Ultrastructure of the Z-organ and Parts of the Female Genital Tract in Xiphinema coxi coxi

Ultrastructure of the Z-organ and associated apophyses in Xiphinema coxi coxi was studied by transmission electron microscopy to determine their structural origin and relationship with other parts of the genital tract. The Z-organ of X. coxi coxi is oval-shaped, ca. 30 µm long and 16 µm wide. It is clearly distinguished from the other parts of the female genital tract by its thick muscular outer wall, epithelium-lined lumen, and 4-5 centrally located apophyses. Each apophysis is continuous with the epithelial lining of the Z-organ, suggesting that it originated from epithelium. The apophyses appear as thickened and densely folded masses forming numerous interlaced pores and (or) chambers containing mucous-like materials and electron-dense crystals. These apophyses are characteristic of a typical Z-organ; no globular structures characteristics of the pseudo-Z-organ were observed. The thickness of the muscular layer of the oviduct and uterus varied with position. The overall Z-organ ultrastructure of this study, including body wall and internal apophyses, was comparable to the typical Z-organ of X. ifacolum. This suggests that X. coxi coxi should be classified as a Xiphinema species that contains the typical Z-organ.     

Morphometric Evidence for Three Juvenile Stages in Some Species of Xiphinema americanum sensu lato

One to two hundred nematodes from each of seven Xiphinema americanum-group populations were measured to determine the range of stylet and body lengths for juveniles and adults. First-stage juveniles were identified by the position of the replacement odontostyle (i.e., the tip of the replacement odontostyle overlapped the base of the odontophore). Nematodes were identified as second stage if the functional odontostyle was the same length as the replacement odontostyle of the first stage. Subsequent stages were similarly identified by establishing the range of corresponding replacement and functional odontostyle lengths. In all populations examined, this procedure created natural divisions that clearly grouped nematodes by stylet and body length. Presumably these groups identified all juvenile and adult stages. Populations of X. americanum, X. rivesi, and X. californicum from the United States had three juvenile stages, but a population of X. pachtaicum from Bulgaria had four juvenile stages.     

Scanning Electron Microscopy of Xiphinema,Longidorus, and Californidorus Stylet Morphology

Stylet ultrastructure of five Xiphinema, four Longidorus, and three Californidorus species was compared by scanning electron microscopy. Morphological differences were seen in the odontophores and odontostyle bases between the genera and some of the species. All Xiphinema studied had well-developed odontophore flanges; the Longidorus species lacked flanges, except for weakly developed ones in L. diadecturus; and none of the Californidorus had flanges. Three sinuses were present in the odontophores of all species. The sinuses varied in length depending upon species. In Xiphinema and Californidorus the odontostyle bases had distinct overlapping collars, but in Longidorus the collars were absent except for L. diadecturus. The odontostyle-odontophore junction from a lateral view appeared as a slanted transverse line in all the species, but in a dorsal view of Xiphinema and Californidorus it was V-shaped. Dorsal longitudinal seams of the odontostyle and odontophore were observed in all the species. The dorsally located odontostyle aperture was ca. 1 μm from the anterior end in all species, except in one Longidorus sp. it was ca. 4 μm from the end.     

Paralongidorus bullatus n. sp. from Groundnut Soils in Niger and Comments on Xiphinema parasetariae Luc

Paralongidorus bullatus n. sp. from groundnut soils in Sador''é Niger is described and illustrated. It is distinguishable from most species of the genus by its body and odontostyle lengths, knob-like head, and digitate ending of the protoplasmic region of the tail. Length of the female body is 4.4-5.5 mm, of the odontostyle 132-156 μm, and of the tail 32-44 μm. Tail terminus is conoid to broadly rounded. Uteri are well developed, without sperms. Males are not found. Xiphinema parasetariae Luc 1958, a species inquirenda, is validated and, based on measurements and tail structures, X. attorodorum Luc 1961 is proposed as a synonym of X. parasetariae. Additional measurements are given for its females and juveniles.     

