites on cell membranes. In order to study the function of the sheath when aphids interact with plants, we used RNA interference (RNAi) to silence the aphid structural sheath protein (SHP) in the pea aphid Acyrthosiphon pisum. The injection of 50 ng of double stranded RNA completely disrupted sheath formation, as confirmed by scanning electron microscopy. Aphid behavior was monitored using the electrical penetration graph technique, revealing that disrupted sheath formation prevented efficient long-term feeding from sieve tubes, with a silencing effect on reproduction but not survival. We propose that sealing the stylet penetration site in the sieve tube plasma membrane is part of a two-step mechanism to suppress sieve-tube occlusion by preventing calcium influx into the sieve tube lumen. The SHP is present in several aphid species and silencing has a similar impact to aphid-resistant plants, suggesting that SHP is an excellent target for RNAi-mediated pest control.     

Feeding Position of Laccifer lucca on Dalbergia balansae and the Influence of Parasitism on Bark Structure

When Laccifer lacca fed in the bark of Dalbergia balansae, the penetration in the bark by a stylet was mainly intracellular, seldom intercellular. Finally, the stylet arrived at the funtional sieve element, and fed in it. The tip of tt,e stylet was at a distance of 0.48–0.78 mm from the surface of periderm. 70.3% of the stylets fed in the zone of newly-differentiated sieve elements. The fed sieve element had P-protein and callose, and exhibited no serious reaction of injury. The parenchyma cells that were pierced through by the stylet and the neighbouring cells Lad obvious reaction of injury, such as: thickened cytoplasm and plasmolysed; dark stained nuclei; smaller starch grains and intracellular deposition of concentrated golden material. The stylet that pierced through the bark was encircled by a stylet sheath consisted of proteins. The stylet sheath looked like a string of beads as a whole. Branching stylet sheath was observed. Some branches even reached far into the xylem, but the stylet finally reached the sieve element. At the same time, the stylet might penetrate through many sieve elements, finally reach newly-differentiated sieve elements. These results suggest that feeding of Laccifer lacca was a process of initiative choice. Two years after collecting shellac by means of skinning instead of cutting the branch, tb.e stylets and styler sheaths still remained in the bark. Several layers of ceils around them were dead and fully imbued with yellow-brown material. Stylers and styler sheaths in the outer cortex were surrounded by bending phellogen and separated from the living cells, forming many cyst-like structures in the periderm. Such bark should not be further used for feeding.     

Systemic antifeedant effects of azadirachtin on the peach-potato aphidMyzus persicae

The electrical penetration graph (EPG) method was used to analyse the feeding behaviour of apterous, adultMyzus persicae (Sulzer) (Homoptera: Aphididae) onNicotiana clevelandii (Gray) seedlings, treated systemically with azadirachtin. A preliminary experiment showed that the effects of tethering aphids for EPG recording were minimal. The percentage of the 9 h recording period devoted to non-penetration activities, and to stylet pathway patterns increased as the azadirachtin concentration in the root treatment increased. The number of probes initiated, and the numbers of sieve tube penetrations also increased with increased azadirachtin concentration. The mean time elapsing between the initiation of the first probe to reach a sieve element and contact with this tissue was not significantly altered by azadirachtin treatment. However, azadirachtin treatment significantly reduced the percentage of probes that reached sieve elements and increased non-penetration activity before and after the first perid of ingestion from the sieve elements. The percentage of the recording period spent in the EPG pattern associated with sieve tube penetration was significantly reduced by an azadirachtin concentration of 300 ppm, and the duration of each individual penetration was significantly reduced by an azadirachtin concentration of 100 ppm. When the total EPG was split into 3 h periods, significant interactions were seen between time period and azadirachtin concentration for the duration of non-penetration, pathway, and sieve tube penetration patterns.     

Symplastic Transfer of Fluorescent Dyes from Mesophyll to Sieve Tube in Stripped Leaf Tissue and Partly Isolated Minor Veins of Commelina benghalensis

We have stripped small (3 × 3 mm) fields of the upper and the opposite lower epidermis of Commelina benghalensis leaves. Pectinase treatment of the resulting chlorenchyma windows produced free-lying viable minor veins with small lumps of mesophyll cells attached. These veins were still connected with the intact remainder of the leaf. Fluorescent dyes were injected into mesophyll cells or mestome sheath cells. Continuous following of the dye from the moment of injection and use of the simple vein system allowed an unhindered and precise assessment of the cell-to-cell route of dye transfer. Disodium fluorescein and Lucifer Yellow CH injected into mesophyll or mestome sheath cells readily moved to the sieve tube. This symplastic dye transfer from mesophyll to sieve tube was also observed after injection into unmacerated stripped leaf tissue. The displacement of fluorescent dyes substantiates a symplastic continuity between mesophyll and sieve tube and therefore supports the possibility of symplastic phloem loading.     

