Use of Low Temperature Scanning Electron Microscopy to Observe Frozen Hydrated Specimens of Nematodes

Frozen hydrated specimens of Pratylenchus agilis and dauer larvae of Steinernema carpocapsae were observed with low-temperature field emission scanning electron microscopy. This new technique provides information about the surface features of nematodes and also allows specimens to be fractured to reveal their internal structure. Furthermore, both halves of fractured specimens can be retained, examined, and photographed either as two-dimensional micrographs or as three-dimensional images for stereo observation (stereology) or quantitative measurements (stereometry). This technique avoids artifacts normally associated with procedures required to prepare nematodes for examination in the transmission and scanning electron microscopes, such as chemical fixation, dehydration, and sectioning or critical point drying.     

Low-Temperature Scanning Electron Microscope Observations of the Meloidogyne incognita Egg Mass: The Gelatinous Matrix and Embryo Development

The root-knot nematode Meloidogyne incognita was cultured monoxenically on excised tomato roots. Galls and egg masses were observed daily using a light microscope. Two phases were distinguished in the gelatinous matrix of the egg mass: a translucent, amorphous material on the surface of the egg mass and a denser, layered phase in which nematode eggs were deposited. Egg masses were also cryofixed, fractured, and observed as frozen, hydrated specimens on a cold stage in a scanning electron microscope (SEM). In the SEM, the layered phase appeared as a meshwork of fibrils that became more loosely associated as the gelatinous matrix aged: Small pearl-like bodies were observed along the fibers of gelatinous matrix. The egg shell surface and several stages of embryo development, including the one-cell stage, initial cleavages, blastula, gastrula, tadpole stage, elongation, and molt of the first-stage juvenile within the egg shell, were observed and photographed with this technique. The developmental events observed were consistent with those described in other nematode species with different techniques.     

Effect of an Ice-Nucleating Activity Agent on Subzero Survival of Nematode Juveniles

Juveniles of five species of nematodes, Caenorhabditis elegans, Panagrellus redivivus, Pratylenchus agilis, Pristionchus pacificus, and Distolabrellus veechi, were added to solutions with (treatment) and without (control) a commercial ice-nucleating activity (INA) agent. Ten-microliter droplets of the solutions containing the juveniles were placed on glass microscope slides and transferred to a temperaturecontrolled freeze plate where the temperature was reduced to -6 to -8 °C. At this temperature, the droplets containing the INA agent froze while those without the agent remained liquid. After 2 minutes, the temperature of the plate was raised to 24 °C, and the slides were examined with a light microscope to determine the viability of the juveniles. The results showed that usually most juveniles (43% to 88%, depending on species) in solutions that did not contain the INA agent (controls) were active, indicating that the juveniles were capable of supercooling and were thereby protected from the subzero temperatures. Alternatively, less than 10% of the juveniles that had frozen for 2 minutes in solutions containing the INA agent remained viable, indicating that inoculative freezing of the solution was lethal to the supercooled juveniles. Our results suggest that, in geographical areas where winter temperatures may not be sufficiently low or sustained to freeze soil, the addition of an INA agent may help induce ice nucleation and thereby reduce the populations of nematode species that are unable to survive when the soil solution is frozen.     

A Comparison of Techniques Useful for Preparing Nematodes for Scanning Electron Microscopy

Second-stage juveniles of Meloidogyne incognita were prepared by several different techniques for scanning electron microscopy (SEM). Sequential fixation in the cold (4-8 C) was superior to rapid fixation at room temperature, glutaraldehyde and glutaraldehyde-formalin were better fixatives than formalin alone, and critical point drying with carbon dioxide or Freon gave similar results that were only slightly better than air drying with Freon. Freeze drying sequentially fixed nematodes from 100% ethanol in liquid propane produced the best preserved specimens with the fewest artifacts. Specimens of various free-living and plant-parasitic nematodes were prepared for SEM by freeze drying. This technique was adequate for most genera but unsatisfactory for a few. Although each genus may require a different procedure for optimum preservation of detail, sequential fixation with glutaraldehyde and freeze drying are comparable and often superior to commonly used techniques for preparing nematodes for SEM.     

