Progress 11/01/01 to 10/31/06
Outputs
To understand the molecular evolutionary mechanisms resulting in the unusual structural features found within the mitochondrial genome of the mosquito-parasitic nematode Romanomermis culicivorax (Enoplea:Mermithidae), a comparative mitochondrial genomics study was undertaken. A methodology was developed to enrich for intact nematode mitochondrial genomes in preparative yields using a rolling circle amplification strategy. Efficient RCA amplifications were conducted on template DNA isolated from individual Thaumamermis cosgrovei nematodes, parasites of the isopod Armadillidium vulgare, revealing that multiple T. cosgrovei mitochondrial DNA haplotypes are maintained in local isopod populations. The magnitude and frequency of mtDNA haplotype variation is unprecedented among metazoan mitochondrial genomes previously analyzed. This observation enabled a genetic analysis that revealed multiply-infected hosts are the result of co-parasitization by members of the same maternal
lineage, and not the result of spatially and temporally independent infections. This result extends in a novel way our understanding of mermithid nematode life histories so necessary in establishing creative nematode biological control strategies for arthropods, including insect pests. Surveys for additional T. cosgrovei haplotypes using RCA led to the discovery a novel mermithid nematode mtDNA haplotype within A. vulgare hosts. This variant was identified as an Agamermis sp. based on both morphological and molecular phylogenetic analyses. Complete sequences of mitochondrial geneomes from T. cosgrovei, Agamermis sp., and the mosquito mermithid nematode Sterlkovimermis spiculatus have been determined. Gene order comparisons between these three mtDNAs and Romanomermis shed little light on the molecular evolutionary history of R. culicivorax mtDNA but have added to our understanding of affinities among insect-parasitic nematodes. However, this comparative study has revealed a number of
unusual features of mermithid mitochondrial genomes when compared to typical metazoan mtDNA organization. These include frequent and large-scale size polymorphism, lengthy repeating sequences, inversion of repeating units, extensive duplication of coding genes, and rapid rearrangement. Characterization and analysis of mermithid mitochondrial genomes has also expanded our understanding of the differences between mtDNAs from the two major nematode taxonomic classes, the Chromodorea and the Enoplea. In contrast to the recurrent mitochondrial gene syntenic relationships typifying the Chromodorea, remarkably divergent mitochondrial gene orders are observed at all taxonomic levels (subfamily, genus, species) among the Enoplea. To place Enoplean mtDNA in a phylogenetic context, a molecular framework was constructed from selected mermithid species based on 18S nuclear rDNA sequences. This effort provided the first molecular phylogeny for this nematode family. The molecular phylogeny was
instrumental in identifying a new mermthid parasitism of the fire ant Solenopsis invicta Buren (Hymenopters: Formicidae) by the nematode Allomermis solenopsi.
Impacts
The broad goal of this research was to ascertain genetic affinities among mermithid nematodes that parasitize a variety of insect hosts. These results will help identify relationships among species that feature two useful phenotypes: broad host-range for simultaneous control of multiple insect hosts and field persistence so repeated application of these nematodes to infested field sites is rendered unnecessary. In turn, this knowledge may be helpful in nematode breeding programs using efficient genetic crosses that would combine useful traits to create an "all purpose" parasite. Broad host-range, self-perpetuating insect pest management of this sort will substantially diminish the cost of employing nematodes as biological control agents. The molecular phylogeny will also be helpful for future identification of mermthid species and provide a useful platform to select appropriate entomopathogenic nematodes for targeted biological control programs.
Publications
- Tang, S. and B. C. Hyman. 2007. Mitochondrial genome haplotype hypervariation within the isopod parasitic nematode Thaumamermis cosgrovei. Genetics, accepted for publication.
- Chen, P., P. A. Roberts, A. E. Metcalf, and B. C. Hyman. 2003. Nucleotide substitution patterning within the Meloidogyne D3 region and its evolutionary implications. Journal of Nematology 35:404-410.