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using ¹⁵N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes. In vitro experiments confirmed the existence of protein kinases in the pure rice phloem sap. The phosphorylation status of the sieve tube sap proteins was affected by the light regime. The possibility that changes in chemical composition or protein modification such as phosphorylation in the sieve tubes might affect plant growth are discussed.Analysis of pure phloem sap collected from rice plants by insect laser technique has shown dynamic changes in the chemical composition and the quality of proteins in the sap.     

The Physiology of the Sieve Tube: PRELIMINARY EXPERIMENTS USING APHID MOUTH PARTS

The willow aphid Tuberolachnus salignus feeds by inserting itsstylets into a single sieve element. If the insect is severedfrom its proboscis whilst feeding, sap from the sieve tube exudesfrom the cut end of the stylet canal and may continue for manyhours. By attaching a capillary tube the rate of exudation canbe measured and the sap analysed. Normally the rate of exudationwas about 1 mm.³ per h. This means a movement of water and sucroseof 100 cm./h. or 100 sieve elements per min. The sap was foundto contain between 5 and 15 per cent, sucrose, up to 0.4 percent, raffinose, no reducing sugars, and about 0.5 per cent,amino-acids. Girdles some distance above and below exuding stylets failedto stop exudation (Tables I and II). This indicates a rapidsealing of the cut ends of the sieve tube and a switch-overin source of supply from the leaves to storage cells in thestem. The use of isolated stem segments and even irrigated stripsof bark followed. Using these, the following points have beenestablished, (a) There is no polarity of movement (Table IV);(b) a minimal length of about 16 cm. of stem or 800–1,000sieve elements are needed to give the full exudation rate (Fig.5); (c) when the D.P.D. on the inside of the bark is raised(Figs. 6, 7, and 8), the rate of exudation declines, but theconcentration of the exudate rises and exudation continues,indicating a maintenance of turgor, even in face of a D.P.D.of 20 atm. or more. Secretion of sugar into the sieve tube continuesalbeit slowly even against a sieve tube concentration of 50per cent. These findings are discussed in relation to current theoriesof translocation and are considered to favour the mass flowhypothesis.     

Physical and chemical interactions between aphids and plants

Aphids feed from sieve tubes deep inside the host plant. Therefore, aphids must be able to recognize their host plant(s) and to direct their stylets which must be long and thin enough to reach and puncture the sieve tubes at a particular site. Sieve tubes in angiosperms are longitudinal arrays of sieve element/companion cell modules which are highly sensitive to disturbance of any kind. The sieve tubes dispose of elaborate sealing mechanisms such as protein plugging and callose sealing which are triggered by a rise in calcium in the sieve tubes. Aphids seem to have developed a range of physical and chemical measures to limit the amount of calcium influx in response to stylet puncturing. Loss of sieve-element turgor pressure induced by stylet insertion is minimized by the minute stylet volume. Turgor-dependent Ca(2+) influx, possibly mediated by mechano sensitive Ca(2+) channels, must therefore be limited. The components of the sheath and watery saliva play a pivotal role in establishing the physical and chemical constraints on the rise of calcium. Most likely, sheath saliva prevents the influx of calcium from the apoplast by sealing the stylet puncture site while watery saliva may prevent plugging and sealing of sieve plates by potential interaction with SE sap ingredients.     

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system.Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy.Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates.Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks.     

The Movement of Sugars into the Sieve Elements of Bark Strips of Willow: I. METABOLISM DURING TRANSPORT

¹⁴C-labelled sugars (sucrose, glucose, and fructose) were suppliedto the cambial surface of bark strips of willow, and in conjunctionwith the aphid stylet technique (Weatherley, Peel, and Hill,1959), the movement into and distribution of activity in thesieve tube sap was examined. As well as sucrose, free hexosesand sugar phosphates were found to contain the ¹⁴C-label, andrapid interconversions occurred between these compounds somewherealong the entry route. Sucrose entry into the sieve elementswas accompanied by at least a partial breakdown into its componenthexoses, and the involvement of a sugar phosphate pool was alsodemonstrated. Possible transformation sites on the entry routebetween the solution bathing the cambial surface and the sieveelement vacuole are discussed.     

