Progress 02/28/05 to 02/27/10
Outputs
OUTPUTS: Activities - Experiments and assessments: In the course of this project, nematodes have been collected and analysed from the following protected areas and research centers in California: Bolsa Chica Wetlands Reserve, Santa Rosa Plateau Ecological Reserve, Shipley-Skinner Reserve, Joshua Tree National Park, UC Carpinteria Salt Marsh Reserve, UC Boyd Deep Canyon Desert Research Center, UC Sweeney Granite Mountains Desert Research Center, UC James San Jacinto Mountain Reserve, the UCR Citrus Research Center and Agricultural Experiment Station, the UC Kearney Agricultural Center. Additional nematode samples have been obtained out of state or outside the country in Yosemite National Park, the Gulf of California (Mexico), Argentina, Brazil, Canada, Moldova, the Philippines, and Ukraine. Polymorphism of the ITS region has been screened by direct sequencing of PCR products across 79 nematode isolates in culture representing 69 species from six different superfamilies, as well as seventeen species isolated directly from soil or sediment samples, including a new root know nematode isolate damaging turf grass in golf courses. Mentoring: three postdoctoral researchers, two graduate students and two undergraduate students have conducted research and received mentoring in the framework of this project. Events - Research results and internet resource developments have been disseminated as six oral presentations and three poster presentations at international conferences, five invited presentations at other colleges including four abroad, and two presentations at UC Riverside. Additionally, I have co-organized and co-instructed a week-long workshop on general nematode systematics and diagnostics in Brazil, as well as a week-long workshop on systematics and diagnostics of plant parasitic nematodes in Argentina. Products - A new internet resource for nematode identification: One of the objectives of this project was the production of new morphological and molecular tools facilitating nematode identification. In the final year of the project, we have populated an image database with diagnostic multifocal microscopy images obtained from 224 specimens representing 149 nematode species sampled broadly across the diversity of the phylum. This dataset provides the core data for a new search and identification system called NemaScope and designed in collaboration with Dr. Eamonn Keogh of UCR's Department of Computer Science and Engineering. It is now ready for substantial expansion during the next five years to include a comprehensive representation of nematode diversity in California as well as across the world, enabling a broader audience with little or no prior expertise to begin learning and applying the process of nematode identification. PARTICIPANTS: Jonathan Chang; University of California Riverside; undergraduate assistant Paul De Ley; University of California Riverside; principal investigator Oleksandr Holovachov; University of California Riverside; Postdoctoral Researcher Cymphonee Robinson; University of California Riverside; undergraduate assistant Irma Tandingan De Ley; University of California Riverside; Assistant Specialist Collaborators: Amit Roy Chowdhury; University of California Riverside; collaborator James Baldwin; University of California Riverside; collaborator Michalis Faloutsos; University of California Riverside; collaborator Eamonn Keogh; University of California Riverside; collaborator Manuel Mundo; University of California Riverside; Senior Research Assistant Steve Nadler; University of California Davis; collaborator Training and professional development: Jonathan Chang, B.Sc., graduation expected June 2010 Ian King, Ph.D., graduation expected Dec 2010 Cymphonee Robinson, B.Sc., graduated June 2009, now M.S., graduation expected June 2011 Melissa Yoder, Ph.D., graduation expected Dec 2011 TARGET AUDIENCES: This research is of direct benefit to scientists investigating ecology, plant protection, systematics and evolution of soil-borne pathogens as well as free-living nematodes. It is also aimed at providing baseline data that will help agricultural regulators and conservation agencies by providing affordable and comprehensive methods for analysing total biodiversity of microscopic organisms in cultivated soils as well as proected natural habitats. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Changes in knowledge: Polymorphic ITS has been detected in 23 of the cultured isolates and six of the species extracted directly from soil or sediment. The species and isolates are currently being scored for likely occurrence of parthenogenesis. Of fourteen species scored as parthenogenetic, eight were polymorphic in the ITS region. Among another fourteen species scored as reproducing sexually, only two exhibited ITS polymorphism. This shows that the link between ITS polymorphism, hybridization and parthenogenesis is not necessarily direct and suggests that some nematode species have either attained parthenogenesis through other processes than hybdridization events, or that some parthenogenetic lineages have existed for a sufficiently long time to allow the process of concerted evolution to remove any trace of polymorphism caused by ancient hybridization events. At the present time it seems unlikely that ITS polymorphism will be easily applicable as a marker of reproductive mode in nematodes, although it is often useful in detecting and analyzing population structure. Conclusions of this survey of ITS polymorphism will be published after sequencing of clones and scoring of reproductive strategies have been completed. Changes in actions: This project has developed and promulgated a safer method of preserving nematodes with a saturated salt solution abbreviated as DESS, it does not include carcinogenic formalin or flammable ethanol and routinely allows nematodes to be used for microscopy as well as molecular analysis. DESS is now being adopted by other nematologists and other laboratories, within as well as outside the US.
