FIG 2 - uploaded by Jonathan Eisenback
Content may be subject to copyright.
A.) A high-resolution megapixel mosaic composed of 22 photomicrographs that are twelve megapixels each of a female of Ditylenchus dipsaci (Kü hn, 1857) Filipjev, 1936 imaged with a digital camera attached to a plano-apo quality 63x oil immersion objective. B.) The mosaic image after the 22 individual photographs have been flattened into a single image and the background has been filled with a uniform color. The insert near the anterior end of the female has been printed at 300 dpi and presented in Fig 3.
Source publication
Multiple images of a whole nematode specimen were taken with a high power oil-immersion objective lens and joined together to form one high-resolution megapixel, mosaic photomicrograph of the entire specimen, with the use of a relatively new mounting technique made with a 4% water agar pad. The agar pad kept the specimen nearly level and lateral, a...
Contexts in source publication
Context 1
... the specimen: The specimen was placed in the field of view and the nematode was examined to insure that it was lying in a lateral position that was nearly level, in a healthy physiological condition, im- mobile, and with morphology typical for the species. Nematodes that were still moving would become para- lyzed in a few minutes. After the correct exposure was selected for the initial picture, the exposure was locked for all of the remaining images. A series of photographs along the entire length of the specimen were made, overlapping each new image by 30-50% of the previous image ( Fig. 2A). The position of the camera was not moved in order to insure that the pictures were taken with the same orientation. Since the specimen was al- ready nearly level, only the most minor adjustments to the focus were necessary in order to keep the medial plane of the specimen in focus. If certain morphologi- cal features occurred outside of the medial plane, the level of focus was changed and the photograph with that feature was imaged after the entire specimen was imaged. Depending upon the length of the specimen and the magnification, the entire nematode was pho- tographed in a series of two to thirty or more images (Fig. ...
Context 2
... the specimen: The specimen was placed in the field of view and the nematode was examined to insure that it was lying in a lateral position that was nearly level, in a healthy physiological condition, im- mobile, and with morphology typical for the species. Nematodes that were still moving would become para- lyzed in a few minutes. After the correct exposure was selected for the initial picture, the exposure was locked for all of the remaining images. A series of photographs along the entire length of the specimen were made, overlapping each new image by 30-50% of the previous image ( Fig. 2A). The position of the camera was not moved in order to insure that the pictures were taken with the same orientation. Since the specimen was al- ready nearly level, only the most minor adjustments to the focus were necessary in order to keep the medial plane of the specimen in focus. If certain morphologi- cal features occurred outside of the medial plane, the level of focus was changed and the photograph with that feature was imaged after the entire specimen was imaged. Depending upon the length of the specimen and the magnification, the entire nematode was pho- tographed in a series of two to thirty or more images (Fig. ...
Context 3
... The finished size of the mosaic was estimated and the canvas was enlarged to fit. The canvas could be in- creased in size as the image was formed or it could be cropped if it was too big for the final image. 4) The intermediate work was saved as a Photoshop file (.psd) to avoid accidentally overwriting the image file. 5) The adjacent photo forming the image was copied and pasted into the working Photoshop file. 6) The opacity of the pasted layer was adjusted to 50%. 7) The semi-transparent layer was moved into position to match the background image, and its opacity was re- stored to 100%. 8) The sharp, overlapping edge of the top photo was erased with the eraser tool set as a brush with soft edges. 9) Steps 6 -8 were repeated for all remaining photos of the specimen (Fig. ...
Context 4
... Finally, a selection was created to crop the mosaic photograph into a rectangular shape that contained the entire specimen (Fig. 2B). 11) The specimen was rotated so that it was in a nearly vertical orientation. 12) All of the layers of the mosaic photomicrograph were flattened into one, and additional editing replaced the blocky, irregular background with the same color (Fig. ...
Context 5
... Finally, a selection was created to crop the mosaic photograph into a rectangular shape that contained the entire specimen (Fig. 2B). 11) The specimen was rotated so that it was in a nearly vertical orientation. 12) All of the layers of the mosaic photomicrograph were flattened into one, and additional editing replaced the blocky, irregular background with the same color (Fig. ...
