Monoclonal Antibodies to Secretory Granules in Esophageal Glands of Meloidogyne Species

Monoclonal antibodies to secretory granules in the dorsal or subventral esophageal glands were generated by injecting BALB/c mice with immunogens from preparasitic second-stage juveniles (J2) of Meloidogyne incognita. Antibodies specific for secretory granules in the J2 subventral esophageal glands or the dorsal gland were identified by indirect immunofluorescence microscopy. Only antibodies that reacted with granules in the J2 dorsal gland reacted with the esophageal gland lobe ofM. incognita adult females. The antibodies also reacted with secretory granules in both types of esophageal glands in M. javanica and M. arenaria J2 but not with granules in esophageal glands of Heterodera glycines J2.     

Ultrastructural Cytochemistry of Secretory Granules of Esophageal Glands of Meloidogyne incognita

Ultrastructural cytochemical tests for several enzymes, proteins, carbohydrates, and nucleic acids were conducted on secretory granules o£ dorsal and subventral esophageal glands of preparasitic second-stage juveniles and the dorsal gland of adult females of Meloidogyne incognita. Secretory granules in the subventral glands of juveniles stained positive for acid phosphatase. Peroxidase, DNase, RNase, cellulase, and nucleic acids were not detected in these granules. Secretory granules in the dorsal gland of adult females stained positive for peroxidase (pH 7.6) in < 50% of the tests, Acid phosphatase, β-glucuronidase, DNase, RNase, polyphenoloxidase, cellulase, and carbohydrates were not detected in dorsal gland granules in adult females. Positive staining with cobalt thiocyanate, a stain for amino groups of basic proteins, occurred in secretory granules in the dorsal gland, ribosomes, and chromatin in adult females. Ribosomes, nuclei, and secretory granules of the dorsal gland of adult females intensely stained when incubated in three reagents specific for nucleic acid.     

Changes in Esophageal Gland Activity During the Life Cycle of Nacobbus aberrans (Nemata: Pratylenchidae)

Electron and light microscopy were used to study the dorsal gland (DG) and the two subventral glands (SvG) of seven developmental phases of Nacobbus aberrans: pre-parasitic second-stage juveniles (J2), parasitic J2, third- (J3) and fourth- (J4) stages, migratory females, young sedentary females, and mature sedentary females. In each developmental phase the level of esophageal gland activity, was estimated by the abundance of organelles associated with secretory pathways, including endoplasmic reticulum, ribosomes, Golgi, multivesicular bodies, and secretory granules. All esophageal glands were metabolically active in all J2 examined, although only in parasitic J2 were there numerous secretory granules in the esophageal gland extensions and ampullae. No evidence of secretory activity was observed in the esophageal glands of the coiled and relatively inactive J3 and J4, nor in migratory females; these stages apparently do not feed. Observations suggest that reserves stored by J2 sustain three ecdyses and the migratory female''s search for a feeding site and induction of a syncytium. Feeding activity is resumed in young and mature sedentary females, in which the DG is highly active and enlarged. The SvG are metabolically active, but with little synthesis of secretory granules, suggesting that in sedentary females the SvG may have physiological roles other than digestion.     

In-situ Hybridization to Messenger RNA in Heterodera glycines

A method is presented for in-situ hybridization to mRNA in second-stage juveniles (J2) of the soybean cyst nematode Heterodera glycines. The protocol was developed using a digoxigenin-labeled RNA probe transcribed from cDNA of a cellulase gene that was known to be expressed in the subventral esophageal glands of H. glycines. Formaldehyde-fixed J2 were cut into sections with a vibrating razor blade to make the inside of the nematodes accessible for probing. These nematode fragments then were hybridized in suspension with riboprobe, and labeled with an alkaline phosphatase-conjugated antibody to digoxigenin. Staining with nitroblue tetrazolium and bromo-chloro-indolyl phosphate revealed a highly specific hybridization signal to mRNA within the cytoplasm of the subventral gland cells, using this specific antisense probe. This in-situ hybridization protocol will be useful for the characterization and identification of esophageal gland secretion genes in plant-parasitic nematodes, among other applications.     