Aphid (Sitobion yakini) investigation suggests thin-walled sieve tubes in barley (Hordeum vulgare) to be more functional than thick-walled sieve tubes

Barley, like most other grasses that have been studied, contains two kinds of sieve tube. The first formed are called thin-walled sieve tubes because of their thin wall compared to the late-formed, and are associated with companion cells. The late-formed are thick-walled sieve tubes, which differentiate next to the metaxylem vessels and lack companion cells. Aphid ( Sitobion yakini (Eastop) feeding was studied using light microscopy to determine if they preferentially feed from thin- or thick-walled sieve tubes in the barley leaf. Penetration of the stylets through the leaf epidermis and mesophyll was largely intercellular, becoming partly intercellular and, partly, intracellular inside the vascular bundle. Sixteen of 19 pairs of stylets (84%), and 293 of 317 (92%) stylet tracks terminated at the thin-walled sieve tubes, suggesting that Sitobion yakini feeds preferentially on the thin-walled sieve tubes which seem to be more attractive to the aphid. These thin-walled sieve tubes are thus probably the most functional in terms of phloem loading and transport.     

Movement of Water and Solutes in Sieve Tubes of Willow in Response to Puncture by Aphid Stylets. Evidence against a mass flow of solution

Experiments are described in which bark strips of willow were sealed to polythene tubes having two compartments. This allowed investigations to be made of the transport along the sieve tubes of tritiated water, ¹⁴C-labelled sugars, and ³²P-phosphates from one compartment, towards a stylet situated in the bark over the other compartment. Although activity from both ¹⁴C and ³²p was detected in the stylet exudate usually within 1 hour from isotope application, tritium activity was never detected even after a period of 8 hours in most experiments, though in certain cases, very low activities were detected after 4 hours. Subsequent experiments in which stylets were sited over both compartments showed that tritium activity moved laterally into the punctured sieve element more rapidly than either ¹⁴C or ³²P. Experiments using both live and dead bark in which stylets were not employed, showed that within 4 hours tritium activity had moved by diffusion along the whole length of a bark strip, therefore after this time tritium activity could have moved into the stylet exudate by a diffusional process. The lack of rapid longitudinal movement of tritiated water along the sieve tubes, indicates that the transport process is unlikely to be a mass flow of solution.     

OBSERVATIONS ON PENETRATION OF LINDEN BRANCHES BY STYLETS OF THE APHID LONGISTIGMA CARYAE

Penetration of the bark of Tilia americana L., the linden tree, by Longistigma caryae (Harr.) is mainly intracellular. Like other aphids, L. caryae secretes a saliva sheath which encloses the path of the stylets, beginning with an external collar of sheath material on the surface of the periderm. Stylet sheaths within the bark gave positive reactions for callose, suggesting that, in reaction to wounding, punctured parenchyma cells secrete callose which diffuses throughout the stylet sheaths. Other, more conspicuous effects of wounding included: proliferation and enlargement of cells of the cortex and dilated rays bordering some stylet sheaths, formation of tylosoids in punctured sieve elements, deposition of massive amounts of callose in penetrated sieve elements and in sieve elements bordering penetrated cells, and stimulation of cambial activity and xylem differentiation. Stylet tips located in living sieve elements projected beyond their sheaths which terminated outside the sieve-element walls. It is suggested that such sieve elements can be considered to be functional. None of the living sieve elements containing stylet tips showed any signs of injury which could be attributed to the presence of the stylets. Stylet tips of feeding aphids were found in living sieve elements of both 1965 and 1966 phloem increments clearly indicating that L. caryae can feed on linden sieve elements more than 1 year of age.     

Estimation of the volumetric elastic modulus and membrane hydraulic conductivity of willow sieve tubes

Severed aphid stylets were used to follow the kinetics of sieve tube turgor and osmotic pressure (π) responses following step changes in water potential applied to the cambial surface of willow (Salix exigua Nutt.) bark strips. The kinetics of the turgor response were monitored with a pressure transducer. In separate experiments, the kinetics of the π response were followed by freezing point determinations on stylet exudate. The sieve tube volumetric elastic modulus in the bark strips was about 21 bars, but may be higher in intact stems. The membrane hydraulic conductivity was about 5 × 10⁻³ centimeters per second per bar; several factors make it difficult to estimate its value accurately. Differences in the turgor pressure (P) and π responses, as well as the relatively more rapid initial turgor response to a water potential (ψ) change, suggested a time-dependent component in sieve tube wall elasticity.