Cluster Analysis of Longidorus Species (Nematoda: Longidoridae), a New Approach in Species Identification

Hierarchical cluster analysis based on female morphometric character means including body length, distance from vulva opening to anterior end, head width, odontostyle length, esophagus length, body width, tail length, and tail width were used to examine the morphometric relationships and create dendrograms for (i) 62 populations belonging to 9 Longidorus species from Arkansas, (ii) 137 published Longidorus species, and (iii) 137 published Longidorus species plus 86 populations of 16 Longidorus species from Arkansas and various other locations by using JMP 4.02 software (SAS Institute, Cary, NC). Cluster analysis dendograms visually illustrated the grouping and morphometric relationships of the species and populations. It provided a computerized statistical approach to assist by helping to identify and distinguish species, by indicating morphometric relationships among species, and by assisting with new species diagnosis. The preliminary species identification can be accomplished by running cluster analysis for unknown species together with the data matrix of known published Longidorus species.     

Molecular Characterization of a Xiphinema hunaniense Population with Morphometric Data of all Four Juvenile Stages

A population of Xiphinema hunaniense Wang and Wu, 1992 with all four juvenile stages was found in the rhizosphere of Pinus sp. in Hangzhou, Zhejiang, China. Morphometrics of 18 females and 35 juveniles of this population are given herein. Detailed morphology and morphometrics of the four juvenile stages are provided. Further comparisons based on morphometrics of the population with previous studies of the females and the first-stage juveniles of X. hunaniense with X. radicicola are given, and morphological variation in X. hunaniense populations are discussed. A revised polytomous key code of Loof and Luc (1990) for X. hunaniense identification is provided, i.e., A1- B4- C4- D4/5- E1- F2(3)- G2- H2-I3- J4- K2- L1. In addition, the sequence of the D2 and D3 expansion region of the 28S rRNA gene was analyzed and compared with sequences of closely related species downloaded from the NCBI database. Cluster analysis of sequences confirmed and supported the species identifications.     

Longidorus biformis n. sp. and L. glycines n. sp. (Nematoda: Longidoridae): Two Amphimictic Species from Arkansas

Two new amphimictic species of Longidorus were found in Arkansas. Longidorus biformis n. sp., found in the rhizosphere of hardwood trees along streams in sandy soil in 14 Arkansas locations, is characterized by its long body (5.42-9.50 mm), wide expanded flattened head end, head width 20.0 to 26.0 µm, odontostyle 96 to 125 µm, guide ring 29 to 38 µm posterior to the anterior end, elongate conoid tail, and c'' = 0.9-2.1. Females with 2 to 11 vetromedian supplement-like structures were found in 2 of 14 populations of this new species. Longidorus biformis n. sp. is closest to L. seinhorsti Peneva, Loof &Brown, 1998 and L. closelongatus Stoyanov, 1964. Among North American species it is closest to L. glycines n. sp. A distinguishing feature of L. biformis n. sp. is the presence of supplement-like organs in some females. Longidorus glycines n. sp., found in soybean microplots at the Main Research Station, Fayetteville, Arkansas, is characterized by its long body (6.14-8.31 mm), wide offset flattened head end, head width 20.3 to 23.3 µm, odontostyle 87.3 to 99.5 µm, guide ring 22.3 to 26.4 µm posterior to the anterior end, short conoid tail with rounded terminus, and c'' = 0.9-1.4. Longidorus glycines n. sp. is closest to L. lusitanicus Macara, 1985. Among North American species it is close to L. biformis n. sp., L. breviannulatus Norton and Hoffman, 1975, and L. crassus Thorne, 1964. Both new species are believed to have four juvenile stages; the first stage was not found for L. biformis n. sp.     