The Immunofluorescent Localization of Subventral Pharyngeal Gland Epitopes of Preparasitic Juveniles of Heterodera glycines Using Laser Scanning Confocal Microscopy

Laser scanning confocal microscopy (LSCM) was used to localize the reactivity of a monoclonal antibody (Sv2) that binds to the subventral pharyngeal glands of preparasitic juveniles of Heterodera glycines. The greater resolution, magnification, and image analysis of LSCM compared with conventional epifluorescent microscopy enabled Sv2 binding to be localized much more precisely to the periphery of the secretory granules. A linear increase of about 55% in fluorescent intensity was found over a 23-μm length of subventral pharyngeal gland just distal to the terminal ampullae. LSCM is a rapid and effective technique for precise immunolocalization of epitopes.     

Ultrastructure of esophageal glands and their secretory granules in the root-knot nematodeMeloidogyne incognita

Summary The dorsal and subventral esophageal glands and their secretory granules in the root-knot nematodeMeloidogyne incognita changed during parasitism of plants. The subventral esophageal glands shrank and the dorsal gland enlarged with the onset of parasitism. While secretory granules formed by both types of glands were spherical, membrane-bound, and Golgi derived, the granules differed in morphology and size between the two types of glands. Subventral gland extensions in preparasitic second-stage juveniles were packed with secretory granules which varied in diameter from 700–1,100 nm and had a finely granular matrix. Within the matrix of each subventral gland granule was an electron-transparent core that contained minute spherical vesicles. The size and position of the core varied within different granules. Few granules were present in the dorsal gland extension in preparasitic juveniles. The matrix of dorsal gland secretory granules formed during parasitism was homogeneous and more electron-dense than the matrix of subventral gland granules. Subventral gland secretory granules of parasitic juveniles and adult females appeared degenerate.     

Ultrastructure of Esophageal Gland Secretory Granules in Juveniles of Heterodera glycines

Ultrastructural observations of the feeding sites of soybean cyst nematode juveniles 3 days after inoculation of soybean roots revealed the presence of feeding tubes in the host cell syncytium. Feeding tubes, which were extruded from the stylet tips, were formed by products of secretory granules that originated in the dorsal esophageal gland and accumulated in the ampulla of the gland extension. Granules traversing the space between the gland cell and the ampulla were regulated in their movement by two sets of sphincter-like muscles located anterior and posterior to the metacorpus pump chamber. Sections through the sphincter muscles revealed obliquely arranged fibers, which in a contracted mode caused microtubules in the gland extension to be tightly packed and devoid of granules.     

Fine Structure of the Esophagus of Males of Sarisodera hydrophila (Heteroderoidea)

The fine structure of the esophagus, including procorpus, metacorpus, isthmus, gland lobe, and esophago-intestinal junction, is examined in males of Sarisodera hydrophila. A cuticle-lined lumen extends most of the length of the esophagus, broadens to form a pump chamber in the metacorpus, and posteriorly is continuous with junctional complexes among four esophago-intestinal cells. These four cells are partially enveloped by the gland lobe which basically consists of three gland cells, one dorsal and two subventral. Each gland cell has an anterior process which opens into the lumen of the esophagus through a cuticle-lined duct. The dorsal gland joins the lumen in the anterior portion of the procorpus, whereas ducts of the subventral glands terminate at the base of the metacorpus pump chamber. The subventral glands are predominant in the posterior portion of the gland lobe and are partially ensheathed by a narrow portion of the dorsal gland which extends to within 5 μm of the posterior terminus of the gland lobe. Contents of the dorsal gland include primarily electron dense granules, although rough endoplasmic reticulum (RER) is predominant posteriorly. Secretory granules within the subventral glands vary in morphology and are evenly distributed throughout the two ceils among other organelles, including RER and a large Golgi apparatus. Innervation of the esophagus includes nerve processes which originate from several perikaryons (cell bodies) located in the anterior portion of the gland lobe. The esophagus of males of S. hydrophila is compared with that of other Heteroderoidea, Heterodera glycines and Meloidogyne incognita.     