- Poinar, G. O. Jr., S. D. Porter, S. Tang and B. C. Hyman. 2007. Allomermis solenopsii sp. new. (Mermithidae: Nematoda) parasitizing the fire ant Solenopsis invicta Buren (Hymenopters: Formicidae) in Argentina. Systematic Parasitology, in press.
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Progress 01/01/05 to 12/31/05
Outputs
We have developed a robust molecular phylogenetic framework for the nematode family Mermithidae using mitochondrial DNA (cytochrome oxidase I) and nuclear (18S and 28S rDNA) sequence data. Nematode taxonomic groups include ROMANOMERMIS, STRELKOVIMERMIS, GASTROMERMIS, ALLOMERMIS, AGAMERMIS, OVOMERMIS and other insect parasitic nematodes of special importance to biological control of arthropod pests. Phylogenies have been constructed within the genus ROMANOMERMIS and between mermithid genera. It appears that the Merithidae form two clades that loosely correlate with aquatic or terrestrial host parasitism. Moreover, the ROMANOMERMIS phylogeny reveals a strong biogeographical structuring along both North-South and East-West transects in North America, and predicts movements of mosquito and insect hosts since glaciation subsided. We have made significant strides in several areas: (i) exploiting our rolling-circle amplification technique to isolate and characterize complete
nematode mitochondrial genomes from additional mermithid nematodes; (ii) determination of the complete double-stranded nucleotide sequence and mitochondrial gene order for a new Mermithid parasite of the common pillbug ARMADILLIDIUM VULGARE, the same host in which THAUMAMERMIS COSGROVEII is propagated (iii) discovery, morphological, and molecular identification (in collaboration with Sanford Porter, USDA, FL and George Poinar, Oregon State University) of two new mermithid nematode parasites of fireants (SOLENOPSIS INVICTA), These Mermithid nematode are obligate parasites with great potential for biological control of this ant.) One species is a new species that we have named ALLOMERMIS SOLENOPSIS; a second new species has been tentatively assigned to the HEXAMERMIS complex within the mermithid sub-family Gastromermithinae. (iv). Successful completion of the life cycle of the Mermithid nematode OVOMERMIS within the laboratory using the Lepidopterin SPODOPTERA EXIGUA (beet army worm) as
a model host. Partial nucleotide sequence of the OVOMERMIS mitochondrial genome suggests multiple haplotypes and we intend to see if haploytype differences play a role in parasitism using S. EXIGUA.
Impacts
The practicality of biological control using insect parasitic nematodes such as the Mermithids is constrained by the limited host-range of these organisms. We anticipate that these studies will enable precise predictions as to which nematodes might attack a broad range of hosts. Broad scale insect pest using "all-purpose" parasitic nematodes will diminish the cost of biological control strategies. The adoption of rolling circle amplification to isolate intact nematode mitochondrial genomes also appears to be of some impact. One referee of our manuscript reporting this success (Tang and Hyman, 2005) stated in his/her review "...the technique could have profound implications in the field of nematode systematics."