Morphological Comparison of Three Host Races of Meloidogyne javanica

A morphological and morphometric comparison using light microscopy and scanning electron microscopy was made of six populations of Meloidogyne javanica belonging to three host races (infective on pepper, peanut, or noninfective on both). The variability of certain morphological characters was studied within these populations, and the reliability of these taxonomic traits was evaluated for usefulness in species identification. The most useful diagnostic characters of M. javanica were head and stylet morphology of males and stylet morphology and perineal patterns of females. Males have an offset head region, usually lacking annulations, and a distinct, narrow head cap with slightly raised labial disc. The stylet has a cone markedly wider than the shaft at the junction and large, transversely ovoid knobs that are offset from the shaft. Females have a robust stylet with a dorsally curved cone and large, transversely ovoid knobs. Perineal patterns are oval to squarish in shape, usually with coarse, broken striae and with conspicuous lateral lines. The host races could not be differentiated on a morphological basis.     

Xiphinema llanosum and Trophurus vultus, Two New Plant Nematode Species from Pasture Soils in Colombia

Xiphinema llanosum n. sp. and Trophurus vultus n. sp. are described and illustrated from grass soils in Llanos Orientales, Colombia. Xiphinema llanosum is a bisexual species. The female body length is 2.3-2.7 mm, odontostyle 86-96 μm, and odontophore 58-65 μm long; vulva at 42-47%; anterior ovary is absent; the anterior uterus and oviduct are similar to the posterior branch but slightly reduced; and the tail is dorsally convex-conoid with a blunt hemispherical terminus. Male body length is 2.06-2.96 mm; spicules are 40-44 μm long; and four (rarely three or five) anterior ventromedian supplementary papillae are present. Trophurus vultus females are 0.52-0.67 mm long; vulva at 56-60%; stylet is 10.5-13.5 μm long; isthmus is as long as the basal esophageal bulb; the tail is subclavate, 1.6-2.2 times anal body width long; and the terminal cuticle thickness is about one-sixth of the tail length.     

Morphological and Molecular Characterization of Longidorus americanum n. sp. (Nematoda: Longidoridae), a Needle Nematode Parasitizing Pine in Georgia

We describe and illustrate a new needle nematode, Longidorus americanum n. sp., associated with patches of severely stunted and chlorotic loblolly pine, (Pinus taeda L.) seedlings in seedbeds at the Flint River Nursery (Byromville, GA). It is characterized by having females with a body length of 5.4-9.0 mm; lip region slightly swollen, anteriorly flattened, giving the anterior end a truncate appearance; long odontostyle (124-165 µm); vulva at 44%-52% of body length; and tail conoid, bluntly rounded to almost hemispherical. Males are rare but present, and in general shorter than females. The new species is morphologically similar to L. biformis, L. paravineacola, L. saginus, and L. tarjani but differs from these species either by the body, odontostyle and total stylet length, or by head and tail shape. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other Longidorus species. Phylogenetic relationships of Longidorus americanum n. sp. with other longidorids based on analysis of this DNA fragment are presented. Additional information regarding the distribution of this species within the region is required.     

Aspects of Biology and Development of Xiphinema americanum and Related Species

Identification of Xiphinema americanum-group nematodes is based on relatively subtle morphological and morphometric differences, many of which overlap. The significance and importance of these separations cannot be assessed without a basic understanding of the biological differences between species. Currently, information is accumulating on Xiphinema biology, development, and genetics that will help to confirm or refute the current systematics of species in this group. Recently, it was demonstrated that Xiphinema species pass through either three or four juvenile stages before becoming adults. This new and fundamental information divides the genus and the X. americanum group into subgroups based on their developmental evolution and provides new insight into the taxonomy and systematic positions of the species.     

Morphological Variation in Xiphinema spp. from New York Orchards

Xiphinema specimens were collected from orchards in southeastern, northeastern, and western New York. Total length, distance of vulva from anterior end, spear length (odontostyle plus odontophore), body diameter at vulva, tail length, anal body diameter, and length and diameter of hyaline tail tip were measured on fixed, glycerol-infiltrated adult females. Most specimens were identified as X. americanum or X. rivesi, but one western New York population was identified as X. californicum (a new record for New York). Multivariate analyses indicated that, with one exception, western New York populations of both X. americanum and X. rivesi were smaller and slimmer than their eastern counterparts. Regional differences were generally larger than differences attributed to host species.     

