Light and Scanning Electron Microscopy of Greenbug Aphid Damage in Wheat Using the Same Section

A method has been developed to enable correlative light microscopy (LM) and scanning electron microscopy (SEM) on the same section of wheat (Triticum aestivum L.) leaves infested by greenbug aphids (Schizaphis gra-minum Rondani). Segments of infested leaf tissue were fixed, embedded in paraffin, sectioned, and affixed to slides by standard histological techniques. Serial sections were viewed by LM as temporary mounts in xylene. Sections of interest were identified and re-embedded in fingernail polish, affixed to aluminum stubs, freed of polish with ethyl acetate or acetone, and sputter-coated for SEM. SEM of re-embedded leaf sections showed excellent preservation of leaf anatomy. The same aphid tracks and regions of cell damage identified by LM were visible. SEM increased resolution and provided a much clearer sense of the three-dimensional relations involved in the interaction between plant and insect.     

Light and electron microscopic structure of golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure)

Summary After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguishable by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pieces of tissue left in a 1 % K₂Cr₂O₇ solution for 13 h before impregnation in a 0.5 % AgNO₃ solution for 2h. Thick sections (30–50 m) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.     

Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.     

中国黄耆属簇毛黄耆亚属的叶表皮特征及其系统学意义

在光学显微镜和扫描电镜下,观察了国产黄耆属簇毛黄耆亚属16种1变种的叶表皮特征。结果表明,气孔器在各种植物的上、下表皮均有分布,多为无规则型,也有不等细胞型;叶表皮细胞形状有不规则形与多边形,表皮细胞垂周壁有平直、浅波状、波状或深波状。叶表皮细胞形状与垂周壁的式样可以分为四种类型,这四种类型与亚属的分组有一定的对应关系。在扫描电镜下可见表皮细胞上有角质层,气孔下陷,气孔的外拱盖及其内缘特征在亚属内都比较一致。表皮细胞角质层的纹饰在个别类群中有一定的变异,对种类的鉴定有一定的意义。     

Comparative morphology of the leaf epidermis in Fritillaria (Liliaceae) from China

The leaf epidermis of 16 species and one putative species of Fritillaria was examined using light microscopy (LM) and scanning electron microscopy (SEM). The results showed that the stomatal and other epidermal features were constant within species. Epidermal cells of Fritillaria under LM were usually polygonal and anticlinal cell walls were straight or curved. In a few species they were irregular, with sinuous anticlinal cell walls. The cuticular membrane of Fritillaria was usually striated, and the wax ornamentations were flaked, granular or concomitant. Based on leaf epidermal characteristics, the subdivision of Fritillaria is discussed, and the statistical t‐test method was used to ascertain the significance level of the differences in the stomata of each species. All orientations of the stomatal poles in Fritillaria were the same, and this phenomenon was named ‘stomatal orientation’. The stomatal characteristics support the origin of section Fritillaria in China from two floristic elements. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 93–109.     

石竹属植物叶表皮特征及其系统学意义

用光学显微镜和扫描电镜,对石竹属12个种的植物叶表皮特征进行观察,统计并测量叶表皮气孔大小、气孔密度及气孔指数。结果表明,石竹属植物叶表皮细胞形态(表面观)为长方形和不规则多边形。乳突只存在于针叶石竹、高石竹和细茎石竹中。按气孔形状可将其分为了3个类型:椭圆形、卵圆形和长方形。研究结果对石竹属的系统分类和种间亲缘关系研究具有一定的参考价值。     

Rapid Three-Dimensional Reconstruction at the Light Microscopic Level and a Technique for Re-Embedding the Same Semithin Sections for Electron Microscopic Examination

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 μm sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.     

Morphology of the Leather Defect Light Flecks and Spots

The skin histology and the scanning electron microscope morphology of the hide defect light flecks and spots after tanning were studied in 11 steers infested with biting lice (Damalinia bovis). Nine steers from herds free of lice were used as controls. Skin biopsies from 6 of the animals in the lice infested group showed mild to moderate hyperkeratosis and moderate perivascular to diffuse dermatitis with infiltration of mainly mononuclear cells and some eosinophilic granulocytes. The steers were slaughtered at an age of 18 to 23 months. Light flecks and spots occurred on all examined hides from the infested group after tanning. No examined hides from the control group demonstrated similar damage. Both light microscopic examination of sections of tanned hide with light flecks and spots and scanning electron microscopy of the same defects showed superficial grain loss and craters with a irregular fibre base encircled by smooth and intact grain.     

