RESUMEN

Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available. Providing a reliable maximum amount of unambiguous genetic information from single nematodes is especially important when identifying damaging, regulated nematodes of importance to trade where a few nucleotide differences in diagnostic markers are significant. There are many possible reasons for difficulty amplifying unpurified nematode DNA for long range PCR followed by direct sequencing. Specimen age, proofreading errors and reagent compatibility during PCR are among those problems. While unsuccessful direct amplification of difficult samples may sometimes be overcome by cloning, a more expensive and time-consuming process. Therefore, long segment PCR of a large 3.5 kb segment of ribosomal DNA was optimized for individual difficult-to-amplify young Litylenchus crenatae mccannii (Anguinidae) nematodes by systematically testing thermostable polymerases, proofreading enzymes and buffers. The combination of thermostable DreamTaq™, proofreading Pfu polymerase, and PicoMaxx™ buffer provided the best results. These nematodes are the subject of surveys currently active at many sites in the northeastern United States. This new, optimized PCR protocol will be useful for diagnostic labs associated with the surveys.Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available. Providing a reliable maximum amount of unambiguous genetic information from single nematodes is especially important when identifying damaging, regulated nematodes of importance to trade where a few nucleotide differences in diagnostic markers are significant. There are many possible reasons for difficulty amplifying unpurified nematode DNA for long range PCR followed by direct sequencing. Specimen age, proofreading errors and reagent compatibility during PCR are among those problems. While unsuccessful direct amplification of difficult samples may sometimes be overcome by cloning, a more expensive and time-consuming process. Therefore, long segment PCR of a large 3.5 kb segment of ribosomal DNA was optimized for individual difficult-to-amplify young Litylenchus crenatae mccannii (Anguinidae) nematodes by systematically testing thermostable polymerases, proofreading enzymes and buffers. The combination of thermostable DreamTaq™, proofreading Pfu polymerase, and PicoMaxx™ buffer provided the best results. These nematodes are the subject of surveys currently active at many sites in the northeastern United States. This new, optimized PCR protocol will be useful for diagnostic labs associated with the surveys.

RESUMEN

Ribosomal DNA has been a reliable source of taxonomic and phylogenetic markers due to its high copy number in the genome and stable variation with few polymorphisms due to the homogenizing effect of concerted evolution. Typically specific regions are amplified through polymerase chain reaction (PCR) with multiple primer pairs that generate often incomplete and overlapping regions between adjacent segments of 18S, ITS1, 5.8S, ITS2, and 28S rDNA nucleotide sequences when combined in tandem. To improve the efficiency of this effort, a strategy for generating all these molecular sequences at once through PCR amplification of a large ribosomal 3.3 to 4.2 kb DNA target was developed using primer 18S-CL-F3 paired with D3B or a new alternative 28S PCR primer (28S-CL-R) and other well-positioned and ribosomal-specific sequencing primers (including novel primers 18S-CL-F7, 18S-CL-R6, 18S-CL-R7, 18S-CL-F8, 5.8S-CL-F1, 5.8S-CL-R1, 28S-CL-F1, 28S-CL-R3, 28S-CL-F3, 28S-CL-R1, and 28S-CL-F2). The D1 region between ITS2 and 28S boundaries and the flanking sequence between 18S and ITS1 boundaries were fully revealed in this large nucleotide segment. To demonstrate the value of this strategy, the long rDNA segment was amplified and directly sequenced in 17 agriculturally important nematodes from the Tylenchida, Aphelenchida, and Dorylaimida. The primers and their positions may be employed with traditional Sanger sequencing and with next-generation sequencing reagents and protocols.Ribosomal DNA has been a reliable source of taxonomic and phylogenetic markers due to its high copy number in the genome and stable variation with few polymorphisms due to the homogenizing effect of concerted evolution. Typically specific regions are amplified through polymerase chain reaction (PCR) with multiple primer pairs that generate often incomplete and overlapping regions between adjacent segments of 18S, ITS1, 5.8S, ITS2, and 28S rDNA nucleotide sequences when combined in tandem. To improve the efficiency of this effort, a strategy for generating all these molecular sequences at once through PCR amplification of a large ribosomal 3.3 to 4.2 kb DNA target was developed using primer 18S-CL-F3 paired with D3B or a new alternative 28S PCR primer (28S-CL-R) and other well-positioned and ribosomal-specific sequencing primers (including novel primers 18S-CL-F7, 18S-CL-R6, 18S-CL-R7, 18S-CL-F8, 5.8S-CL-F1, 5.8S-CL-R1, 28S-CL-F1, 28S-CL-R3, 28S-CL-F3, 28S-CL-R1, and 28S-CL-F2). The D1 region between ITS2 and 28S boundaries and the flanking sequence between 18S and ITS1 boundaries were fully revealed in this large nucleotide segment. To demonstrate the value of this strategy, the long rDNA segment was amplified and directly sequenced in 17 agriculturally important nematodes from the Tylenchida, Aphelenchida, and Dorylaimida. The primers and their positions may be employed with traditional Sanger sequencing and with next-generation sequencing reagents and protocols.