Temperature Effects on Race Determination in Heterodera glycines

Currently there are 16 possible races for Heterodera glycines, and these are differentiated based on ability of a nematode population to develop on a set of four differential soybean genotypes. Because results are based on numbers of nematode females that develop to a specific stage rather than on the reproductive capability of these females, race determinations based on female indices may not represent results obtained after several reproductive cycles of H. glycines. Counting numbers of eggs and juveniles, and then developing corresponding indices, would allow reproduction to be considered in making race determinations. Our objectives were to compare the present race identification scheme for H. glycines based on female indices with those using egg and juvenile indices and to examine the effect of temperature on race designations using female, egg, and juvenile indices. Race designations for H. glycines populations from two locations in Illinois were determined at 20, 27, and 30 °C in a water bath. The numbers of females, eggs, and juveniles (at 19 days) were recorded, and an index based on each life stage was calculated. Race determinations based on female, egg, or juvenile indices were inconsistent when conducted at 20 °C, which demonstrates that this temperature is not suitable for identifying races of H. glycines. However race designations at 27 and 30 °C were consistent for all three indices. This indicates that counting females, eggs, or juveniles should be equally reliable when race determinations are conducted at these two temperatures, and choice of method would depend on investigator preference or research objective.     

Distribution and Morphology of Longidorus breviannulatus Norton &Hoffman, 1975 and Longidorus fragilis Thorne, 1974 (Nematoda: Longidoridae) from North America

In a survey of ecotypes for longidorids, primarily from the rhizosphere hardwood trees growing in sandy soil along stream banks, 828 soil samples were collected from 37 Arkansas counties in 1999-2001. Eight populations of Longidorus breviannulatus were identified from the Arkansas survey samples. A total of 19 populations from California, Illinois, Iowa, Kansas, Michigan, Nebraska, New Jersey, New York, and Wisconsin were identified from the collection of the second author. A few males were found in New York and Nebraska populations and are described herein. Seven populations of L. fragilis were identified in the Arkansas survey samples, and one population was found from Indiana. Four juvenile stages of L. fragilis are present, and data are given for them herein.     

Descriptions of Three New Longidorus Species from Slovakia (Nemata: Longidoridae)

Three new Longidorus species from Slovakia are described. Longidorus carpathicus n. sp. most closely resembles Longidorus silvae but differs by having a longer odontostyle, odontophore, and total stylet; smaller a and c ratios; and longer distance to the guide ring. This new species also resembles L. picenus, L. macrosoma, and L. major but differs by having a narrower lip width. It further differs from L. picenus by having a longer odontostyle and smaller c ratio, and by lacking males; from L. macrosoma by having a longer odontostyle, smaller c ratio, by lacking males, and a more pronounced J1 tail peg; and from L. major by having a shorter body length, longer odontostyle, longer odontophore, and longer J1 tail peg. Longidorus piceicola n. sp. most closely resembles L. eridanicus, from which it differs by having a greater lip width, longer tail, smaller c ratio, larger c'' ratio, shorter hyaline tail length, and a conically rounded vs. hemispherical tail. This new species differs from L. cylindricaudatus by having a larger lip width, longer odontostyle and odontophore, and a greater distance to the guide ring; from L. nevesi by having a shorter body length, longer odontostyle, larger c'' ratio, and shorter hyaline tail length. Longidorus juglansicola n. sp. most closely resembles L. athesinus but differs by its longer body, wider lips, and larger a and c ratios. It closely resembles L. vineacola but differs by its shorter body length, smaller c ratio, and an almost parallel lip outline vs. an expanded lip outline; from L. lusitanicus by a longer odontophore and tail, and an almost parallel lip outline vs. an expanded lip outline.     

