A new technique for studying the stylet tracks of homopteran insects in hand-sectioned plant tissue using light or epifluorescence microscopy

Homopteran insects, such as aphids, psyllids and scales, inject a proteinaceous salivary sheath into their host plant tissue during feeding. This sheath, also referred to as a stylet track, remains in the tissue after the stylets are withdrawn, and is useful for studying plant resistance to insects and plant virus transmission. We describe a new method for studying stylet tracks. Hand microtome sectioned plant material was fixed and cleared in ethanol. The stylet tracks were stained with acid fuchsin and counterstained with aniline blue or fast green. The acid fuchsin stained stylet tracks were pink to red under light microscopy, and orange under TRITC epifluorescence. Stylet tracks in unstained sections autofluoresced under DAPI epifluorescence. This new technique is significantly faster and less complex than previous techniques, and permitted visualization of stylet tracks with light or epifluorescence microscopy within 1 hr of collecting fresh plant material. The technique was also applicable to a broad range of homopterans and plant taxa and provided excellent photomicrographs.     

Fine structure of aphid stylet pathways and its use in host plant resistance studies

Conclusion Although there have been reports, based on light microscope observations, of damage to mesophyll tissue as a result of stylet penetration, we saw no evidence of this in our wax sections. However, the use of the electron microscope revealed that such damage does occur, and has also shown that both inter- and intracellular penetration routes exist, often within the same track, whereas we had formerly believed the stylet pathways of these aphids to be almost entirely intercellular. The intramural-extracellular route of penetration, characterised by the presence of stylets and or saliva between the cell wall and plasmalemma, requires the greater resolution of electron microscopy and cannot be distinguished in light microscope preparations.Our results suggest that an accurate indication of the stylet pathway cannot be obtained from the use of light microscopy alone, and raise serious doubts about the value of previous studies describing the route of stylet penetration. Light microscopy shows the track only at the tissue level and not at the cellular level. We advocate that all future studies of aphid stylet penetration should utilise both light and electron microscope studies because only the higher resolution of the latter technique will indicate the true stylet pathway and the end points of the tracks. Only then can the feeding site be determined with any precision.     

Fine structure of aphid stylet routes in plant tissues in correlation with EPG signals

Abstract. Plant penetration by Aphis fabae (Scopoli) was recorded by the electrical penetration graph (EPG) technique and followed by stylectomy during wave-forms that were suspected of indicating sieve element punctures. The severed stylets in the plant tissue were subsequently processed for transmission electron microscopy (TEM) and sectioned either transverse or longitudinal to the stylets. Two completely serially sectioned probes from the epidermis to the phloem were reconstructed.
In one probe the stylet pathway went to a sieve element and showed many empty branches of salivary sheath material. Breaks in cell walls filled with sheath material demonstrated that the majority of cells bordering the track had been punctured, which supports earlier evidence from EPGs. All types of cells showed punctures and the highest number was found inside the vascular bundle. Very few cells died, which would appear to be important for virus transmission, and in others cellular reactions remained limited to some callose formation. The route of the stylets was intercellular and passed through the secondary wall material. The role of pectinase in intercellular penetration, and previous evidence for intracellular tracks are discussed. Most sieve elements had been punctured but only one was eventually accepted. Thus, reaching a sieve element in a host plant does not automatically imply its acceptance though the reason remains unclear. Gelation of phloem proteins was shown in the stylet canal.
In a second probe, plant cytological and morphological correlations with the EPG were emphasized. Probes by other aphid-plant combinations showed great similarity.     

Xylem feeding by spittlebug nymphs: some observations by optical and cryo-scanning electron microscopy

The feeding of spittlebug nymphs (Philaenus spumarius) from mature xylem vessels was studied by optical and cryo-analytical scanning electron microscopy. Feeding did not produce xylem embolisms and vessels remained liquid-filled during the day. Saliva secreted by the insect forms a hardened lining (salivary sheath) between the stylet bundle and the plant tissues. This sheath is continuous through the hole made by the stylets as they enter a vessel, and it extends into the vessel and along its periphery beyond the breach. The sheath is heterogeneous, with a thin outer layer adjoining the plant tissues and a thicker layer that contacts the stylet bundle. Both layers give positive histochemical reactions for proteins and, in fresh tissues, contain a red, strongly autofluorescent pigment, possibly condensed tannin derived from the plant (which is lost during tissue preparation), and other phenyl propanoid compounds, which are retained and which may produce the intense reaction of the periodic-acid-Schiff's-positive inner layer. It is concluded that the salivary sheath allows the insects to feed from functioning vessels without embolizing them or losing xylem fluid to the surrounding tissues. These findings and others in the entomological literature indicate low daytime tensions in the xylem conduits of the host plants.     