RESUMEN

Millipedes may cause unexpected damage when they are introduced to new locations, becoming invaders that leave behind their old parasites and predators. Therefore, it was interesting to find numerous rhabditid nematodes within the gut of the invasive phytophagous millipede Chamberlinius hualienensis Wang, 1956 (Diplopoda, Paradoxosomatidae) from Hachijojima (Japan) in November, 2014. This millipede originated in Taiwan but was discovered in Japan in 1986. The nematodes were identified as juvenile Oscheius rugaoensis (Zhang et al., 2012) Darsouei et al., 2014 (Rhabditidae), and juvenile and adult Mononchoides sp. (Diplogastridae) based on images, morphometrics, and sequences of 18S and 28S rDNA. A novel short 28S sequence of a separate population of Oscheius necromenus SB218 from Australian millipedes was also included in a phylogenetic comparison of what can now be characterized as a species complex of millipede-associated Oscheius. The only other nematode associates of millipedes belong to Rhigonematomorpha and Oxyuridomorpha, two strictly parasitic superorders of nematodes. These nematode identifications represent new geographic and host associations.Millipedes may cause unexpected damage when they are introduced to new locations, becoming invaders that leave behind their old parasites and predators. Therefore, it was interesting to find numerous rhabditid nematodes within the gut of the invasive phytophagous millipede Chamberlinius hualienensis Wang, 1956 (Diplopoda, Paradoxosomatidae) from Hachijojima (Japan) in November, 2014. This millipede originated in Taiwan but was discovered in Japan in 1986. The nematodes were identified as juvenile Oscheius rugaoensis (Zhang et al., 2012) Darsouei et al., 2014 (Rhabditidae), and juvenile and adult Mononchoides sp. (Diplogastridae) based on images, morphometrics, and sequences of 18S and 28S rDNA. A novel short 28S sequence of a separate population of Oscheius necromenus SB218 from Australian millipedes was also included in a phylogenetic comparison of what can now be characterized as a species complex of millipede-associated Oscheius. The only other nematode associates of millipedes belong to Rhigonematomorpha and Oxyuridomorpha, two strictly parasitic superorders of nematodes. These nematode identifications represent new geographic and host associations.