Publications
- Tang, S. and B.C. Hyman. 2005. Rolling circle amplification of complete nematode mitochondrial genomes. Journal of Nematology 37:236-241. (Was listed as in press last year)
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Progress 01/01/04 to 12/31/04
Outputs
Substantial progress has been made towards our goal of developing a molecular phylogenetic framework for the nematode family Mermithidae using mitochondrial DNA sequence data. This nematode taxonomic group includes ROMANOMERMIS and other insect parasitic nematodes of special importance to biological control of arthropod pests. We have made strides in three areas: (i) developing a rolling-circle amplification technique to isolate intact nematode mitochondrial genomes; (ii) determination of the complete double-stranded nucleotide sequence and mitochondrial gene order for four mermithids DNAs (R. CULICIVORAX, R. NIELSENI, R. IYENGURI, THAUMAMERMIS COSGROVEII) and nearly complete mitochondrial DNA sequence for two other mermithids, MERMIS NIGRESCENS and an OVOMERMIS SP. (iii) discovery of a hypervariable region in the T. COSGROVEII mitochondrial genome that varies among individual nematodes, and (iv) discovery of a potentially new parasitic nematode of the terrestrial
isopod ARMADILLIDIUM, the same host in which T. COSGROVEII propagates. From a phylogenetic perspective, mitochondrial gene order (synteny) analysis has revealed profound differences between the two major nematode taxonomic classes. Lengthy repeating units, often containing mitochondrial genes, appear to be an architectural feature of Enoplid mitochondrial genomes that include the family Mermithidae, but are not present within the Chromodorid mitochondrial DNAs sequenced to date. Mitochondrial gene order, and hence transcriptional organization, is similar among the Chromodorea, but is highly diverse among Enoplean mitochondrial genomes; no two Enoplean nematode species share the same syntenic relationships among their mitochondrial genes. Even inter-specific comparisons of mitochondrial gene order within ROMANOMERMIS sp. show considerable variation. Computer-assisted modeling enables a deduction of the most parsimonious steps leading variation in mitochondrial gene order and the
molecular evolution of the complex R. CULCIVORAX mitochondrial genome. Adjacency of several gene pairs have been conserved within mitochondrial genomes representing the mermithid nematodes, other Enoplids (TRICHINELLA SPIRALIS), XIPHINEMA AMERICANUM) and more distant representatives of the class Chromodorea, a data set that virtually spans the phylum Nematoda. This discovery enables predication of ancestral nematode mitochondrial gene clusters, furthers our ability to deduce possible ancestral gene order(s), and define the more contemporary molecular events leading to nematode mitochondrial gene order diversity. Using our extensive DNA sequence data set that we collected for the gene order studies, a robust phylogeny for the mermithid nematodes has been developed based on nucleotide sequence alignments among mitochondrial genes. Phylogenies have been constructed within the genus ROMANOMERMIS and between mermithid genera. The ROMANOMERMIS phylogeny reveals a strong biogeographical
structuring along both North-South and East-West transects in North America and predicts movements of mosquito and insect hosts since glaciation subsided. Basal mermithid genera have been identified from these analyses.
Impacts
The long term importance of our work is to overlay host preference on a robust phylogeny of mermithid nematodes in order to understand evolution of insect parasitism. The practicality of biological control using insect parasitic nematodes such as the Mermithids is constrained by the limited host-range of these organisms. We anticipate that these studies will enable precise predictions as to which nematodes might attack a broad range of hosts. Broad scale insect pest using "all-purpose" parasitic nematodes will diminish the cost of biological control strategies. The adoption of rolling circle amplification to isolate intact nematode mitochondrial genomes also appears to be of some impact. One referee of our manuscript reporting this success (Tang and Hyman, accepted for publication) states in his/her review "...the technique could have profound implications in the field of nematode systematics."
Publications
- Tang, S. and B.C. Hyman. 2005. Rolling circle amplification of complete nematode mitochondrial genomes. Journal of Nematology, in press.
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Progress 01/01/03 to 12/31/03
Outputs
In our previous report, we described the development of two robust phylogenetic trees intended to understand affinities within the nematode family Mermithidae.. This nematode taxonomic group includes ROMANOMERMIS, a potentially useful biological control agent for mosquitoes, and other insect-parasitic nematodes. We continue to make progress in several related areas: (i) further validation of tree topology using Baysian methodologies to analyze mitochondrial cytochrome oxidase I (COI) and nuclear 26S rDNA D3 sequence alignments within the genus ROMANOMERMIS and among genera in the mermithid family. Our studies include the species R. CULICIVORAX, R. NIELSENI, R. IYENGARI, R. COMMUNENSIS and R. KIKTOREAK. These ROMANOMERMIS species represent isolates along a north/south transect that ranges from Northern Canada to Mexico. Our results have unmasked likely speciation events along this cline and signal mosquito host migrations and additional vectoring into these regions.