Stylet penetration and survival of three psyllid species on adult leaves and `waxy' and `de-waxed' juvenile leaves of Eucalyptus globulus

The stylet probing behavior and survival of Ctenarytaina eucalypti Maskell, C. spatulata Taylor and Glycaspis brimblecombei Moore (Homoptera, Psyllidae) on adult leaves and `waxy' (untreated) and `de-waxed' (rubbed) juvenile leaves of the heteroblastic plant Eucalyptus globulus Labillardiere were compared. Psyllids were confined within clip-on cages and after 15 days the leaf tissue under each cage was sectioned and stained to characterize and quantify stylet tracks. Across all leaf treatments and psyllid species 1438 stylet tracks were observed in 7239 leaf sections and 571 of these stylet tracks reached the vascular tissue. Survival and the number of stylet tracks of C. eucalypti on the different leaf types did not differ. In contrast, C. spatulata survived significantly longer and produced significantly more stylet tracks on adult leaves and `de-waxed' juvenile leaves than on `waxy' juvenile leaves. Although G. brimblecombei survived equally on all treatments, it produced more stylet tracks on adult leaves and `de-waxed' juvenile leaves than on `waxy' juvenile leaves. For all three psyllid species, there was a positive correlation between survival and number of stylet tracks reaching the vascular tissue. Stylet tracks of all species were generally intercellular from the leaf epidermis to the vascular tissue. Oil glands were avoided by all species, as indicated by the repeated branching and formation of stylet tracks at the periphery of the glands. In `de-waxed' juvenile leaves, C. spatulata and G. brimblecombei produced stylet tracks that entered the leaf at the adaxial surface and exited at the abaxial surface. This pattern was rarely observed on adult leaves, and never occurred with C. eucalypti in any leaf type. We conclude that (1) the epicuticular wax on juvenile leaves reduced stylet probing by C. spatulata and G. brimblecombei, (2) there were apparently no internal physical barriers in either juvenile or adult leaves to prevent the stylets of any of the psyllid species from reaching the vascular tissue, (3) the psyllids avoided oil glands in both leaf types, and (4) C. spatulata and G. brimblecombei may lack cues to orient their stylets towards the vascular tissue in the juvenile leaves.     

A simple staining technique to demonstrate the sporozoite refractile globule in coccidian parasites

A staining technique was developed for locating the sporozoites of Eimeria spp. in both mammalian and avian hosts. Bouin-fixed tissues embedded in Paraffin wax and cut at a thickness of 3 micrometers were stained by a controlled sequence of Ehrlich's haematoxylin, acid fuchsin and aniline blue-orange G-acetic acid which took only 30 min to perform. The refractible globule of the sporozoite stained a brilliant crimson whilst the tissue had an overall blue colour, thus enabling rapid scanning of material under low-power magnification. The granules of eosinophils and heterophils also stained a brilliant crimson and their significance in immunological responses indicates a wider application for this technique.     

Stylet penetration of Cacopsylla pyri; an electrical penetration graph (EPG) study

Detailed information on plant penetration activities by pear psylla Cacopsylla pyri L. (Hemiptera Psyllidae) is essential to study phytoplasma transmission of “Candidatus Phytoplasma pyri” responsible of pear decline disease (PD) and to trace and evaluate resistant traits in new pear tree selections for advanced breeding programs. The electrical penetration graph technique or (full) EPG may relevantly contribute to this knowledge. C. pyri EPG waveforms were characterized on basis of amplitude, frequency, voltage level, and electrical origin. Additionally, stylet tracks and the putative location of stylet tips in the plant tissue were histologically related to EPG waveforms by light and transmission electron microscopy observations after stylectomy. More than one waveform occurred in the same tissue: PA, PB, PC1 and PC2 were all detected in the mesophyll, and PE1 and PE2 were both recorded in the phloem. Waveform PE1 was always preceded by transient waveform PD, as previously described in other psyllids. Interestingly, no brief intracellular punctures (potential drop waveforms) were observed during plant penetration, opposite of what is usually recorded in aphids and other Sternorrhyncha.     

A New and Permanent Staining Method for Starch Granules Using Fluorescence Microscopy

A fluorescence technique has been developed for observing starch granules in plant tissues. Sections are stained with a mixture of dyes which we have named F.A.S.G.A. from the initials of the Spanish names of its components (fucsina, alcian blue, safranina, glicerina, agua), and viewed by epifluorescence microscopy. The starch granules fluoresce greenish yellow, allowing the degradative state to be observed. Cell structures which do not fluoresce are also differentiated. The stain permits identification of other structures when examined by visible light microscopy and is relatively resistant to fading over time.     