Two Simple Methods for the Collection of Individual Life Stages of Reniform Nematode,Rotylenchulus reniformis

The sedentary semi-endoparasitic nematode Rotylenchulus reniformis, the reniform nematode, is a serious pest of cotton and soybean in the United States. In recent years, interest in the molecular biology of the interaction between R. reniformis and its plant hosts has increased; however, the unusual life cycle of R. reniformis presents a unique set of challenges to researchers who wish to study the developmental expression of a particular nematode gene or evaluate life stage–specific effects of a specific treatment such as RNA-interference or a potential nematicide. In this report, we describe a simple method to collect R. reniformis juvenile and vermiform adult life stages under in vitro conditions and a second method to collect viable parasitic sedentary females from host plant roots. Rotylenchulus reniformis eggs were hatched over a Baermann funnel and the resultant second-stage juveniles incubated in petri plates containing sterile water at 30°C. Nematode development was monitored through the appearance of fourth-stage juveniles and specific time-points at which each developmental stage predominated were determined. Viable parasitic sedentary females were collected from infected roots using a second method that combined blending, sieving, and sucrose flotation. Rotylenchulus reniformis life stages collected with these methods can be used for nucleic acid or protein extraction or other experimental purposes that rely on life stage–specific data.     

Two New Species of Actinolaimidae Thorne, 1939 (Nemata: Dorylaimida) from China

Two new species of Actinolaimidae are described from China. Trachactinolaimus brevicaudatus n. sp. is 3.4-4.4 mm long; a = 40-60, b = 3.5-5.2, c = 20-21 in female and 34-39 in male; odontostyle length is 29-33 µm; spicules are 67-77 µm long; and the stoma has four onchia with numerous mural denticles. The female has a longitudinal vulva, and the male has 20 to 23 contiguous ventromedian supplements. Egtitus sinensis n. sp. is 1.7-2.2 mm long; a = 24-33, b = 3.1-3.9, c = 16-19 in female and 0.7-0.9 in male; odontostyle length is 25-29 µm; spicules are 55-56 µm long; and the prerectum 53-77 µm long. The cardia is short and blunt conoid, 13-19 µm long. The male has 12 to 13 ventromedian supplements at intervals of 2-3 µm.     

Stepwise and Canonical Discriminant Analysis of Longidorus Species (Nematoda: Longidoridae) from Arkansas

During a 1998-to-2001 survey from Arkansas, nine distinct species of Longidorus were found including five new species. Morphometrics of these nine species were used in a stepwise and canonical discrimination to select a subset of characteristics that best identified each species. Student''s t test was applied to compare Longidorus breviannulatus Norton &Hoffman, 1975; L. crassus Thorne, 1974; L. diadecturus Eveleigh &Allen, 1982; L. fragilis Thorne, 1974; L. biformis Ye &Robbins, 2004; L. glycines Ye &Robbins, 2004; L. grandis Ye &Robbins, 2003; L. paralongicaudatus Ye &Robbins, 2003; and L. paravineacola Ye &Robbins, 2003 to examine interspecies variation and test for the most useful morphometric characters in species discrimination. Most of the morphometric characters were useful to differentiate species, but species identification could not be based on a single character because the morphometric character ranges often overlap. Stepwise discriminant analysis indicated that the guide ring position, head width, tail length, body length, odontostyle length, and anal body width were the most important variables. These were used to generate canonical variables in discriminating the species. The first three canonical variables accounted for 95% of the total variance. The scatterplots by the first three canonical variables grouped and separated the Longidorus species from Arkansas. Stepwise and canonical discriminant analyses were useful for examining the groupings and morphometric relationships of the nine Longidorus species.     

Differential Expression of Hox and Notch Genes in Larval and Adult Stages of Echinococcus granulosus

This investigation aimed to evaluate the differential expression of HoxB7 and notch genes in different developmental stages of Echinococcus granulosus sensu stricto. The expression of HoxB7 gene was observed at all developmental stages. Nevertheless, significant fold differences in the expression level was documented in the juvenile worm with 3 or more proglottids, the germinal layer from infected sheep, and the adult worm from an experimentally infected dog. The notch gene was expressed at all developmental stages of E. granulosus; however, the fold difference was significantly increased at the microcysts in monophasic culture medium and the germinal layer of infected sheep in comparison with other stages. The findings demonstrated that the 2 aforementioned genes evaluated in the present study were differentially expressed at different developmental stages of the parasite and may contribute to some important biological processes of E. granulosus.