Our observations were generally not supportive of the idea that sieve tubes might osmoregulate. However, the bark strip system may not be suitable for addressing that question.

Separate measurements of ψ, P, and π demonstrate that the relationship predicted by the fundamental cell water potential equation, ψ = P − π, is applicable within experimental error (± 0.4 bar) to sieve tube water relations.

     

Bemisia tabaci （Hemiptera： Aleyrodidae） nymphal feeding in cotton （Gossypium hirsutum） leaves

We used brightfield electron microscopy （BEM）, differential interference contrast microscopy （DICM）, scanning electron microscopy （SEM）, transmission electron microscopy （TEM）, and confocal laser scanning microscopy （CLSM） to investigate the stylet pathways of Bemisia tabaci during nymphal feeding behavior in cotton leaves beginning with penetration of the abaxial leaf surface and ending with stylets in sieve tubes in phloem tissues. Most nymphal stylets within salivary sheaths penetrating leaf tissues made complex turns and developed more than one salivary sheath branch before ending in sieve tubes. The external morphology of the salivary sheaths and their routes between and through leaf cells are described during the present study. Results showed the presence of the stylet within the sieve tubes. B. tabaci nymphs may remove stylets and feed in different sieve tubes. Ten short movies showing the progression of the stylet penetrations from adaxial surface to the sieve tubes are attached to Figures 8-15. The report and movies can be viewed from the internet. Download the movies to a local drive in your computer first for fast upload. The movies are posted on the website http：//www.ars.usda.gov/Services/docs.htm？docid=14629. The movies can be used as a teaching aid in biology classes.     

Stylet biogenesis in Bactericera cockerelli (Hemiptera: Triozidae)

The discovery of ‘Ca. Liberibacter solanacearum’, causal agent of certain solanaceous and apiaceous crop diseases, inside the functional (intrastadial) and pharate stylet anatomy of the potato psyllid prompted elucidation of the mechanism of stylet replacement as a novel exit portal in the transmission pathway. In Hemiptera, presumptive (formative) stylets, secreted during consecutive pharate instars, replace functional stylets lost with the exuviae. In potato psyllids, each functional stylet has a hollow core filled with a cytology that extends out of the core to form a hemispherical aggregate of cells, the ‘end-cap’, somewhat resembling a golf ball on a tee. A tightly folded mass of extremely thin cells, the ‘matrix’, occurs inside the end-cap. Micrograph interpretations indicate that during the pharate stage, the end-cap apolyses from the core and ‘deconstructs’ to release and expand the matrix into a long, coiled tube, the ‘atrium’. Cells that were in contact with the inner walls of the functional stylet core maintain their position at the apex of the tube, and secrete a new stylet, apex first, the growing length of which descends into the tube until completed. They then despool from the coils into their functional position as the exuviae is shed.     

Some observations on the role of ATP in sieve tube translocation

Summary Using the aphid stylet technique ¹⁴C ATP was shown to be readily taken up into the sieve elements of willow. At the same time this compound was found to be metabolised during uptake resulting in labelled ADP and AMP appearing in the stylet exudate. Longitudinal movement of labelled ATP was also found to occur.Measurement of the levels of ATP and ADP in stylet exudate showed that both were present in high concentrations. The ratio ATP/ADP varied between 2.0 and 5.3.The effect of certain inhibitors of oxidative phosphorylation (oligomycin and DNP) and glycolysis (fluoride) on the rate of stylet exudation was studied. All three inhibitors caused a cessation of exudation but this did not occur until several hours after inhibitor application. Oligomycin and DNP had no effect on the concentration of ATP in the sap. Fluoride however, appeared in some cases to reduce the ATP concentration to a low level an hour or more before exudation finally stopped.Incorporation of ³²Pi into organic phosphate esters present in stylet exudate was found to occur within 15 minutes of the application of the tracer to a bark strip. Labelling of organic phosphates also took place, at a slower rate, when ³²P inorganic phosphate was incubated with stylet exudate.     