RESUMEN

The 18S small subunit (SSU) ribosomal DNA sequence is one of the most useful molecular loci for identification and phylogeny reconstruction of agriculturally important nematodes. Various pairs of universal primers have been developed in the past to amplify short and long nematode sequences. However, certain nematode taxa were not readily amplified and/or sequenced with the existing primer tools. Frequently, the center region of a roughly 1,000 nucleotide segment would be lost. Therefore new primers were developed based on a very large 276 taxon alignment of 124 agriculturally important nematode species, and tested on problematic nematode taxa such as Aphelenchoides, Bursaphelenchus, Ditylenchus, and Panagrolaimus. New primers and protocols are provided for successful generation of sequences useful in future investigations of nematode systematics.The 18S small subunit (SSU) ribosomal DNA sequence is one of the most useful molecular loci for identification and phylogeny reconstruction of agriculturally important nematodes. Various pairs of universal primers have been developed in the past to amplify short and long nematode sequences. However, certain nematode taxa were not readily amplified and/or sequenced with the existing primer tools. Frequently, the center region of a roughly 1,000 nucleotide segment would be lost. Therefore new primers were developed based on a very large 276 taxon alignment of 124 agriculturally important nematode species, and tested on problematic nematode taxa such as Aphelenchoides, Bursaphelenchus, Ditylenchus, and Panagrolaimus. New primers and protocols are provided for successful generation of sequences useful in future investigations of nematode systematics.

RESUMEN

A thrips insect Caliothrips sp. (Thysanoptera: Panchaetothripinae) from persimmon fruit (Ebenaceae: Diospyros sp.) from an unknown origin, possibly Asia, was intercepted in a passenger bag in November 2012 at the Peace Arch Border Crossing from Canada to Blaine, WA, by a USDA-APHIS-PPQ port inspector. Nematodes were attached to the abdomen of the female insect and sent to us in saline. Seven nematodes (five females, two males) were measured and these and others were processed for permanent slides. An adult female and a female juvenile were prepared for PCR. Morphologically these nematodes belonged to the Trichodorus sparsus group, and the 28S rDNA D2-D3 sequence showed greatest similarity to Trichodorus paragiennensis (94%) and T. giennensis (93%), with greatest morphological similarity to the latter species. Among other morphological differences, the innermost uterus width is wider than in related species. Trichodorus spp. are normally found in soil, so this is the first population seen in the atypical habitat of an insect. Morphological and molecular characteristics of Trichodorus sp. are presented, but a putative new species name is not currently advisable because of relatively poor condition of specimens. Ecological associations are also discussed.

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An unusual population of cyst nematode was found in soils collected from a Powell Butte, OR field with a cropping history including potato, wheat, other crops, and significant weed presence. These nematodes could not be placed with certainty into any known species and exhibited some unique morphological features in some specimens. Compared with Globodera pallida, the cyst body length was slightly longer and the second-stage juvenile stylet length was slightly shorter. In some individuals, the J2 stylet knob height was greater and the tail annules were more prominent than in G. pallida, and the tail abruptly narrowed, with a slight constriction near the posterior third of the hyaline terminus. Compared with G. rostochiensis, the hyaline tail terminus had a larger number of refractive bodies, and cysts of this population had a smaller Granek's ratio and fewer cuticular ridges between the anus and vulva. In some individuals, the tail termini of second-stage juveniles were more bluntly pointed, and the stylet knobs were more anteriorly directed with greater height. Unlike G. tabacum, the cyst wall often lacked a network-like pattern and, in some individuals, the juvenile tail terminus distinctly narrowed after a constriction. Molecularly, the population was distinct from G. pallida, G. rostochiensis, and G. tabacum. Multiplex polymerase chain reaction of the internal transcribed spacer (ITS) rDNA region gave results similar to G. tabacum; however, ITS restriction fragment length polymorphism patterns were observed to have individual bands in common with G. rostochiensis and G. pallida. Phylogenetic analysis based on ITS1 and -2 rDNA sequences showed greatest similarity to populations from Argentina and Chile; together, they form a moderately supported clade, distinct from G. rostochiensis, G. tabacum, G. "mexicana," European type G. pallida, and several G. pallida populations from South America.