Publications
- Holovachov, O., Tandingan De Ley, I., Mundo-Ocampo, M., Gingold, R., De Ley, P. 2009. Nematodes from the Gulf of California. Part 3. Three new species of the genus Diplopeltoides Gerlach, 1962 (Nematoda: Diplopeltoididae) with overviews of the genera Diplopeltis Gerlach, 1962 and Diplopeltula Gerlach, 1950. Russian Journal of Nematology. Vol. 17: 1 p.43-57.
- Holovachov, O., Bostrom, S., Tandingan De Ley, I., Nadler, S.A., De Ley, P. 2010. Description of Penjatinema novaezeelandiae sp. n. (Rhabditida: Cephalobidae) from New Zealand - a second species of a rare genus. Journal of Nematode Morphology and Systematics. In press.
- King, I.W., Mundo-Ocampo, M., De Ley, P. 2010. Nematodes from the Gulf of California. Part 4. Xyala finneyi sp. n. (Nematoda: Xyalidae). Nematology. In press.
- Giblin-Davis, R.M., Kanzaki, N., Williams, D., De Ley, P., Schierenberg, E., Ragsdale, E.J., Zeng, Y., Center, B.J. 2010. Ultrastructure and life history of Myolaimus byersi n. sp.; a phoretic associate of the crane fly Limonia schwarzi (Alexander) (Limoniidae) in Florida. Nematology. In press.
- Derycke, S., De Ley, P., Tandingan De Ley, I., Holovachov, O., Rigaux, A., Moens, T. 2010. Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae). Zoologica Scripta. In press.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In collaboration with Dr Kelley Thomas in the University of New Hampshire, we have analysed during 2008 the genome-scale DNA sequence data collected in 2007 with first generation Innova/Solexa massively parallel sequencing of five phylogenetically distant nematode species (Plectus minimus, Romanomermis culicivorax, Thoracostoma microfenestratum, Diplolaimelloides meyli and Hemicycliophora sp.). Although the data allow searching for relatively conserved known genes, an unusually high error rate in the sequence fragments was found to prevent assembly of more divergent sequences de novo. The experiment was therefore repeated in 2008 with the competing technology developed by Roche as the 454 system, and this has produced a complete new dataset for Romanomermis culicivorax which is currently being analysed. Additionally, we are exploring a different strategy by subjecting direct DNA extracts from sediment samples to 454 metagenomic sequencing with conserved ribosomal DNA primers. In collaboration with other researchers in the US and UK, a series of 12 sediment samples (including 4 from southern California and 1 from the Sea of Cortez) was collected from different coastal and marine sediments along the Pacific and Atlantic Oceans. Initial results show that molecular diversity is substantially higher than evident from microscopy surveys of the same sediments, and this discrepancy is at least in part due to higher levels of intraspecific polymorphism than detected or suspected before on the basis of smaller-scaled surveys. PARTICIPANTS: The research reported here was conducted in collaboration with Dr. Kelley Thomas of the Hubbard Center for Genome Studies at the University of New Hampshire. My own research team consisted of Assistant Specialist Irma Tandingan De Ley and Postdoctoral Researcher Oleksandr Holovachov, who contributed to the culturing of nematodes, preparation and analyses, as well as graduate students Ian King and Melissa Yoder, and undergraduate Vanessa Garcia, who participated in the collection and preparation of soil and sediment samples, and respectively in startup and maintenance of nematode cultures in our laboratory collection. TARGET AUDIENCES: This research is of direct benefit to scientists investigating ecology, plant protection, systematics and evolution of soil-borne pathogens as well as free-living nematodes. It is also aimed at providing baseline data that will help agricultural regulators and conservation agencies by providing affordable and comprehensive methods for analysing total biodiversity of microscopic organisms in cultivated soils as well as proected natural habitats. PROJECT MODIFICATIONS: Our emphasis in 2008 has temporarily shifted from targeted PCR of a specific single locus in individual nematodes, to a metagenomic approach as well as massive multigene sequencing. This change was made because of the unprecedented opportunities provided by new sequencing technologies. At the same time, the knowledge gained by these advances is allowing us to improve our original approaches and we are now returning to our more targeted PCR-based research of ITS polymorphism, and we have now commenced a comprehensive survey of ITS diversity in our laboratory collection of nematode cultures (output to be summarized in our 2009 annual report).