(ii) ) We have obtained nucleotide sequence and determined mitochondrial gene order for the entire THAUMAMERMIS COSGROVEII mitochondrial genome. We have mapped and determined the transcriptional organization of the expected 12 protein coding genes and two ribosomal RNA genes about the mitochondrial DNA molecule. We have also mapped five of 22 anticipated tRNA genes. The T. COSGROVEII mitochondrial gene order is significantly different from all other nematodes for which complete mitochondrial genome sequence is available. However, similar to its Adenophorean mermithid relatives, this mitochondrial DNA contains duplicated copies for several mitochondrial genes, though the amplified genes differ from those found in the R. CULICIVORAX mitochondrial genome. This year we have also finished the complete nucleotide sequence for the R. CULICIVORAX mitochondrial DNA molecule and have completed 70% of the related R. IYENGURI mitochondrial DNA molecule. R. IYENGURI carries repeated mitochondrial
genes that differ from R. CULICIVORAX and T. COSGROVEII. By understanding comparative mermithid mitochondrial gene order, insights into molecular events sponsoring the generation of genetic diversity can be achieved. By overlaying this information on the molecular phylogenies established using the COI and D3 regions will enable an understanding of how host-preference evolved. In turn, this scientific information will assist furthering the use of entomophagous nematodes as efficient biological control agents.
Impacts
Specific information on the evolution of nematode parasitism among insect hosts will help define the utility of nematodes as efficient biological control agents. By using DNA sequences derived from both the nuclear and mitochondrial genomes, precise predictions as to which nematodes might attack a broad range of hosts will be enabled. Our expectation remains viable goal, namely that one or several nematodes will be identified that can simultaneous parasitize and destroy multiple insect pests. Broad scale insect pest management of this sort will diminish the cost of employing nematodes as biological control agents.
Publications
- Chen, P. P. A. Roberts, A. E. Metcalf and B. C. Hyman. 2003. Nucleotide substitution patterning within the Meloidogyne rDNA D3 region and its evolutionary implications. Journal of Nematology, in press (Dec. 2003 issue, not yet in print).
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Progress 01/01/02 to 12/31/02
Outputs
In the past year, we have made steady progress towards our goal of developing a molecular phylogenetic framework for the nematode family Mermithidae. This nematode taxonomic group includes ROMANOMERMIS and other insect-parasitic nematodes. We have made strides in three areas: (i) deducing interspecific relationships using mitochondrial cytochrome oxidase I (COI) DNA sequences within the genus ROMANOMERMIS. Our studies include the species R. CULICIVORAX, R. NIELSENI, R. IYENGARI, R. COMMUNENSIS and R. KIKTOREAK. These ROMANOMERMIS species represent isolates along a north/south transect that ranges from Northern Canada to Mexico. Our results have unmasked likely speciation events along this cline and signal mosquito host migrations and additional vectoring into these regions. (ii) Using the polymerase chain reaction (PCR), we have amplified a DNA segment from the nuclear 26S ribosomal DNA D3 region as well as the aforementioned COI locus from from eight different
mermithid nematode genera that represent a wide range of insect hosts (pillbugs, mosquitos, spiders, leafhoppers, grasshoppers, spiders, and mites) and incorporate a worldwide sampling. We have complete, double-stranded nucleotide sequence from all 16 PCR products (two loci x 8 nematodes) and have performed phylogenetic analysis on aligned DNA sequences using three different methodologies (Maximum Parsimony, Maximum Likelihood, and Kimura 2-parameter distance). All methodologies generate congruent trees. Our studies continue to reveal that the mermithid nematodes appear to co-evolve with their insect hosts. Nematodes that parasitize aquatic insect hosts form a clade distinct from related mermithids that replicate within terrestrial insect hosts. We have learned that COI and D3 gene sequences from two different isolates of the mermithid THAUMAMERMIS COSGROVEII (from California and New Zealand) cluster together to the exclusion of all other mermithids tested despite being derived from
two different arthropods, suggesting a host switch upon introduction of this nematode to the Americas. This result furthers our interests in the evolution of insect parasite host preference. (iii) We have obtained nucleotide sequence and determined mitochondrial gene order for 50% of the T. COSGROVEII mitochondrial DNA molecule. We have learned that the gene order in T. COSGROVEII mtDNA is ND3-COII-COI-COIII-ND1-CytB, a gene architecture significantly different from the one other available mermithid mtDNA gene order, that of R. CULICIVORAX, established in our laboratory some time ago. These gene orders also differ from all other nematode mitochondrial genomes described to date. Understanding comparative mermithid mitochondrial gene order and overlaying this information on the molecular framework established using the COI and D3 regions are significant because this information will enable an understanding of how host-preference evolved. In turn this scientific information will assist
furthering the use of entomophagous nematodes as efficient biological control agents.