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RESUMEN

A new Parasitorhabditis species with males and females was discovered from the southern pine beetle Dendroctonus frontalis and its galleries in loblolly pine, Pinus taeda, growing in Mississippi. Females of the new species have a cupola-shaped tail with a small spike; males possess a 2 + (3+2) + 3 ray pattern on the tail fan with ray 10 reaching the margin, and a distinctive stomatal tooth. Parasitorhabditis frontali n. sp. has some similarities to P. hylurgi Massey, 1974 from Hylurgops pinifex in New York, USA, P. terebranus Massey, 1974 from D. terebrans (Olivier, 1795) in Texas USA, P. ligniperdae Fuchs, 1915 from Hylergops ligniperda (Fabricius, 1787) and P. dendroctoni Rühm, 1956 from D. micans (Kugelann, 1794) in Europe, P. ateri Fuchs, 1915 isolated from the beetle Hylastes ater (Paykull, 1800) in Germany, and P. malii Devdariani and Kakulia,1970 from Scolytus mali (Bechstein, 1805) within the republic of Georgia. Morphometrics for 44 species of Parasitorhabditis are provided to update older keys. Parasitorhabditis frontali n. sp. was initially grown on Malt Extract (ME) agar with its own microbial contaminants that included a bacterium and fungus. The nematode also grew and reproduced after slices of ME agar with nematodes and microbial contaminants were transferred to water agar. It was killed by E. coli on NGM agar plates commonly used to raise other Rhabditida. Drawings of diagnostic anatomy and low-temperature SEM images of bodies, heads, and tails are provided for cultured specimens from pine beetle frass.

RESUMEN

Three stunt nematode species, Tylenchorhynchus leviterminalis, T. dubius and T. claytoni were characterized with segments of small subunit 18S and large subunit 28S rDNA sequence and placed in molecular phylogenetic context with other polyphyletic taxa of Telotylenchidae. Based upon comparably sized phylogenetic breadth of outgroups and ingroups, the 28S rDNA contained three times the number of phylogenetically informative alignment characters relative to the alignment total compared to the larger 18S dataset even though there were fewer than half the number of taxa represented. Tail shapes and hyaline termini were characterized for taxa within these subfamily trees, and variability discussed for some related species. In 18S trees, similar terminal tail thickness was found in a well-supported clade of three Tylenchorhynchus: broad-tailed T. leviterminalis branched outside relatively narrow-tailed T. claytoni and T. nudus. Terminal tail thickness within Merliniinae, Telotylenchinae and related taxa showed a mosaic distribution. Thick-tailed Trophurus, Macrotrophurus and putative Paratrophurus did not group together in the 18S tree. Extremely thickened tail termini arose at least once in Amplimerlinius and Pratylenchoides among ten species of Merliniinae plus three Pratylenchoides, and three times within twelve taxa of Telotylenchinae and Trophurinae. Conflicting generic and family nomenclature based on characters such as pharyngeal overlap are discussed in light of current molecular phylogeny. Contrary to some expectations from current taxonomy, Telotylenchus and Tylenchorhynchus cf. robustus did not cluster with three Tylenchorhynchus spp. Two putative species of Neodolichorhynchus failed to group together, and two populations of Scutylenchus quadrifer demonstrated as much or greater genetic distance between them than among three related species of Merlinius.

RESUMEN

The identity of a newly discovered population of pale potato cyst nematode Globodera pallida associated with potato in eastern Idaho was established by morphological and molecular methods. Morphometrics of cysts and second-stage juveniles were generally within the expected ranges for G. pallida with some variations noted. The Idaho population and paratype material from Epworth, Lincolnshire, England, both showed variations in tail shape, with bluntly rounded to finely pointed tail termini. Compared to literature values for the paratypes, second-stage juveniles of the Idaho population had a somewhat shorter mean body length, and cysts had a slightly higher mean distance from the anus to the nearest edge of the fenestra. PCR-RFLP of the rDNA ITS region, sequence-specific multiplex PCR and DNA sequence comparisons all confirmed the identity of the Idaho population as G. pallida. The ITS rDNA sequence of the Idaho isolate was identical to those from York, England, and the Netherlands. Species-specific primers that can positively identify the tobacco cyst nematode Globodera tabacum were also developed, providing a new assay for distinguishing this species from G. pallida and the golden potato cyst nematode Globodera rostochiensis.