Variation in rates of leaf abscission between plants may affect the distribution patterns of sessile insects

Summary At Sand Lake, Leon County, Florida, mines of Stilbosis quadricustatella, a leaf-mining moth, occur on sand live oak trees (Quercus geminata) over a broad range of densities. Some trees have fewer than 2% of their leaves mined (lightly infested), others up to 70% (heavily infested). Similar levels of infestation are maintained on the same trees year after year. There are no significant negative correlations of miner density per tree with any mortality factor that might explain miner preferences for certain trees. Nor is there a positive correlation with host leaf quality as measured by foliar nitrogen or by total or individual amino acid concentrations of host leaves. Egg-transfer experiments showed that larvae from eggs transferred to lightly infested trees were more likely to be killed by leaf abscission than were those that were transferred to and developed on heavily infested trees. This is the first demonstration that variation in rates of leaf abscission could be an important cause of the observed distribution pattern of sessile insects between conspecific host plants.     

A combined light and scanning electron microscopy study

The uppermost Eocene Florissant Formation, Rocky Mountains, Colorado, has yielded numerous insect, vertebrate and plant fossils. Three previous comprehensive palynological studies investigated sections of lacustrine deposits of the Florissant Formation and documented the response of plant communities to volcanic eruptive phases but overall found little change in plant composition throughout the investigated sections. These studies reported up to 150 pollen and spore phenotypes. In the present paper, we used a taxonomic approach to the investigation of dispersed pollen and spores of the Florissant Formation. Sediment samples from the shale units containing macrofossils were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The general picture of the palynoflora is in agreement with previous studies. However, the combined LM and SEM investigation provides important complementary information to previous LM studies. While a fairly large amount of previous pollen determinations could be confirmed, the purported taxonomic affinities of several pollen phenotypes need to be revised. For example, pollen referred to as Podocarpus or Podocarpidites sp. belongs to the Pinaceae Cathaya, Malus/Pyrus actually belongs to Dryadoideae, pollen of the form genus Boehlensipollis referred to as Proteaceae/Sapindaceae/Elaeagnaceae or Cardiospermum belongs to Sapindaceae but not to Cardiospermum, and pollen of Persicarioipollis sp. B with previously assumed affinities to Polygonaceae actually belongs to Thymelaeaceae. Pandaniidites and one type of Malvacipollis cannot be linked with Pandanaceae and Malvaceae. A few taxa are new records for Florissant (Ebenaceae: Diospyros; Mernispermaceae; Trochodendraceae: Tetracentron). In general, SEM investigations complement the LM palynological studies and improve the identification of dispersed pollen and spores and enable integration of data from dispersed fossil pollen into a wide range of comparative morphological, taxonomic, evolutionary, biogeographic and phylogenetic studies.     

Host plant changes produced by the aphid Sipha flava: consequences for aphid feeding behaviour and growth

Induced plant responses may affect the behaviour and growth of the attacking herbivore insect. The aphid Sipha flava (Forbes) produces reddish spots on the infested leaf of its host plant Sorghum halepense (L.). In order to assess the consequences on the aphid of this presumptive induced plant response, we studied the feeding behaviour and growth of S. flava on previously infested and non-infested leaves of S. halepense. Considering that the reddish pigment could play a defensive role, its effect on aphid survival was determined in artificial diets. In addition, changes in the histology of the leaf and the chemical nature of the induced pigment were also studied. Aphids devoted a significantly shorter total time to non-penetration activities in infested than in non-infested leaves. Time before the first phloem ingestion tended to be shorter in infested leaves. The mean relative growth rate of S. flava nymphs was significantly higher on infested than on non-infested leaves. Survival of aphids on diet containing the reddish extract was not significantly different from that on the control diet. Infestation of S. halepense by S. flava produced a reddish coloration in the leaf, which was identified as an anthocyanin by UV-visible spectrometry. Light microscopy showed that only mesophyll cells of previously infested plants presented swelled, dispersed, and heterogeneously stained chloroplasts with a higher accumulation of starch granules, no grana arranged in stacks, and reduction in the amount of inner membranes (thylakoids), relatively to chloroplasts of non-infested leaves. Scanning electron micrographs of leaf surface revealed reduced presence of crystalline epicuticular waxes of epidermal cells in infested leaves as compared to non-infested ones. The main conclusion is that the attack of S. flava to S. halepense leaves induced plant susceptibility where aphid feeding behaviour and growth were both enhanced on previously infested leaves.     