Impacts
Specific information on the evolution of nematode parasitism among insect hosts will help define the utility of nematodes as efficient biological control agents. By using DNA sequences derived from both the nuclear and mitochondrial genomes, precise predictions as to which nematodes might attack a broad range of hosts will be enabled. Our expectation is still a viable goal, namely one or several nematodes will be identified that can simultaneous parasitize and destroy multiple insect pests. Broad scale insect pest management of this sort will diminish the cost of employing nematodes as biological control agents.
Publications
- Hyman, B. C. 2003. Nucleic Acids and Proteins: Modern Linguistics for the Genomics and Bioinformatics Era. Pp. 5-24 in S.A. Krawetz and D. D. Womble, eds. Introduction to Bioinformatics: A Theoretical and Practical Approach. Humana Press, New Jersey.
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Progress 01/01/01 to 12/31/01
Outputs
In the past year, we have made significant strides in our long term goal to develop a molecular framework for the nematode family Mermithidae, which includes ROMANOMERMIS and other insect-parasitic nematodes. Using the polymerase chain reaction (PCR), we have successfully obtained a DNA segment from the nuclear 26S ribosomal DNA D3 region, and a mitochondrial DNA product from the mitochondrial cytochrome oxidase I (COI) locus. These DNA segments have been obtained from eight different mermithid nematodes that represent a wide range of insect hosts (pillbugs, mosquitos, spiders, leafhoppers, grasshoppers and mites) and incorporate a worldwide distribution. We have obtained complete, double-stranded nucleotide sequence from all 16 PCR products (two loci x 8 nematodes) and have performed phylogenetic analysis on aligned DNA sequences using three different methodologies (Maximum Parsimony, Maximum Liklihood, and Kimura 2-parameter distance). We have developed a molecular
framework for affinities among these mermithid nematodes. Our studies have revealed that the mermithid nematodes appear to co-evolve with their insect hosts. Nematodes that parasitize aquatic insect hosts form a clade distinct from related mermithids that replicate within terrestrial insect hosts. The evolution of host preference presents a new area for study that we plan to pursue in my laboratory in the near future. We have also begun to establish the gene order within THAUMAMERMIS COSGROVEII and ROMANOMERMIS NIELSENI mitochondrial genomes by successfully establishing cloned DNA fragment libraries and screening them for mtDNA clones using heterologous probes derived from another mermithid nematode, R. CULICIVORAX. We have obtained several lengthy mtDNA clones from each species and have determined that they contain the COI gene and the 16S mitochondrial rRNA gene. We have been able to employ these DNA sequences to develop PCR primers to that are being used to amplify whole
mitochondrial genomes from these mermithids using long-distance PCR methodology . This procedure is conducted in the anticipation of determining the entire nucleotide sequences of these mtDNAs. These mtDNA gene orders will then be compared to that of R. CULICIVORAX mtDNA, which was established in our laboratory some time ago. Understanding comparative gene order and overlaying this information on the molecular framework established using the COI and D3 regions are significant because this information will enable an understanding of how host-preference evolved. In turn this scientific information will assist furthering the use of entomophagous nematodes as efficient biological control agents.