RESUMEN

In 2006, a cyst nematode was discovered in tare dirt at a potato (Solanum tuberosum) processing facility in eastern Idaho. The nematode was found during a routine survey conducted jointly by the Idaho State Department of Agriculture and the USDA Animal and Plant Health Inspection Service through the Cooperative Agricultural Pest Survey program. Extensive additional sampling from two suspect fields led to the identification of the same nematode in a 45-acre (18.2-ha) field located in northern Bingham County. The morphology of cysts and second-stage juveniles and molecular analyses established the identity of the species as the pale cyst nematode Globodera pallida (Stone 1973) Behrens 1975. Morphological characters used for identification included cyst shape, characteristics of cyst terminal cone including nature of fenestration, cyst wall pattern, anal-vulval distance, number of cuticular ridges between anus and vulva, and Granek's ratio (1,4). The second-stage juvenile morphologies critical for identification were the following: body and stylet length, shape of stylet knobs, shape and length of tail and hyaline tail terminus, and number of refractive bodies in the hyaline part of tail (1,4). Diagnosis as G. pallida was clearly confirmed by two molecular tests. First, PCR-RFLP (restriction fragment length polymorphism) profiles of a ribosomal DNA fragment using restriction enzymes RsaI, TaqI, and AluI (2) were consistent with a G. pallida control and not G. rostochiensis. Second, the ribosomal DNA region that extends from the 3' end of the 18S ribosomal subunit and includes all of ITS1, 5.8S, and ITS2 to the 5' end of the 28S ribosomal subunit was used to generate sequence for the most accurate species determination. Sequences obtained from three individual juveniles were compared with those from several Globodera species (3), revealing unequivocal similarity to G. pallida. This detection represents a new country record for G. pallida in the United States. Collection of additional information regarding distribution of this nematode within the region is underway. References: (1) J. G. Baldwin and M. Mundo-Ocampo. Heteroderinae, Cyst- and Non-cyst-forming Nematodes. Pages 275-362 in: Manual of Agricultural Nematology. W. R. Nickle, ed. Marcel Dekker, New York, 1991. (2) V. C. Blok et al. J. Nematol. 30:262, 1998. (3) L. A. Pylypenko et al. Eur. J. Plant Pathol. 111:39, 2005. (4) A. R. Stone. Nematologica 18:591, 1973.

RESUMEN

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 microm); 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.

RESUMEN

An unusual population of Meloidogyne hapla, earlier thought to be an undescribed species, was found causing large galls, without adventitious roots, and substantial damage to coffee in Maui, Hawaii. Only in Brazil had similar damage to coffee been reported by this species. Unlike M. exigua from South and Central America, this population reproduced well on coffee cv. Mokka and M. incognita-susceptible tomato but poorly on tomato with the Mi resistance gene. Characterization included SEM images, esterase isozymes, and five DNA sequences: i) the D3 segment of the large subunit (LSU-D3 or 28S) rDNA, ii) internal transcribed spacer (ITS-1) rDNA, iii) intergenic spacer (IGS) rDNA, iv) the mitochondrial interval from cytochrome oxidase (CO II) to 16S mtDNA, and v) the nuclear gene Hsp90. Sequences for ITS-1, IGS, and COII were similar to other M. hapla populations, but within species ITS-1 variability was not less than among species. One LSU-D3 haplotype was similar to a previously analyzed population with two minor haplotypes. Hsp90 exhibited some variation between Maryland and Hawaiian populations distinct from other species. Females were narrow with wide vulval slits, large interphasmidial distances, and more posterior excretory pores; 20% of perineal patterns had atypical perivulval lines. Males had a low b ratio (<12 microm). Juveniles had a short distance between stylet and dorsal gland orifice. Juvenile body length was short (<355 microm) and was different between summer and winter populations.