Impacts
Analysis of 2007 data obtained with an early implementation of Innova/Solexa Gene Analyzer technology has revealed flaws in sequencing accuracy that needed addressing and have been mitigated in the latest upgrades to the system. Our results with the main technological alternative, the 454 system, have provided better accuracy and generated the first large-scale genomic dataset for the biocontrol organism Romanomermis culicivorax. These data will be highly useful to the greater scientific community by helping identify not only diagnostic and phylogenetic markers but also the infectivity and virulence factors that underlie effectiveness of R. culicivorax in mosquito control applications. Processing of our experimental metagenomic sample series is still being completed at this time, but analyses of the first six completed samples indicates that this technique will be very useful for qualitative diagnostics, however the data are not directly useful for quantitative analyses due to mechanisms of amplification bias that may boost signal for less abundant species while depressing it for some of the dominant species in a sample. We are currently working on methods that will overcome these limitations, among others by applying multigene primer pairs to identify artefactual outliers in a given analysis. Because of the rapidly decreasing cost of metagenomic sequencing, this approach promises to substantially improve our ability to detect parasitic nematodes and other pest organisms in soil or sediment samples.
Publications
- Holovachov O., Tandingan De Ley I., Mundo-Ocampo M., Baldwin J.G. & De Ley P. 2008. Nematodes from the Gulf of California, part 1: the genera Ceramonema Cobb, 1933, Pselionema Cobb in Cobb, 1933 and Pterygonema Gerlach, 1954. Nematology 10: 347-373.
- Holovachov O., Mundo-Ocampo M., Tandingan De Ley I., & De Ley P. 2008. Nematodes from the Gulf of California. Part 2. Ceramonema nasobema sp. n. (Nematoda: Ceramonematidae). Nematology 10: 835-844.
- Holovachov O., Bostrom S., Mundo-Ocampo M., Tandingan De Ley I., Yoder M., Burr. A.H.J. & De Ley P. In press. Morphological and molecular characterisation of Hemiplectus muscorum Zell, 1991 (Nematoda: Plectida). Nematology: - .
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Progress 01/01/07 to 12/31/07
Outputs
During 2007 we continued our investigations of of the aquatic plant parasite genus Hirschmanniella and obtained new isolates from California, Florida, Belgium and the Ukraine. The new material confirms our proposal last year of a local new species (H. santarosae Tandingan De Ley et al., 2006) as it reveals that a morphologically disjunct isolate from tha Santa Maria river in California separates H. santarosae phylogenetically from its former sister species H. pomponiensis, which it resembles much more than the isolate from Santa Maria. This is all the more interesting because of the geographic position of the Santa Maria river in between Pomponio State Beach and the Santa Rosa Plateau, both type localities of their respective eponymous species, indicating that species along the California coastline may well have radiated locally from a common ancestor. In collaboration with visiting postdoctoral researcher Sofie Derycke from Ghent University, Belgium, we have sampled
extensively along Southern California beaches and in northern Baja California to obtain specimens of two genera of marine nematodes: the seaweed specialist Thoracostoma and the more generalized intertidal nematode Camacolaimus. Preliminary analysis of cytochrome oxidase I and ITS sequences confirms that two distinct species of Thoracostoma are present sympatrically in giant kelp holdfasts washed ashore between Santa Maria and Ensenada. In addition to this work focusing on Hirschmanniella, Camacolaimus and Thoracostoma, we have collaborated closely with Dr. W. Kelley Thomas at the University of New Hampshire to test out a potentially revolutionary approach to multigene sequencing from minute amounts of tissue, based on the aforementioned Illumina/Solexa Gene Analyzer platform. We have collected and coordinated supply of the following phylogenetically distant nematode species to Dr. Thomas' laboratory: Plectus minimus from culture, Romanomermis culicivorax kept in culture in the
Department of Nematology at UCR, Thoracostoma microfenestratum giant kelp collected along the shores of southern California and Hemiplectus muscorum from mosses collected near Vancouver, Canada. Successful reverse transcription was obtained by Dr. Thomas for quantities of nematodes ranging from a few hundred specimens in the case of P. minimus (an extremely small nematode) to a single adult of T. microfenestratum and a single postparasitic juvenile of R. culicivorax (both much larger nematodes). Processing of the cDNA with the Gene Analyzer has produced an estimated 14 million sequence fragments per species, and these data are currently being assembled for further analysis.