Context 6
... resulting high-resolution megapixel mosaic photomicrograph of Ditylenchus dipsaci (Kü hn, 1857) Filipjev, 1936 (Figs. 2-3) is the most detailed photo- graph that has been made of a whole nematode; it is more than 616 megapixels in size. At 300 dpi, the pho- tograph is 80 cm wide by 194 cm long. An insert of the whole specimen shown in Fig. 2B is presented at 300 dpi in Fig. 3. Many details of the specimen are clearly visible including the morphology of the stylet, the opening of the dorsal pharyngeal gland, and the nucleoli and nu- clei of the oocytes. The photograph of the entire speci- men contains many additional morphological details. Because of the large size of the imaging sensor in modern cameras (12.8 megapixels in this case), these photographs can be reduced in size by 50% and still retain many of the morphological details that were contained in the original ...
Context 7
... resulting high-resolution megapixel mosaic photomicrograph of Ditylenchus dipsaci (Kü hn, 1857) Filipjev, 1936 (Figs. 2-3) is the most detailed photo- graph that has been made of a whole nematode; it is more than 616 megapixels in size. At 300 dpi, the pho- tograph is 80 cm wide by 194 cm long. An insert of the whole specimen shown in Fig. 2B is presented at 300 dpi in Fig. 3. Many details of the specimen are clearly visible including the morphology of the stylet, the opening of the dorsal pharyngeal gland, and the nucleoli and nu- clei of the oocytes. The photograph of the entire speci- men contains many additional morphological details. Because of the large size of the imaging sensor in modern cameras (12.8 megapixels in this case), these photographs can be reduced in size by 50% and still retain many of the morphological details that were contained in the original ...
Context 8
... 1900) Dougherty, 1953. Specimens mounted on an agar pad are nearly always level and they lie in a lateral position. This agar pad technique was slightly modified to make preparations of nematodes that are level and lying in a natural lateral position (Fig. 1). The water agar prevents the nematodes from being flat- tened by the weight of the coverslip and it contains a narcotizing agent that causes paralysis instead of an unnatural death caused by heat or harsh fixatives. As a result, the specimens can be photographed at high resolution with very fine details, reminiscent of fresh, living nematodes (Figs. 2-3). Furthermore, students who have never mounted a nematode before usually achieve success on the very first attempt; whereas, other techniques require several days or weeks to ...
Citations
... The supernatant was removed, and the nematodes were used for morphological analyses. For morphological characterization nematodes were mounted in water agar slides [33] or fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method [34,35]. Fresh or fixed nematodes were photographed with a DP73 digital Olympus camera mounted on an Bx51 Olympus microscope. ...
The beech leaf disease nematode, Litylenchus crenatae subsp. mccannii, is recognized as a newly emergent nematode species that causes beech leaf disease (BLD) in beech trees (Fagus spp.) in North America. Changes of leaf morphology before emergence from the bud induced by BLD can provoke dramatic effects on the leaf architecture and consequently to tree performance and development. The initial symptoms of BLD appear as dark green, interveinal banding patterns of the leaf. Despite the fast progression of this disease, the cellular mechanisms leading to the formation of such aberrant leaf phenotype remains totally unknown. To understand the cellular basis of BLD, we employed several types of micros-copy to provide an exhaustive characterization of nematode-infected buds and leaves. His-tological sections revealed a dramatic cell change composition of these nematode-infected tissues. Diseased bud scale cells were typically hypertrophied and showed a high variability of size. Moreover, while altered cell division had no influence on leaf organogenesis, induction of cell proliferation on young leaf primordia led to a dramatic change in cell layer architecture. Hyperplasia and hypertrophy of the different leaf cell layers, coupled with an abnormal proliferation of chloroplasts especially in the mesophyll cell layers, resulted in the typical interveinal leaf banding. These discrepancies in leaf cell structure were depicted by an abnormal rate of cellular division of the leaf interveinal areas infected by the nematode, promoting significant increase of cell size and leaf thickness. The formation of symptomatic BLD leaves is therefore orchestrated by distinct cellular processes, to enhance the value of these feeding sites and to improve their nutrition status for the nematode. Our findings thus uncover relevant cellular events and provide a structural framework to understand this important disease.
... The easiest and most efficient way to make a temporary slide is to use a pad of 5% water agar (Fig. 5.5) (Eisenback, 2012). The pad is made by placing three slides on the laboratory bench top with the two outer slides containing a layer of thick tape. ...