RESUMEN

A root-knot nematode Meloidogyne thailandica n. sp. was discovered on roots of ginger (Zingiber spp.) intercepted from Thailand in October 2002 by the U.S. Department of Agriculture Animal and Plant Health Inspection Service at the port of San Francisco. Comparison by light microscopy (LM) and scanning electron microscopy (SEM) to five other morphologically related species (M. incognita, M. arenaria, M. microcephala, M. megatyla, and M. enterolobii) revealed that the new species differs from these by one or more of the following: body, tail and hyaline tail length, shape of head, tail and tail terminus of second-stage juveniles; stylet length and shape of spicules in males; perineal pattern, stylet length and shape of knobs in females. The distinctive perineal pattern is oval to rectangular, with smooth to moderately wavy and coarse striae, and with characteristic radial structures present underneath the pattern area; the dorsal arch is high, sometimes round to rectangular, and striae in and around the anal area form a thick network-like pattern interrupted by lateral lines and large phasmids. Second-stage juveniles have a long, slender tail and long, gradually tapering hyaline tail region ending in a rounded terminus. Male spicules commonly have an acutely angled shaft with a bidentate terminus. Molecular data from the ribosomal large subunit D3 expansion segment revealed four haplotypes, two of which were unique and distinguish M. thailandica n. sp. from M. arenaria, M. incognita, and M. javanica.

RESUMEN

A root-knot nematode, Meloidogyne floridensis n. sp., is described and illustrated from peach originally collected from Gainesville, Florida. This new species resembles M. incognita, M. christiei, M. graminicola, and M. hispanica, but with LM and SEM observations it differs from these species either by the body length, shape of head, tail and tail terminus of second-stage juveniles, body length and shape of spicules in males, and its distinctive female perineal pattern. This pattern has a high to narrowly rounded arch with coarsely broken and network-like striae in and around anal area, faint lateral lines interrupting transverse striae, a sunken vulva and anus, and large distinct phasmids. Molecular data from ribosomal IGS illustrate that M. floridensis n. sp. is different from the mitotic species M. arenaria, M. incognita, and M. javanica. Data from RAPDs confirm it and suggest that this new species lies in an intermediate phylogenetic position between the previous species and the meiotic species M. hapla, M. fallax, and M. chitwoodi. Differential host tests based on annual crops and on Prunus accessions are reported.

RESUMEN

Faces of lesion nematodes Pratylenchus teres (populations RTB and JK) and P. zeae or the bacterivore Distolabrellus veechi were observed on frozen specimens with low-temperature scanning electron microscopy and as chemically fixed, critical-point dried specimens with conventional scanning electron microscopy. Amphidial secretions were preserved in chemically fixed but not cryofixed lesion nematodes. Overhanging liplets of chemically fixed D. veechi may be artifactual because they appeared as variably filled, mostly empty membranes when cryofixed. The diagnostically useful lips of the frozen lesion nematodes exhibited six sectors of variable prominence that were absent in chemically fixed specimens. This variability may be due to different degrees of muscle contraction captured during cryofixation, which occurs in milliseconds. This is the first evidence that rarely observed lip sectors in Pratylenchus may be something other than an artifact of shrinkage.

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RESUMEN

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 degrees 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 degrees 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.