Temperature-Dependent Development of Pasteuria penetrans in Meloidogyne arenaria

Pasteuria penetrans is a promising biological control agent of plant-parasitic nematodes. This study was conducted to determine effects of temperature on the bacterium''s development in Meloidogyne arenaria. Developmental stages of P. penetrans were viewed with a compound microscope and verified with scanning electron microscopy within each nematode at 100 accumulated degree-day intervals by tracking accumulated degree-days at three temperatures (21, 28, and 35 °C). Five predominant developmental stages of P. penetrans were identified with light microscopy: endospore germination, vegetative growth, differentiation, sporulation, and maturation. Mature endospores were detected at 28, 35, and >90 calendar days at 35, 28, and 21 °C, respectively. The number of accumulated degree-days required for P. penetrans to reach a specific developmental stage was different for each temperature. Differences were observed in the development of P. penetrans at 21, 28, and 35 °C based on regression values fitted for data from 100 to 600 accumulated degree-days. A linear response was observed between 100 to 600 accumulated degree-days; however, after 600 accumulated degree-days the rate of development of P. penetrans leveled off at 21 and 28 °C, whereas at 35 °C the rate decreased. Results suggest that accumulated degree-days may be useful only in predicting early-developmental stages of P. penetrans.     

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.     

Scanning Electron Microscope Study of the Root-knot Nematode (Meloidogyne incognita) on Tomato Root

This study examines the types of structural information that can be gained by utilizing the scanning electron microscope (SEM) and a cryofracture technique to examine the host-parasite interaction. Roots of tomato, Lycopersicon esculentum cv. Marglobe, were cultured aseptically and inoculated with the root-knot nematode, Meloidogyne incognita. Twenty-four hours to four weeks after inoculation, developing galls were removed from the cultures and processed for SEM observation. The cryofracture technique was used to reveal internal structural features within the developing galls. The results illustrate structural details concerning penetration of the roots, differentiation of syncytia, and development of the nematodes beginning with the second-stage larvae and ending with adult egg-laying females.     

Unique advantages of using low temperature scanning electron microscopy to observe bacteria

Summary Electron microscopy (EM) has greatly helped to elucidate our understanding of bacterial structure and function. However, several recent studies have cautioned investigators about artifacts that result from the use of conventional EM preparation procedures. To avoid these problems, the use of low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leaves (Spinacia oleracea L. cv. New Jersey), which were naturally infected or inoculated with bacteria, were used as the experimental material. 1 cm segments of the infected leaves were plunge frozen in liquid nitrogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hydrated leaf segments were transferred onto agar medium to determine whether preparation for LTSEM was nondestructive to the bacteria. The other tissue segments were chemically fixed by freeze-substitution. The results indicated that after cryopreparation and observation in the LTSEM: (i) viable bacteria, which were recovered from the leaf sample, could be cultured on agar medium for subsequent study, and (ii) the frozen samples could be freeze substituted and embedded so that transmission electron microscopic (TEM) observations could be carried out on the same specimen. In conclusion, frozen, hydrated leaf tissue infected with bacteria can be observed using LTSEM and then can be either processed for TEM observation to obtain further structural details or recovered to culture the pathogenic bacteria for supplementary studies.Abbreviations EPS extracellular polysaccharide - EM electron microscopy - LTSEM low temperature scanning electron microscopy - SEM scanning electron microscopy - TEM transmission electron microscopy - TSA tryptic soy agar - TSB tryptic soy broth Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Axenizing and Culturing Endomigratory Plant-Parasitic Nematodes using Pluronic F127, Including its Effects,on Population Dynamics of Pratylenchus penetrans