Impacts
Specific information on the evolution of nematode parasitism among insect hosts will help define the utility of nematodes as efficient biological control agents. By using DNA sequences derived from both the nuclear and mitochondrial genomes, precise predictions as to which nematodes might attack a broad range of hosts will be enabled. The expectation is that one or several nematodes will be identified that can simultaneous parasitize and destroy multiple insect pests. Broad scale insect pest management of this sort will diminish the cost of employing nematodes as biological control agents.
Publications
- Hyman, B. C. 2002. Nucleic Acids and Proteins: Modern Linguistics for the Genomics and Bioinformatics Era. Humana Press, in press. (Contributions to a new textbook)
- Metcalf, A.E,, Nunney, N. and Hyman, B.C. 2001.Geographic patterns of genetic differentiation within the restricted range of the endangered Stephens' kangaroo rat DIPODOMYS STEPHENSI. Evolution 55:1233-44.
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Progress 01/02/00 to 12/31/00
Outputs
To address the evolutionary relationships among the four major MELOIDOGYNE species, nucleotide variation within the D3 26S rDNA region was examined. Our collection of M. HAPLA isolates represents a worldwide distribution, while our M. ARENARIA, M. JAVANICA and M. INCOGNITA samples are a more localized collection. Using D3A an D3B primers designed from C. ELEGANS, a 350 bp D3 region was PCR amplified from genomic DNA template and double-stranded nucleotide sequence obtained. Phylogenetic analysis using Maximum Parsimony, Maximum likelihood and Kimura 2-parameter distance methodologies all placed M. HAPLA in a separate clade from M. INCOGNITA, M. JAVANICA and M. ARENARIA. This resolution provides strong support for a division between varying reproductive modes employed by root-knot nematode which include facultative parthenogenesis (automixis) and obligate parthenogenesis (apomixis). These affinities are likely driven by the presence of a 3bp insertion at position 204
in M. HAPLA isolates. Branching of the three apomictic species appears to be a recent event, as M. INCOGNITA, M. JAVANICA and M. ARENARIA all share a common D3 haplotype. In the automictic species M. HAPLA, single individuals contain 2 different haplotypes. The differences within M. HAPLA individuals are greater than between individuals. Our isolates of M. JAVANICA appear to have fixed only one haplotype, while M. INCOGNITA and M. ARENARIA maintain more than one haplotype in an isolate. In this apomictic group, M. INCOGNITA and M. ARENARIA have greater differences within species than between species. The D3 nucleotide polymorphism level within MELOIDOGYNE is further investigated by comparison to D3 sequences from published studies. We have also begun to address phylogenetic relationships among representatives of the Mermithid nematodes. Our collection of Mermithid nematodes encompasses obligate parasites of both aquatic and terrestrial arthropods. We applied the same phylogenetic
methodologies (above) to nucleotide sequence obtained from both the nuclear D3 rDNA region and the mitochondrial cytochrome oxidase subunit I locus. Identical affinities are obtained when the nuclear and mitochondrial loci are individually analyzed or catenated into a lengthy sequence. We find that mosquito mermithid parasites (ROMANOMERMIS, STRELKOVIMERMIS) form a clade separate from nematodes parasitizing grasshoppers and leaf hoppers (MERMIS, HEXAMERMIS, AGAMERMIS). A third clade is represented by HELEIDOMERMIS and THALMAMERMIS, parasites of the biting midge and pillbug respectively. As the pillbug was introduced into the Americas from Europe and European pillbugs are not parasitized, our data suggest a host switch occurred, possibly involving transfer from midge hosts.