A new and permanent staining method for starch granules using fluorescence microscopy

A fluorescence technique has been developed for observing starch granules in plant tissues. Sections are stained with a mixture of dyes which we have named F.A.S.G.A. from the initials of the Spanish names of its components (fucsina, alcian blue, safranina, glicerina, agua), and viewed by epifluorescence microscopy. The starch granules fluoresce greenish yellow, allowing the degradative state to be observed. Cell structures which do not fluoresce are also differentiated. The stain permits identification of other structures when examined by visible light microscopy and is relatively resistant to fading over time.     

Differentiation of Enamel and Dentine Particles

A method, based on the periodic acid-leucofuchsin reaction, is described, by which dentine particles may be selectively stained with neutral solutions. The method is suitable for pulverized teeth, containing particles with dimensions greater than 1-2μ. It can be applied also to sections. The dentine is stained red by treatment with a buffered neutral solution of periodic acid followed by a dilute solution of unreduced fuchsin; the excess fuchsin being removed by washing with water. The enamel is either unstained or stained a light pink. The loss of material caused by the usual acidic solutions is reduced to negligible proportions.     

Silver Impregnation of Alzheimer's Neurofibrillary Changes Counterstained for Basophilic Material and Lipofuscin Pigment

A method is described in which selective silver staining of Alzheimer's neurofibrillary changes is combined with staining of cell nuclei, Nissl material, and lipofuscin granules. Formalin fixed, paraffin embedded sections of human autopsy tissue are silver stained according to a method proposed by Gallyas. Lipofuscin is stained by crotonaldehyde fuchsin following performic acid oxidation. Nissl substance is visualized by either Darrow red or gallocyanin-chrome alum staining. Architectonic units showing the specific pathology and the neuronal types prone to develop the neurofibrillary changes can be recognized using this technique.     

Silver impregnation of Alzheimer's neurofibrillary changes counterstained for basophilic material and lipofuscin pigment

A method is described in which selective silver staining of Alzheimer's neurofibrillary changes is combined with staining of cell nuclei, Nissl material, and lipofuscin granules. Formalin fixed, paraffin embedded sections of human autopsy tissue are silver stained according to a method proposed by Gallyas. Lipofuscin is stained by crotonaldehyde fuchsin following performic acid oxidation. Nissl substance is visualized by either Darrow red or gallocyanin-chrome alum staining. Architectonic units showing the specific pathology and the neuronal types prone to develop the neurofibrillary changes can be recognized using this technique.     

Stains for A, B, and D cells in fetal rat islets.

Ten techniques often used for identification of A, B, and D cells in adult islets of Langerhans were applied to fetal rat pancreas. Modifications were tried with many of these techniques. Two indole methods (xanthydrol and postocoupled benxylidene reactions) and a cryostat technique using o-phthaladehyde failed to stain fetal islets. Phosphotungstic acid hematoxylin and lead hematoxylin lightly stained fetal A cell granules in Helly's fixed tissue. The Grimelius silver nitrate technique stains adult rat A cells but failed to stain fetal cells. A modification of this technique stained fetal A cells and a possible 4th cell type. The specificity of this method was confirmed by restaining stained cells with a fluorescent antibody technique and with pseudoisocyanin. B cells, as previously reported, were readily stained by the aldehyde fuchsin technique. Fetal D cells were not stained by the Hellerstrom-Hellman alcoholic silver nitrate method, nor did they display pseudoisocyanin metachromasia after acid hydrolysis; they did fluoresce brightly with this technique when viewed with UV light. It was thus possible to distinguish the three usual cell types, plus a possible fourth type, in the fetal rat pancreas.     

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.     

唾液成分在刺吸式昆虫与植物关系中的作用

近年来,人们对刺吸式昆虫唾液成分的研究,揭示出其在刺吸式昆虫与植物关系中的重要作用。对多数刺吸式昆虫而言,他们取食时会分泌胶状和水状两种唾液,其中胶状唾液会在取食早期分泌形成唾液鞘来围绕并保护口针,通过直接和间接的作用来帮助取食;而水状唾液中则包含了果胶酶、纤维素酶、多酚氧化酶、过氧化物酶、碱性磷酸酯酶、蔗糖酶等组分,来帮助刺吸式昆虫对植物穿刺、消化食物、解毒次生物质并破坏植物的防御反应。有趣的是,唾液成分同时还可以诱导植物的防御反应,包括诱导植物的伤信号引起直接防御反应和诱导植物产生挥发物吸引植食者的天敌引起间接防御反应。并且,许多刺吸式昆虫取 食能够特异性地引发植物的病理反应,有研究推测刺吸式昆虫唾液中多聚半乳糖醛酸酶、碱性磷酸酯酶、蔗糖酶、多酚氧化酶等成分可能是某些植物特定病理反应的激发子,但是目前还没有定论,同时许多刺吸式昆虫唾液中的氨基酸和蛋白酶还是引起植物虫瘿的原因之一。 迄今的研究表明,刺吸式昆虫会根据不同的寄主植物和不同的生理需要,通过唾液组分的改变,来达到取食和发育的目的。对刺吸式昆虫唾液成分和作用机理的研究,可以为揭示刺吸式昆虫致害机理特别是传毒机理、指导害虫有效治理、阐明其与植物的协同进化等提供一定的思路。     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeffs iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Modified elastic tissue-Masson trichrome stain