This book is extensively illustrated, and addresses both fundamental traditional techniques and new methodologies. The chapters aim to provide an introduction to basic techniques for laboratory and field work with plant-parasitic and free-living soil-dwelling nematodes. The coverage highlights areas that have expanded and/or become more widespread over recent years, such as techniques used in diagnostic laboratories, including computerized methods to count and identify nematodes, and the use of entomopathogenic nematodes as environmentally acceptable control systems for some insect pests. The use of molecular techniques is relevant to many areas of work on nematodes and basic information on current molecular methodologies and their various applications is included.
... The easiest and most efficient way to make a temporary slide is to use a pad of 5% water agar (Fig. 5.5) (Eisenback, 2012). The pad is made by placing three slides on the laboratory bench top with the two outer slides containing a layer of thick tape. ...
This book is extensively illustrated, and addresses both fundamental traditional techniques and new methodologies. The chapters aim to provide an introduction to basic techniques for laboratory and field work with plant-parasitic and free-living soil-dwelling nematodes. The coverage highlights areas that have expanded and/or become more widespread over recent years, such as techniques used in diagnostic laboratories, including computerized methods to count and identify nematodes, and the use of entomopathogenic nematodes as environmentally acceptable control systems for some insect pests. The use of molecular techniques is relevant to many areas of work on nematodes and basic information on current molecular methodologies and their various applications is included.
... Perineal patterns were made as described by Hartman and Sasser (1985) and observed according to Eisenback (2010). Whole specimens prepared for light microscopy (LM) were mounted on 4% water agar blocks (Eisenback, 2012). They were photographed with a Nikon D300 DSLR camera attached to a Dialux 22 Leitz microscope. ...
Females, males, and second-stage juveniles of Meloidogyne kikuyensis were examined by light and scanning electron microscopy. The morphology of M. kikuyensis was typical for species of the genus in general, but differed in several characters, appearing to be in a more primitive state. The head morphology of males and second-stage juveniles of most species of root-knot nematode is made up of a large labial disk surrounded by the fused pairs of the sub-dorsal and sub-ventral lips, but in M. kikuyensis, the labial disk is surrounded by six distinct lips. Second-stage juveniles appear to develop similarly to that of other members of the genus. The division of the egg seems to be quite different from typical species in that two small, highly refractive cells, are set-aside early in embryogenesis. Elucidation of the mitochondrial nucleotide sequence for the cytochrome oxidase subunit II and the large subunit of the ribosomal RNA gene (COII-16S rRNA) and the ITS1 region implicated M. kikuyensis is in a basal position when compared to other species of the genus.
... The slides were observed under a laser scanning microscope (Zeiss LSM 710, Germany) using the differential interference contrast (DIC) method. Measurements of the dorsal and subventral gland cells were performed from 10 individuals for each of the life stages (juveniles and adults), mounted using an agar pad technique, as described by Eisenback (2012). Statistical significance was tested using Mann-Whitney U-test analysis (STATISTICA v12.0) (Mann and Whitney, 1947). ...
The migratory endoparasitic nematode, Bursaphelenchus xylophilus, which is the causal agent of pine wilt disease, has phytophagous and mycetophagous phases during its life cycle. This highly unusual feature distinguishes it from other plant-parasitic nematodes and requires profound changes in biology between modes. During the phytophagous stage the nematode migrates within pine trees, feeding on the contents of parenchymal cells. Like other plant pathogens, B. xylophilus secretes effectors from pharyngeal gland cells into the host during infection. We provide the first description of changes in the morphology of these gland cells between juvenile and adult life stages. Using a comparative transcriptomic approach and an effector identification pipeline we identify numerous novel parasitism genes which may be important for mediating interactions of B. xylophilus with its host. In-depth characterisation of all parasitism genes using in situ hybridisation reveals two major categories of detoxification proteins, those specifically expressed in either the pharyngeal gland cells or the digestive system. These data suggest that B. xylophilus incorporates effectors in a multilayer detoxification strategy in order to protect itself from host defence responses during phytophagy.
This article is protected by copyright. All rights reserved.