Pollen morphology of some species belonging to Codonoprasum and Allium sections of Allium (Liliaceae-Alliaceae) genus

Pollen morphology of 14 Allium L. species grown in Turkey, that belong to the sections Codonoprasum and Allium, were investigated under LM (light microscopy) and by SEM (scanning electron microscopy). However, the pollens of 5 species were investigated under TEM (transmission electron microscopy). Detailed pollen morphological characteristics are given for Allium in the family on the basis of the results presented here together with data from the literature. The genera Allium homogeneous in both aperture type and exine ornamentation. It is suggested that some palynological characters, such as aperture type and the presence of an operculum, could be of taxonomic value at the section level.     

栒子属（Cotoneaster Medikus）植物叶表皮微形态特征及其分类学意义

在光学显微镜和扫描电镜下,对栒子属（Cotoneaster Medikus）2组15种植物的叶表皮特征进行了观察,发现疏花组（Sect. Cotoneaster）植物叶表皮细胞多为多边形,垂周壁一般为平直—弓形,气孔均为无规则型;单花组（Sect. Uniflos）植物叶表皮细胞常不规则型,垂周壁多浅波状,气孔除无规则型（anomocytic）外还兼有十字型（staurocytic）、四分体型（tetracytic）和等三体型（isotricytic）。依据气孔周围角质膜特征等叶表皮性状,把15种植物分为W型、S型和I型,其中W型是S型的特化类型,疏花组的种类为W型或S型,单花组的种类为I型。叶表皮微形态特征可以为栒子属植物组及种的分类学处理提供解剖学资料。     

The effect of the cassava green mite Mononychellus tanajoa on the growth and yield of cassava Manihot esculenta in a seasonally dry area in Kenya

The effect of the cassava green mite Mononychellus tanajoa on the growth and yield of cassava Manihot esculenta was studied over a 10-month period in two field trials near Lake Victoria in Kenya. One plot was maintained free of mites by means of acaricide, while the other was artificially infested.The highest population density of M. tanajoa occurred during the dry season. A maximum leaf area index (LAI) of about 2 was reached at the onset of the dry season. The total leaf area of mite infested plants was reduced compared with uninfested plants during the dry spell. During the following rainy season infested plants recovered and attained the same leaf area as uninfested plants. A multiple regression model predicting the leaf area showed that 58% of the seasonal variation could be explained by plant age, soil water, and leaf injury.The net growth rate of infested plants was lower than that of uninfested plants. Maximum values of 21 (infested plants) and 49 (uninfested plants) g m^-2 week^-1 were attained at the onset of the second rainy season. No difference was found between uninfested and infested plants with respect to net assimilation rates per unit leaf area during the dry season. The net assimilation rates reached a maximum almost at the same time as the growth rates, but the infested plants peaked slightly earlier and at a lower level than the uninfested plants. M. tanajoa did not affect the relative allocation of dry matter into stems and storage roots, but the absolute allocation of dry matter declined with increasing mite injury. Thus, after 10 months the dry matter of infested plants was reduced by 29% and 21% for storage roots and stems, respectively, compared with the uninfested plants.     

The metareticulate pollen morphology of Alternanthera Forssk. (Gomphrenoideae,Amaranthaceae) and its taxonomic implications

Alternanthera (Amaranthaceae) is a diverse genus largely restricted to the American Tropics that belongs to the alternantheroid clade containing C₄ and C₃–C₄ intermediate species. This research focuses on the study of pollen characters by studying 13 species, representatives of the two major clades and subclades of Alternanthera. General palynological comparisons were conducted with light microscopy (LM), scanning electron microscopy (SEM) and with confocal laser scanning microscopy (CLSM) for exine ultrastructure. Twenty-five characters were measured and described for Alternanthera and among these, 14 pollen characters were used to discriminate pollen groups using cluster analysis and canonical analysis of principal coordinates (CAP). Pollen form and ornamentation, pores number, spines length, number of ektexinous bodies and nanospines on the ektexinous bodies on pore membranes, arrangement of nanopores and spines on structural elements, and metareticula form were taxonomically important and therefore used to construct the first palynological key to the alternantheroid clade species. Our study indicates that the seemingly subtle morphological variation of pollen is useful for recognising three main pollen types within Alternanthera. The much needed palynological terminology for describing the mesoporium in the metareticulate pollen of Amaranthaceae is provided.     