Impacts
Our research shows that substantial diversity exists among (presumably) native isolates of nematodes from the genus Hirschmanniella. The accurate diagnosis of Hirschmanniella species is of importance to agriculture, because the genus includes important parasites of rice. Due to their difficult morphological identification, invasive species could easily slip under the radar of quarantine into the country from other continents. The diagnostic precision provided by molecular data such as ours permits much more reliable identification. First experiments with Innova/Solexa Gene Analyzer technology have shown that it is possible to obtain an enormous amount of sequence data from very few nematode specimens, down to a single individual nematode in some cases. This opens up far-reaching possibilities for characterizing gene expression and a multitude of diagnostic markers from very small numbers of nematodes, which will substantially increase the accuracy of quarantine
diagnosis and identification of nematode strains with complex host ranges and virulence levels.
Publications
- Meldal, B.H.M., Debenham, N.J., De Ley, P., Tandingan De Ley, I., Vanfleteren, J.R., Vierstraete, A.R., Bert, W., Borgonie, G., Moens, T., Tyler, P.A., Austen, M.C., Blaxter, M., Rogers, A.D., Lambshead, P.J. 2007. An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa. Molecular Phylogenetics and Evolution. Vol. 42: 622-636.
- Mundo Ocampo, M., Lambshead, P.J.D., Debenham, N., King, I.W., De Ley, P., Baldwin, J.G., Tandingan De Ley, I., Rocha-Olivares, A., Waumann, D., Thomas, W.K., Packer, M., Boucher, G. 2007. Biodiversity of littoral nematodes from two sites in the Gulf of California. Hydrobiologia. Vol. 586: 179-189.
- Eyualem, A., Decraemer,, W., De Ley, P. Global diversity of nematodes(Nematoda) in freshwater 2007. Hydrobiologia. (Accepted 07/12/2007. 12 galley
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Progress 01/01/06 to 12/31/06
Outputs
During 2006 we focused in greater detail on three particular components of our project. (i) We have completed our testing of a solution of 20% dimethyl sulfoxide and 0.25M disodium EDTA, saturated with NaCl, pH 8.0 (abbreviated here as DESS) for various applications in the preservation for combined morphological and molecular analyses of nematodes. DESS can be used to preserve individual nematodes, nematode suspensions or entire soil/sediment samples. Preserved material can be stored for months at room temperature, shipped by mail, or carried in luggage. Morphological quality is comparable to formalin and much better than ethanol fixation. Specimens can be transferred from DESS to glycerin with traditional protocols. Unlike formalin-preserved material, routine PCR can be performed on individual nematodes after any of these procedures, with success rates and amplification sizes comparable to fresh specimens. Scanning electron microscopy (SEM) of DESS-preserved
nematodes yields results comparable to formalin-preserved specimens. Preservation quality is maintained for at least two years and PCR can still be performed after at least one year. DESS solution clearly simplifies a wide range of applications, due to its suitability for combined morphological and molecular analyses, as well as its less hazardous properties. We expect it to become a staple in the nematological repertoire. (ii) A new species of the aquatic plant parasite genus Hirschmanniella was described from the largest vernal pool in the Santa Rosa Plateau Ecological Reserve, Murrieta, California, USA. The cryptic new species is morphologically very close to H. pomponiensis and H. gracilis; it can be distinguished by a more anterior excretory pore position, by its laterally expanded stylet knobs, and by the distance from phasmid to tail tip. Analysis of rDNA sequences shows that the new species is genuinely distinct, phylogenetic analysis places it in sibling relationship with H.
pomponiensis. Although many more isolates and species will need to be studied before informative biogeographic analyses can be performed, the presently available sequence data suggest that some Hirschmanniella lineages have diversified independently on either side of the Atlantic. (iii) Nematodes are the most abundant metazoans on earth, but their identification is extremely difficult and labor intensive for novices. New approaches are needed to reduce the time necessary for identifications, by using a process of point-and-click visual matching. In collaboration with colleagues from UCR's Department of Computer Science & Engineering, we have produced a substantially improved version of the proof-of-concept prototype we created in 2005. This new tool is called NemaScope and can be accessed at https://www.cs.ucr.edu/bradyj/nematodes/. The image database component of NemaScope currently houses an estimated 750 to 1500 high-definition DIC pictures and multifocal images, representing the
six most important nematode body parts needed for genus identifications. The identification key component at this point has approximately 150 different nematode genera.