Plant penetration by pea aphids (Acyrthosiphon pisum) of different plant range

Plant penetration by the stylets of six clones of the pea aphid, Acyrthosiphon pisum, on Vicia faba (acceptable to all clones) and Pisum sativum (acceptable to 3/6 clones) was investigated by the DC electrical penetration graph technique. In a 10 h recording period, 93% of 144 aphids exhibited sustained feeding on phloem sap. Significant interclonal differences were observed for the incidence of potential drops (indicative of brief punctures of plant cells) and the duration of waveform E1 (insect salivation into a sieve element). In addition, the total duration of the sieve element phase and the duration of completed bouts of sustained feeding differed between the two test plants, in a fashion varying between clones. However, these differences could not be related to the acceptability of plants to the different aphid clones. The duration of the stylet pathway phase preceding the first sustained feeding on phloem sap did not vary significantly with either aphid clone or plant. It is concluded that the resistance of P. sativum to certain A. pisum clones does not arise from factors impeding either stylet penetration through the plant tissues or the maintenance of feeding from the sieve elements. It is proposed that host plant affiliation of A. pisum may be mediated primarily by specific olfactory or gustatory cues, before the aphid initiates stylet penetration of the plant.     

The Movement of Sugars and Phosphates into the Sieve Elements of Bark Strips of Willow

On the basis of the distribution of radioactivity between sucrose,organic phosphates, and hexose in sieve-tube exudate obtainedvia severed stylets, it has been postulated (Peel and Ford,1968), that two pathways exist for the movement of sugars fromthe bathing solution into the sieve elements of bark strips.These pathways were designated the direct and the indirect.Theexperiments described in the present paper show that the distributionof radioactivity in stylet exudate when labelled sugars aremoving by the indirect pathway may be influenced by the simultaneoustransport of other solutes. Phosphates moving by either thedirect or indirect pathway can influence the activity distributionin stylet exudate, as can sugars moving by the direct pathway.     

Forisome performance in artificial sieve tubes

In the legume phloem, sieve element occlusion (SEO) proteins assemble into Ca(2+)-dependent contractile bodies. These forisomes presumably control phloem transport by forming reversible sieve tube plugs. This function, however, has never been directly demonstrated, and appears questionable as forisomes were reported to be too small to plug sieve tubes, and failed to block flow efficiently in artificial microchannels. Moreover, plugs of SEO-related proteins in Arabidopsis sieve tubes do not affect phloem translocation. We improved existing procedures for forisome isolation and storage, and found that the degree of Ca(2+)-driven deformation that is possible in forisomes of Vicia faba, the standard object of earlier research, has been underestimated substantially. Forisomes deform particularly strongly under reducing conditions and high sugar concentrations, as typically found in sieve tubes. In contrast to our previous inference, Ca(2+)-inducible forisome swelling certainly seems sufficient to plug sieve tubes. This conclusion was supported by 3D-reconstructions of forisome plugs in Canavalia gladiata. For a direct test, we built microfluidics chips with artificial sieve tubes. Using fluorescent dyes to visualize flow, we demonstrated the complete blockage of these biomimetic microtubes by Ca(2+)-induced forisome plugs, and concluded by analogy that forisomes are capable of regulating phloem flow in vivo.     

Ultrastructure of compatible and incompatible interactions in phloem sieve elements during the stylet penetration by cotton aphids in melon

Resistance of the melon line TGR‐1551 to the aphid Aphis gossypii is based on preventing aphids from ingesting phloem sap. In electrical penetration graphs (EPGs), this resistance has been characterized with A. gossypii showing unusually long phloem salivation periods (waveform E1) mostly followed by pathway activities (waveform C) or if followed by phloem ingestion (waveform E2), ingestion was not sustained for more than 10 min. Stylectomy with aphids on susceptible and resistant plants was performed during EPG recording while the stylet tips were phloem inserted. This was followed by dissection of the penetrated leaf section, plant tissue fixation, resin embedding, and ultrathin sectioning for transmission electron microscopic observation in order to study the resistance mechanism in the TGR. The most obvious aspect appeared to be the coagulation of phloem proteins inside the stylet canals and the punctured sieve elements. Stylets of 5 aphids per genotype were amputated during sieve element (SE) salivation (E1) and SE ingestion (E2). Cross‐sections of stylet bundles in susceptible melon plants showed that the contents of the stylet canals were totally clear and also, no coagulated phloem proteins occurred in their punctured sieve elements. In contrast, electron‐dense coagulations were found in both locations in the resistant plants. Due to calcium binding, aphid saliva has been hypothesized to play an essential role in preventing/suppressing such coagulations that cause occlusion of sieves plate and in the food canal of the aphid's stylets. Doubts about this role of E1 salivation are discussed on the basis of our results.