Evaluation of Morphological Variability in Meloidogyne arenaria

Seven populations, representing cytological race A (triploid, 3n = 51-56) and the two host races (infective and noninfective on peanut) of Meloidogyne arenaria were studied with light microscopy (LM) and scanning electron microscopy (SEM). Characteristics of root-knot nematodes, recently recommended as useful taxonomic traits, were reexamined among these populations, and their variability both within and between populations was ascertained. We found that stylet morphology of females and head and stylet morphologies of males and second-stage juveniles were the most reliable characters for identification. The two host races of M. arenaria could not be distinguished morphologically. Two of the populations could be separated consistently from the remainder but were not sufficiently divergent to be considered new species. These two variant populations were similar; neither produced males in culture, and they differed from the typical populations in female perineal patterns (LM) as well as in cephalic structure (SEM) and tail shape (LM) of second-stage juveniles. In morphometric studies, most characters of the variant populations differed significantly from those of the typical M. arenaria.     

Assessing photosystem I and II distribution in leaves from C4 plants using confocal laser scanning microscopy

Images of chlorophyll fluorescence emitted at wavelengths above and below 700 nm were recorded from leaf sections of C₄ species using confocal laser scanning microscopy (LSM). We investigated species exhibiting both NAD-malic enzyme (NAD-ME) C₄ photosynthesis and NADP-malic enzyme (NADP-ME) C₄ photosynthesis. Comparing LSM fluorescence of leaf sections with flow-cytometrically determined fluorescence from individual chloroplasts revealed that LSM fluorescence was distorted by the optical properties of leaf sections. Leaf section fluorescence, when corrected by transmission data derived from light transmission images, agreed with flow cytometry data. The corrected LSM fluorescence yielded information on the distribution of the individual photosystems in the C₄ leaf sections: PSII concentrations in bundle sheath cells were elevated in NAD-ME species but diminished in most of the NADP-ME species investigated. The NADP-ME species, Arundinella hirta, however, showed normal PSII and increased PSI concentration in bundle sheath chloroplasts. Finally, a gradient of PSI was observed within the bundle sheath cells from Euphorbia maculata.     

Grapevine leaf gas exchange, plant growth, yield, fruit quality and carbohydrate reserves influenced by the grape leafhopper,Empoasca vitis

The impact of the grape leafhopper,Empoasca vitis, on leaf gas exchange, plant growth, yield, fruit quality and carbohydrate reserves of the grapevines,Vitis vinifera L., was studied. Gas exchange was measured on the discolored (red) and the green parts of infested main leaves and on leaves from uninfested vines. Photosynthesis and mesophyll conductance were severely reduced on main leaves showing leafhopper feeding symptoms. The stomatal conductance of the red leaf section of infested main leaves was lower than on undamaged control leaves. Additionally, the red leaf section of infested main leaves showed lower transpiration rates when compared to the green parts of the same leaves and to undamaged control leaves. Gas exchange processes of lateral leaves were not affected by leafhopper feeding. Leafhopperload on main leaves was correlated to visual damage symptoms. At 71.8 leafhopper-days per leaf up to 40% of the main leaf area of the infested plants was discolored from the borders towards the center. Lateral leaves showed no feeding symptoms. Shoot diameter, pruning weight and carbohydrate reserves in the wood were not affected by leafhoppers. Lateral leaf area growth was significantly stimulated on plants infested by leafhoppers. No decrease in yield and fruit quality with leafhopper-loads up to 71.8 leafhopper-days per leaf were observed.     

Rapid,in situ detection of Agrobacterium tumefaciens attachment to leaf tissue

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging‐based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum‐infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post‐infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012