A non-chemical technique for surface sterilizing plant-parasitic nematodes for aseptic cultures is described. The method is most applicable to nematodes with active migratory infective stages and requires only a few starting specimens. Rate of achieving a primary aseptic culture with the technique ranged from 60%-100% depending on the conditions of the specimens collected for culturing. Aseptic cultures of species of Meloidogyne, Rotylenchuluz, Pratylenchus, and Radopholus initiated with the method remained contamination-free after 12 months of maintenance in tomato root explant or alfalfa callus cultures. Further studies of Pluronic F127, a polyol gel medium employed in the technique to confine the spread of contaminating bacteria or fungi associated with the nematodes, showed that the polyol gel was a suitable support medium for culturing corn root explant, alfalfa callus tissues, and consequently Pratylenchus species including P. agilis, P. brachyurus, P. scribneri, and P. penetrans. During the course of 10 months, P. penetrans reared in polyol-base medium followed a standard biological growth curve, multiplied to a higher population density, maintained a similar female-to-male ratio, and possessed a similar tendency to reside inside or outside host tissues as did P. penetrans reared in agar-base medium. The percentages of P. penetrans juveniles in the sub-populations residing outside or inside the host tissues reared in polyol-base medium also were similar to and fluctuated temporally in like manner as those reared in agar-base medium. Members of these sub-populations from the polyol- or agar-base were equally infective and reproductive after 9 months of culturing.     

Morphological Comparison of Head Shape and Stylet Morphology of Second-stage Juveniles of Meloidogyne Species

Head shape and stylet morphology of second-stage juveniles of one population each of M. incognita, M. javanica, M. arenaria, and M. hapla were compared by light microscopy. Excised stylets of each species were also compared by scanning electron microscopy (SEM). Differences in head morphology were observed only between M. hapla and the other three species. In SEM, differences in stylet size, shape, and relative distance of the dorsal esophageal gland orifice to the base of the stylet were evident. Differences in stylet morphology between M. incognita and M. javanica could not he detected by light microscopy, but M. arenaria and M. hapla could be distinguished from each other and from the other two species. Head shape and styler morphology of second-stage juveniles are considered useful taxonomic characters.     

Leptosomatides brevicaudatus n. sp. and a Redescription of Leptosomatides marinae Platonova, 1967 (Enoplida: Leptosomatidae)

The free-living marine nematodes Leptosomatides brevicaudatus n. sp. and L. marinae were described and redescribed, respectively, from material collected in the northwest Pacific. Leptosomatides brevicaudatus n. sp. from Simushir Island differs from L. marinae in the ratio c8 (body length divided by tail length measured on the chord) and the length of the spicules. Leptosomatides marinae is redescribed from light microscopy (LM) observations of the type specimens and LM and scanning electron microscopy (SEM) observations of specimens from Hokkaido, Japan. It appears to be impossible to distinguish among some species of Leptosomatides because they are either insufficiently described or known only from females. Secondary sexual characters of males are essential for purposes of identification.     

Morphological Comparison and Taxonomic Utility of Copulatory Structures of Selected Nematode Species

Spicules of 9 Meloidogyne, 2 Heterodera, 3 Globodera, and 12 other plant-parasitic, insect-parasitic, and free-living nematodes were excised and examined using scanning electron microscopy (SEM). Gubernacula of some of the species were also excised, and their structure was determined. The two spicules of all species examined were symmetrically identical in morphology. The spicule typically consisted of three parts: head, shaft, and blade with dorsal and ventral vela. The spicular nerve entered through the cytoplasmic core opening on the lateral outer surface of the spicule head and generally communicated with the exterior through one or two pores at the spicule tip. Spicules of Xiphinema sp. and Aporcelaimellus sp. were not composed of three typical parts, were less sclerotized, and lacked a cytoplasmic core opening and distal pores. Spicules of Aphelenchoides spp. had heads expanded into apex and rostrum and had very arcuate blades with thick dorsal and ventral edges (limbs). Gubernaculum shapes were stable within a species, but differed among species examined. The accessory structures of Hoplolaimus galeatus consisted of a tongue-shaped gubernaculum with two titillae at its distal end and a plate-like capitulum terminating distally in two flat, wing-like structures. A comparison of spicules of several species of Meloidogyne by SEM and light microscopy revealed no striking morphological differences.     