Impacts
(i) Our new method of preserving nematode samples in DESS solution will make it much easier for farmers and farm advisers to collect and send soil samples to diagnostic laboratories, which will be able to identify nematodes with both traditional microscopy as well as the latest molecular assays. DESS solution is much less hazardous than traditional fixatives such as formalin, and we hope that its use will lead to substantial reductions in exposure to carcinogenic fixatives in nematological laboratories. (ii) The accurate diagnosis of Hirschmanniella species is of importance to agriculture, because the genus includes important parasites of rice. Due to their difficult morphological identification, invasive species could easily slip under the radar of quarantine into the country from other continents. The diagnostic precision provided by molecular data such as ours permits much more reliable identification. (iii) The development of NemaScope is an important new step in
the creation of an online nematode identification resource that will allow both experts and beginners to quickly and accurately identify nematodes. Our aim is to involve a wide diversity of users, including e.g. farmers and high school students, and thereby generate a much broader awareness of the importance and diversity of nematodes. The algorithms and software we are developing will be of much wider usefulness beyond nematodes, as it can be used to organize and identify images of complex shapes and objects of any kind.
Publications
- Tandingan De Ley, I., M. Mundo-Ocampo, M. Yoder, and P. De Ley. In Press. Nematodes from vernal pools in the Santa Rosa Plateau Ecological Reserve, California I. Hirschmanniella santarosae sp. n. (Nematoda: Pratylenchidae), a cryptic new species closely related to H. pomponiensis Abdel-Rahman & Maggenti, 1987. Nematology.
- Yoder, M., I. Tandingan De Ley, I. W. King, M. Mundo-Ocampo, J. Mann, M. Blaxter, L. Poiras and P. De Ley. 2006. DESS: a versatile solution for preserving morphology and extractable DNA of nematodes. Nematology 8, 367-376.
- De Ley, P. 2006. A quick tour of nematode diversity and the backbone of nematode phylogeny. In WormBook (eds. The C. elegans Research Community). WormBook. http://www.wormbook.org
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Progress 01/01/05 to 12/31/05
Outputs
During the first year of this project, we have concentrated on the following five of six components in our project: (i) We have sampled, extracted, video captured and preserved 200 nematode specimens from Baja California (with extramural support provided by an NSF Biodiversity Surveys and Inventories award) and conducted a first small-scale survey of nematode diversity in the Agricultural Experiment Station at UC Riverside. We also extensively tested and adapted a specimen preservation protocol that was not previously used in nematology, based on a solution including dimethyl sulfoxide (DMSO). The solution was found to secure both good quality DNA as well as excellent morphological character preservation, thereby substantially facilitating the collection, transportation and processing of nematodes for population genetics studies combined with video microscopy. (ii) We have gathered additional cultured strains for hybridization experiments and ITS sequencing, expanding
our culture collection to a present total of 37 viable strains. Ten isolates among these strains in culture were subjected to PCR, ITS polymorphism was encountered in two Plectus species among these. (iii) A total of 25 mutation accumulation lines of Caenorhabditis elegans were subjected to PCR and sequencing of ITS, the locus was found to be homogeneous within and among lines indicating that concerted evolution is fully active in this hermaphroditic species when cultured under conditions of nutrient excess. (iv) PCR and sequencing are in progress of additional taxa, including strains of Oscheius and Romanomermis that are reportedly hybridizing in laboratory conditions. (v) Video images were used to construct a working prototype of our proposed video-based key to the identification of nematode genera. The prototype includes images of 72 genera and is now available online at http://faculty.ucr.edu/pdeley/scalefree.html ; sets of video clips were generated for an additional 33 genera
that will be incorporated in the next major development version.
Impacts
We have established that concerted evolution of ribosomal loci is active in Caenorhabditis elegans, the most widely researched model for nematode genetics. Our research is also substantially expanding the molecular databases for a wide range of nematodes sampled from various natural and agricultural habitats in Southern California and Baja California. This contributes significantly to the development of molecular techniques for assessment of soil quality and for diagnosis of phytoparasitic nematodes. One particularly important development is the adaptation to nematode research of a preservation protocol that gives excellent results both in DNA sequencing and morphological vouchering of specimens. This technique greatly facilitates the use of molecular techniques on soil samples collected from agricultural fields as well as threatened natural habitats. We have also produced a first prototype of an easy-to-use new tool for morphological identification of nematodes.
Publications
- De Ley, P., I. Tandingan De Ley, K. Morris, E. Abebe, M. Mundo-Ocampo, M. Yoder, J. Heras, D. Waumann, A. Rocha-Olivares, A.H.J. Burr, J.G. Baldwin and W.K. Thomas. 2005. An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. Philosophical Transactions of the Royal Society of London B, 360 : 1945-1958.
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