Electron Microscopy of the Stomatostylet and Esophagus of Criconemoides curvatum

The stomatostylet of Criconemoides curvature consists of three parts: tooth cone, shaft, and knobs. The tooth cone constitutes the outer conical covering and inner lining of the anterior half of the stylet lumen. The tooth cone is easily separated from the shaft by treating an isolated styler with 0.5% sodium hypochlorite. The posterior half of the shaft is cylindrical, tapering anteriorly to form the shaft extension, wedged between the inner and outer tooth cone. The shaft extension extends to the stylet lumen orifice, which is subterminal and ventral. Six ducts enter the shaft through the junction between the shaft and knobs. They extend anteriad toward the tip of the shaft extension. Cytoplasmic connections between the ducts and the cells surrounding the stylet occur near tile junction between the shaft and the basal knobs. Ribosome and membranous structures are observed in these ducts. The esophagus of the adult female consists of a fused procorpus and metacorpus with a large valve possessing thickened cuticular walls at the anterior and posterior ends. The dorsal esophageal gland reservoir is composed of many honeycomb-like compartments made up of two types of differing electron density. The subventral esophageal glands, however, consist of only one type of granules. Both dorsal and subventral esophageal glands open into the esophageal lumen through trachea-like branched multiple canals.     

Heterodera achilleae n. sp. (Nematoda: Heteroderidae) from Yarrow in Yugoslavia

Heterodera achilleae n. sp., a member of the H. rostochiensis group, is described and illustrated from roots of yarrow, Achillea millefolium L. in Sarajevo, Yugoslavia. This new, round-cyst species differs from closely related species especially as follows: (1) from H. leptonepia, by having stouter larvae (a = 21), with longer styler (25 μ), and with outlet of dorsal esophageal gland averaging 5.7 μ from base o f styler; (2) from H. millefolii, in having excretory pore at base of neck and small, straight vulval slit of 5 μ; (3) from H. rostochiensis, in having a B/A ratio (Granek''s ratio) of 1.6 ; (4) from H. tabacum, by longer female stylet, two annules on female head, and males with outlet of dorsal esophageal gland further back (5.7 μ). In addition, H. achilleae n. sp. differs from the latter three species in having prominent longitudinal striae on the anterior half, or more, of cysts and females.     

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.     

Penetration Rates by Second-stage Juveniles of Meloidogyne spp. and Heterodera glycines into Soybean Roots

The rates of soybean root penetration by freshly hatched second-stage juveniles (J2) of Meloidogyne arenaria, M. hapla, M. incognita, M. javanica, and Heterodera glycines races 1 and 5 were examined over a period of 1 to 240 hours. Heterodera glycines entered roots more quickly than Meloidogyne spp. Penetration by most nematodes was accomplished within 48 hours. The increases in penetration after 48 hours were insufficient to warrant further assessments. Penetration of J2 into roots of soybean seedfings in a styrofoam container was as good or better than in a clay pot. Thus, rapid and accurate root-penetration assessments can be made at 48 hours after inoculation.     

Population Density and Spatial Pattern of Heterodera glycines in Relation to Soybean Phenology

Population dynamics of Heterodera glycines (SCN) were influenced by initial nematode population density in soil, soybean root growth pattern, soil type, and environmental conditions in two field experiments. Low initial populations (Pi) of SCN increased more rapidly during the growing season than high Pi and resulted in greater numbers of nematodes at harvest. Egg and juvenile (J2) populations increased within 2-6 weeks after planting when early-season soil temperatures were 20 C and above and were delayed by soil temperatures of 17 C or below in May and early June. Frequencies of occurrence and number of nematodes decreased with increasing depth and distance from center of the soybean row. Spatial pattern of SCN paralleled that of soybean roots. Higher clay content in the subsoil 30-45 cm deep in one field restricted soil penetration by roots, indirectly influencing vertical distribution of SCN. Shoot dry weight was a good indicator of the effect of SCN on seed yield. Root dry weight was poorly correlated with soybean growth and yield. The relationship of yield (seed weight) to Pi was best described by a quadratic equation at one site, but did not fit any regression model tested at the second site.     