Identification of Meloidogyne Species on the Basis of Head Shape and,Stylet Morphology of the Male

Head shape and stylet morphology of males of 90 populations of M. arenaria, M. hapla, M. incognita, and M. javanica from geographic regions of the world were compared by light microscopy (LM). In addition, stylets of one population each of M. arenaria, M. incognita, and M. javanica and three different chromosomal forms of M. hapla race A and two of race B were excised and examined with a scanning electron microscope (SEM). Differences among species occurred in both head and stylet morphology. Head morphology differed in size and shape of the head cap, annulation of the head region, and width of the head region relative to the first body annule. Differences in stylets occurred in size and shape of the cone, shaft, and knobs. All populations of M. hapla, except one, had similar head morphology, but stylet morphology was different between cytological races A and B. Populations of M. javanica varied with respect to the presence of head annulations. Head shape and stylet morphology of males are recommended as additional characters useful in the identification of root-knot nematodes.     

Surface Coat of Meloidogyne incognita

The nematode surface coat is defined as an extracuticular component on the outermost layer of the nematode body wall, visualized only by electron microscopy. Surface coat proteins of Meloidogyne incognita race 3 infective juveniles were characterized by electrophoresis and Western blotting of extracts from radioiodine and biotin-labeled nematodes. Extraction of labeled nematodes with cetyltrimethylammonium bromide yielded a principal protein band larger than 250 kDa and, with water soluble biotin, several faint bands ranging from 31 kDa to 179 kDa. The pattern of labeling was similar for both labeling methods. Western blots of unlabeled proteins were probed with a panel of biotin-lectin conjugates, but only Concanavalin A bound to the principal band. Nematodes labeled with radioiodine and biotin released ¹²⁵I and biotin-labeled molecules into water after 20 hours incubation, indicating that surface coat proteins may be loosely attached to the nematode. Antiserum to the partially purified principal protein bound to the surface of live nematodes and to several proteins on Western blots. Differential patterns of antibody labeling were obtained on immuno-blots of extracts from M. incognita race 1, 2, and 3; Meloidogyne hapla race 2; and Meloidogyne arenaria cytological race B.     

Nematodes in Dryland Field Crops in the Semiarid Pacific Northwest United States

Soils and roots of field crops in low-rainfall regions of the Pacific Northwest were surveyed for populations of plantparasitic and non-plant-parasitic nematodes. Lesion nematodes (Pratylenchus species) were recovered from 123 of 130 non-irrigated and 18 of 18 irrigated fields. Pratylenchus neglectus was more prevalent than P. thornei, but mixed populations were common. Population densities in soil were affected by crop frequency and rotation but not by tillage or soil type (P < 0.05). Many fields (25%) cropped more frequently than 2 of 4 years had potentially damaging populations of lesion nematodes. Pratylenchus neglectus density in winter wheat roots was inversely correlated with grain yield (r² = 0.64, P = 0.002), providing the first field-derived evidence that Pratylenchus is economically important in Pacific Northwest dryland field crops. Stunt nematodes (Tylenchorhynchus clarus and Geocenamus brevidens) were detected in 35% of fields and were occasionally present in high numbers. Few fields were infested with pin (Paratylenchus species) and root-knot (Meloidogyne naasi and M. chitwoodi) nematodes. Nematodes detected previously but not during this survey included cereal cyst (Heterodera avenae), dagger (Xiphinema species), and root-gall (Subanguina radicicola) nematodes.     