RESUMEN

The potential of different bacterial-feeding Rhabditida to consume isolates of Burkholderia cepacia with known agricultural biocontrol ability was examined. Caenorhabditis elegans, Diploscapter sp., Oscheius myriophila, Pelodera strongyloides, Pristionchus pacificus, Zeldia punctata, Panagrellus redivivus, and Distolabrellus veechi were tested for growth on and preference for Escherichia coli OP50 or B. cepacia maize soil isolates J82, BcF, M36, Bc2, and PHQM100. Considerable growth and preference variations occurred between nematode taxa on individual bacterial isolates, and between different bacterial isolates on a given nematode. Populations of Diploscapter sp. and P. redivivus were most strongly suppressed. Only Z. punctata and P. pacificus grew well on all isolates, though Z. punctata preferentially accumulated on all isolates and P. pacificus had no preference. Oscheius myriophila preferentially accumulated on growth-supportive Bc2 and M36, and avoided less supportive J82 and PHQM100. Isolates with plant-parasitic nematicidal properties and poor fungicidal properties supported the best growth of three members of the Rhabditidae, C. elegans, O. myriophila, and P. strongyloides. Distolabrellus veechi avoided commercial nematicide M36 more strongly than fungicide J82.

RESUMEN

The invariant development of free-living nematodes combined with the extensive knowledge of Caenorhabditis elegans developmental biology provides an experimental system for an analysis of the evolution of developmental mechanisms. We have collected a number of new nematode species from soil samples. Most are easily cultured and their development can be analyzed at the level of individual cells using techniques standard to Caenorhabditis. So far, we have focused on differences in the development of the vulva among species of the families Rhabditidae and Panagrolaimidae. Preceding vulval development, twelve Pn cells migrate into the ventral cord and divide to produce posterior daughters [Pn.p cells] whose fates vary in a position specific manner [from P1.p anterior to P12.p posterior]. In C. elegans hermaphrodites, P(3-8).p are tripotent and form an equivalence group. These cells can express either of two vulval fates (1 degree or 2 degrees) in response to a signal from the anchor cell of the somatic gonad, or a nonvulval fate (3 degrees), resulting in a 3 degrees-3 degrees-2 degrees-1 degree-2 degrees-3 degrees pattern of cell fates. Evolutionary differences in vulval development include the number of cells in the vulval equivalence group, the number of 1 degree cells, the number of progeny generated by each vulval precursor cell, and the position of VPCs before morphogenesis. Examples of three Rhabditidae genera have a posterior vulva in the position of P9-P11 ectoblasts. In Cruznema tripartitum, P(5-7).p form the vulva as in Caenorhabditis, but they migrate posteriorly before dividing. Induction occurs after the gonad grows posteriorly to the position of P(5-7).p cells. In two other species, Mesorhabditis sp. PS 1179 and Teratorhabditis palmarum, we have found changes in induction and competence with respect to their presumably more C. elegans-like ancestor. In Mesorhabditis, P(5-7).p form the vulva after migrating to a posterior position. However, the gonad is not required to specify the pattern of cell fates 3 degrees-2 degrees-1 degree-2 degrees-3 degrees. Moreover, the Pn.p cells are not equivalent in their potentials to form the vulva. A regulatory constraint in this family thus forces the same set of precursors to generate the vulva, rather than more appropriately positioned Pn.p cells.

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Phylogenetic characters for Heteroderinae Luc. et al., 1988 are evaluated in Meloidodera which is believed to have primarily ancestral characters. Phasmid ultrastructure is observed in second-stage juveniles (J2), third-stage juvenile males, fourth-stage juvenile males, and fifth-stage males of Meloidodera floridensis and M. charis. Phasmid secretion occurs inside the egg before the J1-J2 molt. Before J2 hatch, concentric lamellar membranes occur within the sheath and socket cells. Some membranes become lamellae of the sheath cell plasma membrane; others become multilamellar bodies. During early molting, plasma membrane lamellae disappear and a distal dendrite segment appears in a rudimentary canal. After the molt, the distal dendrite is not present within the canal. The phylogenetic utility of phasmid features is discussed. In both species the ampulla shape and size between molts are stable features in juveniles and males. The posthatch J2 sheath cell receptor cavity may vary in a species specific manner, but comparative morphology requires precise timing after hatch.