Impacts
Understanding the co-evolution of mermithid parasites and their hosts have profound implications for predicting the efficacy of entomopathogenic nematodes. Moreover, elucidating relationships among the root-knot nematodes will ultimately suggest more efficient control strategies.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
In the past year we have made progress in two independent areas aimed at describing genetic variation within MELOIDOGYNE HAPLA (a root-knot nematode) and within the Mermithid nematodes (obligate parasites of insects). Root-knot nematodes: Within M. HAPLA, we have analyzed intraspecific variability among a world-wide collection of seven isolates (all race A) in collaboration with Dr. Phillip Roberts. WE have used PCR amplification to isolate the hypervariable D3 expansion region of the nuclear 28S ribosomal RNA gene. After cloning and subsequent nucleotide sequencing, we find there are two forms of the D3 expansion segment that differ from each other by three nucleotide substitutions. Experiments are in progress to determine whether individual nematodes contain one or both types. That these rDNA forms are found in isolates representing a world-wide distribution suggests that nucleotide polymorphisms are ancient and preceded mechanical distribution of M. HAPLA
throughout the world. This unexpected level of nucleotide polymorphism suggests that molecular-based assays for M. HAPLA races may be developed. We will investigate this possibility in the upcoming year. Mermithid nematodes: We have obtained nucleotide sequence for a mitochondrial DNA locus (cytochrome oxidase subunit 1) and a nuclear locus (the D3 rDNA expansion segment) from 10 nematode genera representing obligate parasites of both terrestrial and aquatic hosts. Preliminary phylogenetic analysis has revealed a separation into clades that appear to parallel a preference for aquatic or land-dwelling insects. We hope to use this molecular framework to examine changes in mitochondrial gene order in the coming year. The utility of this project can be found in the use of entomophagic nematodes as biological control agents. This project is important as it will enable us to examine the evolution of parasitism among Mermithid nematodes, including how host-preference evolved. In turn, this
approach may sponsor predictions as to the efficacies of novel biological control strategies using combinations of entomopathogenic nematodes. We have recently initiated a third project that involves the molecular cloning of plant carbamoyl-phosphate synthetase (CPSase), a key enzyme in plant nitrogen metabolism that is a likely target for expression alteration during nematode infestation of plant roots. We have successfully cloned several members of an alfalfa (MEDICAGO SATIVA) gene family. DNA sequence analysis has revealed an unexpected bacterial-like gene architecture. Examination of how members of these gene family members are regulated at the level of transcription and translation during nematode infection and other plant stresses will provide new strategies towards plant protection.
Impacts
Each of these projects is aimed at better understanding insect and plant parasitism by nematodes. In turn, this knowledge should provide novel strategies to protect plants from nematode and insect pests.
Publications
- Zhou, Z., Metcalf, A.E., Lovatt, C.J., and Hyman, B.C. 2000. Alfalfa (Medicago sativa) carbamoylphosphate synthetase gene structure records the deep lineage of plants. Gene 243:105-114.
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Progress 01/01/98 to 12/01/98
Outputs
Analysis of mitochondrial DNA size polymorphism in the form of variable number tandem repeats (mtVNTRs) has become an increasing popular methodology for addressing questions in molecular ecology. When detected by PCR, mtVNTR analysis can provide a sensitive, rapid and cost-effective measure of genetic variability that may be exploited in studies of population differentiation and biogeography. Despite the emergence of this approach, including our own analysis of (Meloidogyne) root-knot nematode populations, there has been little critical evaluation of its success or utility as a practical tool. We have spent the past year providing this critical analysis. We have identified problematic methodological, theoretical and interpretive factors that can influence the utility of mtVNTR analysis. The reliability of the technique was evaluated with respect to both detection of alleles and scoring of intra individual allele frequencies. We found the potential problems of the
technique do not raise serious practical concerns, but the methodology is seriously flawed by the difficulty of reliably assessing allele frequencies, of assaying only germline tissue, and our ignorance of the mechanisms generating mtVNTR polymorphism. The utility of the technique to resolve broader questions in the molecular ecology of root-knot nematodes should be treated cautiously. We have also initiated a study to examine the phylogenetic relationships among the mermithid nematodes, obligate parasites of many insects. We have successfully isolated and obtained nucleotide sequence for ribosomal DNA encoding.