Immunocytochemical localization of secretory acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis

Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.     

Vittatidera zeaphila (Nematoda: Heteroderidae), a new genus and species of cyst nematode parasitic on corn (Zea mays)

A new genus and species of cyst nematode, Vittatidera zeaphila, is described from Tennessee. The new genus is superficially similar to Cactodera but is distinguished from other cyst-forming taxa in having a persistent lateral field in females and cysts, persistent vulval lips covering a circumfenestrate vulva, and subventral gland nuclei of the female contained in a separate small lobe. Infective juveniles (J2) are distinguished from all previously described Cactodera spp. by the short stylet in the second-stage juvenile (14-17 μm); J2 of Cactodera spp. have stylets at least 18 μm long. The new species also is unusual in that the females produce large egg masses. Known hosts are corn and goosegrass. DNA analysis suggests that Vittatidera forms a separate group apart from other cyst-forming genera within Heteroderinae.     

Response of Soybean in Cyst Nematode-Infested Soils at Three Soil Water Regimes

Large pot (2 years) and field experiments (1 year) were conducted to determine the response of susceptible soybean Glycine max (L.) Merr. cultivars (Essex and Hutcheson) grown in soybean-cyst-nematode (SCN), Heterodera glycines-infested soils at three soil water regimes. The soil water regimes were irrigation whenever soil water potential ([psi]s) 0.30-m deep was i) -30 kPa (I-30) or ii) - 50 kPa (I-50), and iii) no irrigation. Cyst nematode levels in the pot experiment were either 0 or 20,000 second-stage juveniles (J2) per pot. The field experiment was conducted on soil naturally infested with a population of 145 to 475 cysts L⁻¹ of soil. All growth parameters studied were drastically affected in the presence of SCN under nonirrigated conditions for the large pot tests; however, SCN did not influence growth parameters in the field experiment. Seed yield was lowest in the no irrigation treatment when all treatments were compared in both the pot and field experiments. The infested no irrigation treatment in the pot experiment had the lowest yield among soil water treatments.     

Effect of Within-field Variation in Soil Texture on Heterodera glycines and Soybean Yield

The influence of soil texture on Soybean yield in the presence of Heterodera glycines was investigated by comparing yields of susceptible cultivars with a resistant cultivar for 2 years. Soybean yield was negatively correlated with increasing sand content (P = 0.05). Yields of susceptible cultivars were suppressed with increasing sand content. Final nematode population densities were lowest in plots with greatest sand content. Soybean infection by SCN, as determined by the number of cysts 30 days after planting, was not consistently related to soil texture over 2 years. Initial nematode population density was positively related to soybean yield the first year and negatively related to soybean yield the second, probably a result of greater yield suppression by H. glycines in plots with greater sand content.     

Description of Non-Type Seinura winchesi from Mushroom Compost (Nematoda: Seinuridae)

Non-type material identified as Seinura winchesi, and deposited in the collection of the Nematology Department, Rothamsted Experimental Station, England, is described. The material was collected from mushroom compost in Leeds, England, in 1957. Females of this population are characterized by a set-off head, knobless stylet (20-21 µm), two rows of oogonia-oocytes, and absence of a postuterine sac. The median bulb is oblong and, at times, is constricted in the middle. The body is 565 to 675 µm long and tapers posteriorly to a pointed terminus. The cuticle is finely annulated, and there are three incisures in the lateral field. The ovary is outstretched and overlaps the esophageal gland lobes. A spermatheca is present, and spermatozoa are visible. The vulva is posteriorly located (V = 77-80), and a flap is absent. Males are 550 to 680 µm long with a spicate tail that bears three pairs of subventral papillae. The spike is short (14-18 µm), about one-half the tail length. The testis is 360 to 412 µm long, not reflexed, and at times overlaps the esophageal gland lobes. Spermatogonia-spermatocytes are in two rows. Spicules are 14 to 15 µm long with a prominent apex, and small rostrum. A bursa and gubernaculum are absent.