Pratylenchus penetrans is a common and important agricultural pest in Wisconsin, a USA state with a diverse agriculture. We compared populations from around the state to each other and to data published for populations around the world to gain insight on the variability of features important for identification of this cosmopolitan species. Thirteen isolates from samples collected in soybean fields in ten Wisconsin counties were established in monoxenic cultures. Analysis of morphological features revealed the least variable feature for all isolates collectively was vulva percentage. Features less variable within than among isolates were body width, lip region height, and stylet length. Some isolates showed only the smooth tail tip phenotype and others had a mix of smooth and annulated tail phenotypes. A suite of features provided sufficient pattern to group isolates into four clusters according to hierarchical agglomerative clustering and canonical discriminative analyses, but not with enough distinction to be useful for classification. Haplotype analysis based on the COI mitochondrial gene of the 13 cultured isolates, 39 Wisconsin field populations, and published sequences representing five additional USA states and six countries revealed 21 haplotypes, 15 of which occurred in Wisconsin. Ten haplotypes represented in Wisconsin were shared with populations from Europe, South America, Africa, or Asia. Five haplotypes were unique to Wisconsin, six were unique to The Netherlands, and one was unique to Japan suggesting that even more COI diversity will be revealed when more COI sequences for P. penetrans become available. The maximum pairwise sequence variation was 6% and the SNPs did not alter amino acids, indicating cryptic biodiversity within the species worldwide. The cosmopolitan to localized scale of distribution of COI haplotypes could be due to frequent and ongoing dispersal events, facilitated by life history traits and the broad host range of P. penetrans. Regions of diverse agriculture, like Wisconsin, show promise for studying this important pest and our study confirms the utility of the COI mtDNA gene for studying variation within a species.
Meloidogyne aegracyperi n. sp. is described from roots of purple nutsedge in southern New Mexico, USA. Mature females are small (310-460 µm), pearly white, with their egg masses completely contained inside root galls. The neck is often at a 90 to 130° angle to the protruding posterior end with the perineal pattern. The distance of the dorsal esophageal gland orifice (DGO) to the base of the stylet is relatively long (4.0-6.1 µm), and the excretory pore is level with the base of the stylet. The anterior portion of the rounded lumen lining of the metacorpus contains 3 to 10 small vesicles. The perineal pattern has a rounded dorsal arch with a tail terminal area that is smooth or marked with rope-like striae. Only two males were found. The body twists 90° throughout its length. The DGO to the base of the stylet is long (3.0-3.3) µm. The cephalic framework of the second-stage juvenile is weak, and the stylet is short (10.1-11.8 µm). The DGO to the base of the stylet is long (3-5 µm). The tail is very long (64-89 µm) and the hyaline portion of the tail is very narrow, making the tail finely pointed. Eggs are typical for the genus and vary in length (85.2-99.8 µm) and width (37.1-48.1 µm), having a L/W ratio of (2.1-2.6). Maximum likelihood phylogenetic analyses of the different molecular loci (partial 18S rRNA, D2-D3 of 28S rRNA, internal transcribed spacer (ITS) rRNA, cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA gene fragments and partial Hsp90 gene) placed this nematode on an independent branch in between M. graminicola and M. naasi and a cluster of species containing M. chitwoodi. M. fallax, and M. minor. Greenhouse tests showed that yellow and purple nutsedge were the best hosts, but perennial ryegrass, wheat, bentgrass, and barley were also hosts.
The first plant parasitic nematode to cause alarm in Virginia and West Virginia was the wheat gall nematode, Anguina tritici, which was found in Rockingham County, Virginia in 1917 where it had been present for 10 years or more, causing a loss of 25–50%. A quarantine was threatened for Virginia, West Virginia and Georgia, but was not realized. It was eventually eradicated by using crop rotation and clean seed. Root knot nematodes became the second recognized plant parasitic nematode, and is the most prevalent and widespread plant nematode in these two states. They affect most of the major field crops such as tobacco, cotton, wheat, tomato, potato, peanut and pumpkin; damage crops that support livestock including alfalfa, soybean and corn and cause injury to crops that improve the quality of life like vegetables, grapes, fruit, nursery, greenhouse, floriculture and turfgrass. Although a bulletin was published warning about the occurrence of soybean cyst nematode in North Carolina and strict measures were put in place to prevent its spread to Virginia, it was too late and it rapidly became another important nematode. However, soybean cyst nematode is not a problem in West Virginia, because soybeans are not widely grown. In West Virginia, root knot, lesion, and dagger nematodes are the three most important genera. Fortunately, in both states, agriculture has adapted very well to the demands placed on it by growers, government and society. As both states continue to lose vast areas of farmland to urban sprawl, and less available fertile agricultural land remains for farming and more pressure is placed on utilizing sustainable practices, plant parasitic nematodes will remain an important factor that must be considered when implementing sustainable agricultural practices.