Impacts
(N/A)
Publications
- LUNT, D. H., L. E. WHIPPLE and B. C. HYMAN. 1998. Mitochondrial DNA variable number tandem repeats (VNTRs): utility and problems in molecular ecology. Molecular Ecology 7:1441-1455.
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Progress 01/01/97 to 12/01/97
Outputs
Mitochondrial genome analysis: Absence of covariance of the 63 bp and 102 bp mtDNA VNTRs. Studies previously reported focused upon the use of a 63 bp variable number tandem repeat (VNTR) mitochondrial DNA sequence to examine population differentiation among Meloidogyne isolates. We concluded that this locus was undergoing mutation to different allele sizes at a rate that did not provide sufficient signal for reliable analysis. As such, we recognized that development of additional markers were required given the limited level of resolution provided by 63 bp VNTR analysis for the populations under study. Thus, we initiated a study to evaluate genetic variation of the 102 bp repeat, another VNTR co-resident in the Meloidogyne mitochondrial genome. We find that both VNTRs exhibit very different distributions of repeat copy numbers. Inspection of repeat copy number at the 63 bp locus reveals a much broader distribution (1-13 repeats) then that of the 102 bp repeats (4-6).
The confined "scatter" at the 102 bp locus suggests a diminished rate of change to different allele sizes for the 102 bp VNTR. As such, this locus may ultimately provide some measure of differentiation among population hierarchies at a level of resolution not currently obtainable with the 63 bp VNTR locus. We have also been actively engaged in mechanistic studies that focus upon how genetic variability is generated within mtDNA VNTRS. Meloidogyne mitochondrial genomes of unusual size were characterized by DNA sequence analysis. Based on these nucleotide sequences,.
Impacts
(N/A)
Publications
- HYMAN, B.C. and WHIPPLE, L.E. 1996. Application of mitochondrial DNA polymorphism to Meloidogyne molecular population biology. Journal of Nematology 28:268-276.
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Progress 01/01/96 to 12/30/96
Outputs
Since my appointment in July, 1996, we have made progress on two projects aimed at describing genetic variation in MELOIDOGYNE INCOGNITA (the root-knot nematodes) and NACOBBUS ABERRANS (the false root-knot nematode). For M. INCOGNITA, we have studied genetic variability at a highly polymorphic 63 base pair tandemly repeat sequence within the mitochondrial genome. For N. ABERRANS, we have evaluated a panel of nuclear loci accessed by isozyme, RAPD and rDNA analysis. Progress on each project is summarized as follows: (1) Genetic diversity among conspecific MELOIDOGYNE isolates is important in understanding their diverse phenotypes, including virulence behavior on different hosts. Genetic variability within and among J2-stage individuals from six M. INCOGNITA isolates was assessed via a polymerase chain reaction (PCR) assay designed to measure size variation within a 63 base pair mitochondrial DNA tandem repeat array. Among the isolates assayed, tandemly repeated 63 bp
VNTRs ranged from one to 21 copies. Hierarchical analysis of allele frequencies revealed that 60% of the total genetic diversity measured for these six isolates resides within individual nematodes. When the three most genetically related isolates were evaluated, there appeared to be a relationship between genetic diversity of each isolate and the history of its propagation on resistant or susceptible host plants.
Impacts
(N/A)
Publications
- HYMAN, B.C. and WHIPPLE, L.E. 1996. Application of mitochondrial DNA polymorphism to Meloidogyne molecular population biology. Journal of Nematology 28:268-276.
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