A combined elastic tissue-Masson technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeff's iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

An investigation of preferential feeding habit in fourAsclepiadaceae by the Aphid,Aphis nerii B. de F.

Summary Penetration of the stems ofAraujia sericofera, Asclepias curassavica, Cynanchum ellipticum andSarcostemma viminale by stylets of the aphidAphis nerii was studied with light and differential interference contrast microscopes. Of a total of 118 stylets and 446 stylet tracks observed in cross-sections of stems of the fourAsclepiadaceae, 97 stylets and 372 stylet tracks terminated within the internal primary phloem. Of the remainder, 15 stylets and 74 tracks terminated within the external primary phloem. 22 stylets and 179 of the stylet tracks penetrated the external phloem on the way to the internal phloem. Of these, only four stylets and 32 of the stylet tracks showed signs of attempted probes of the external phloem. It is suggested thatAphis nerii may obtain not only its essential food requirements but also cardiac glycosides as a basis for chemical aposematism.     

Influences of leaf age and type,non‐host volatiles,and mineral oil deposits on the incidence,distribution, and form of stylet tracks of Diaphorina citri

Psyllids produce saliva that gels to form a protective sheath around their stylets. This saliva can be visualized as stylet tracks, and we have used the presence of tracks to study the feeding behaviour of Diaphorina citri Kuwayama (Hemiptera: Liviidae). Both single and branched tracks were produced by nymphs and adults, and the tracks made by males and females did not differ. Tracks dissipated from calamondin leaves within a 7‐day post‐feeding period after adults fed for 2 days and numbers of tracks declined with increasing maturity of calamondin leaves. In the six host plants studied, most psyllids fed abaxially from midribs and most probes traversed or terminated in the tissue (midrib, lateral vein, minor vein, or mesophyll) above which the stylets entered the leaf or leaflet. The number of tracks and landings were recorded on the six hosts in choice tests. More tracks were found in leaves or leaflets of orange jasmine, wampee, and trifoliate orange than in sour orange, calamondin, and lemon. Orange jasmine is considered a preferred host of D. citri, and this is in agreement with the number of tracks found in this study, but not the number of landings. Trifoliate orange is considered a poor host of D. citri, which, in this study, is reflected in the low number of landings, but not in the numbers of tracks. Our results indicate that the presence of adult psyllids on a plant may not reflect its host status, and that the presence of stylet tracks should also be determined in host preference studies. When calamondin leaves were paired with leaves of guava, billygoat weed, or greenleaf desmodium, the presence of volatiles from these plants reduced feeding by adults on calamondin and suggests that understoreys of billygoat weed or desmodium in orchards may also reduce feeding. A negative, exponential relationship between numbers of tracks per leaf and the concentrations of an agricultural mineral oil applied to leaf surfaces was found. This reduction in feeding, in conjunction with reductions in oviposition, has practical implications for suppressing psyllid populations in orchards.     

Uvitex2B: a rapid and efficient stain for detection of arbuscular mycorrhizal fungi within plant roots

The study of arbuscular mycorrhiza often requires the staining of fungal structures using specific dyes. Fluorescent dyes such as acid fuchsin and wheat germ agglutinin conjugates give excellent results, but these compounds are either hazardous or very expensive. Here, we show that a safer and inexpensive dye, Uvitex2B, can be efficiently used to stain intraradical fungal structures formed by the arbuscular mycorrhizal fungus Glomus intraradices in three plant species: carrot, Casuarina equisetifolia, and Medicago truncatula. The intensity and stability of Uvitex2B allow the acquisition of high-quality images using not only confocal laser scanning microscopy but also epifluorescence microscopy coupled with image deconvolution. Furthermore, we demonstrate that Uvitex2B and β-glucuronidase staining are compatible and can thus be used to reveal arbuscular mycorrhizal structures in the context of promoter activation analysis.