Position of Pharyngeal Gland Outlets in Monhysteridae (Nemata)

In this study an attempt was made to determine the position of the outlets and nuclei of the pharyngeal glands in four monhysterid genera. Five Eumonhystera spp., seven Monhystera spp., and eight Monhystrella spp. were studied under the light microscope. Longitudinal sections of an undescribed Monhystera sp. and cross sections of Geomonhystera disjuncta were also studied under the scanning and transmission electron microscope, respectively. The results of the light microscopic studies were inconclusive about the position of the outlets but showed a number of nuclei in the basal part of the pharynx. The scanning and transmission electron microscopic studies revealed five pharyngeal glands and their outlets; their position was as follows: dorsal gland outlet at the base of buccal tooth, first pair of ventrosublateral gland outlets halfway along the pharynx, and second pair of ventrosublateral gland outlets close to the base of the pharynx. It is concluded that at least three, and possibly five, nuclei are in the basal part of the pharynx. This pattern, in the position of the outlets and nuclei, is similar to that in Caenorhabditis elegans (Maupas, 1900) Dougherty, 1953 and may well be the basic plan in the Class Chromadorea (including Secernentia as a subclass).     

Morphology of Second-stage Juveniles and Males of Globodera tabacum tabacum,G. t. virginiae,and G. t. solanacearum (Nemata: Heteroderinae)

Morphological comparisons with light microscopy and scanning electron microscopy were made among second-stage juveniles (J2) and males of several isolates of the three subspecies of the tobacco cyst nematode complex, Globodera tabacum sspp. tabacum, virginiae, and solanacearum. Observations focused on the anterior region, (including head shape, lip pattern, and stylet morphology) and the tail region (including tail shape in J2 and spicules in males). The three subspecies could not be separated on the basis of any of these characters.     

Structure of the Cuticle of Ceramonema carinatum (Chromadorida: Ceramonenatidae)

The cuticle of Ceramonema carinatum (Chromadorida: Ceramonematidae) is described and illustrated from scanning and transmission electron microscopy. Each of ca. 200 annules is composed of a single ring with eight external flat faces (plates), which are divided by longitudinal ridges formed by pairs of parallel upstanding vanes. Vanes and plates overlap those of the adjacent annules. Longitudinal ridges extend from the cephalic capsule to the tail spike. On the cephalic capsule a simple ridge extends each of the eight ridges to a position just anterior to the amphid. Cuticular plates are formed from the electron-dense cortical layer and contain lacunae filled with fine fibrils. The vanes are denser, with laminations on a central core. In the annular grooves between the plates there is an electron-lucent layer, which it is suggested, by comparison with other nematodes, is the basal layer. An epicuticle overlies the cortical plates, the vanes, and the interannular lucent layer. Cuficular structure is compared with that of other Ceramonematidae and related nematodes.     

Cactodera salina n. sp. from the Estuary Plant, Salicornia bigelovii,in Sonora,Mexico

Cactodera salina n. sp. (Heteroderinae) is described from roots of the estuary plant Salicornia bigelovii (Chenopodiaceae), in Puerto Pefiasco, Sonora, Mexico, at the northern tip of the Sea of Cortez. The halophyte host is grown experimentally for oilseed in plots flooded daily with seawater. Infected plants appear to be adversely affected by C. salina relative to plants in noninfested plots. Cactodera salina extends the morphological limits of the genus. Females and cysts have a very small or absent terminal cone and deep cuticular folds in a zigzag pattern more typical of Heterodera and Globodera than of Cactodera spp. Many Cactodera spp. have a tuberculate egg surface, whereas C. salina shares the character of a smooth egg with C. amaranthi, C. weissi, and C. acnidae. Only C. milleri and C. acnidae have larger cysts than C. salina. Face patterns of males and second-stage juveniles, as viewed with scanning electron microscopy, reveal the full complement of six lip sectors as in other Cactodera spp. Circumfenestrae of C. salina are typical for the genus.