Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: Signature features of Enoplean nematode mitochondrial DNAs (mtDNAs) include lengthy sequence duplications, inversions, and a striking absence of gene order conservation. Within the family nematode Mermithidae, comparative mitochondrial genomics has enabled modeling the molecular evolution of diverse mtDNA architectures within a more confined taxonomic unit. Rolling circle mtDNA amplification facilitated determination of complete nucleotide sequences for eight mermithid mitochondrial genomes including three ROMANOMERMIS and two THAUMAMERMIS congeners. Diverse mitochondrial genome organizations were observed at the subfamily, genus, and species levels indicating a rapid rate of mtDNA rearrangement is occurring in the mermithid lineage. The first molecular phylogeny for the Mermithidae was constructed using nuclear 18S rDNA sequences for the purpose of mapping mtDNA rearrangements onto a framework that may help identify ancestral mtDNA forms. The topology of the molecular framework was reminiscent of hypothesized affinities provided by Gafurov (1997) based on morphology and life history traits. This molecular phylogeny has also proven useful for characterizing new mermithid parasitism of terrestrial arthropods. Previous studies have shown that the mermithid nematode THAUMAMERMIS COSGROVEI, parasitizing local populations of the common terrestrial isopod ARMADILLIDIUM VULGARE (Latr.), carried numerous mitochondrial DNA (mtDNA) haplotypes. Recent surveys for additional haplotypes using rolling circle mtDNA amplification revealed the presence an unusual RFLP variant within a mermithid whose macroscopic appearance was strikingly similar to that of T. COSGROVEI. Exposure of this nematode to distilled water resulted in spontaneous body fragmentation, whereas T. COSGROVEI remained intact under identical hypotonic conditions. Guided by a morphology-based phylogeny that divides the nematode family Mermithidae into seven subfamilies (Gafurov, 1997), a Maximum Likelihood molecular phylogenetic analysis employing mitochondrial COI and nuclear 18S and 28S rDNA nucleotide sequences consistently yielded a single tree after a series of full heuristic searches. The subfamily Agamermethinae was resolved as monophyletic, and within this clade the tested nematode was a sister taxon to AGAMERMIS. Morphological characters from juvenile and adults also support a tentative assignment as AGAMERMIS (Cobb, Steiner and Christie, 1923). The complete nucleotide sequence of the AGAMERMIS sp. mitochondrial genome has been determined; unlike the hypervariable T. COSGROVEI mitochondrial genome, only a single AGAMERMIS sp. haplotype is found. We infer an ancient, large duplication encompassing the mitochondrial genes for NADH dehydrogenase subunits 3, 4 and 6, (ND3, ND4, ND6) and the small mitochondrial rRNA (rrnS), followed by random loss of duplicate gene copies. These observations provide the first report of any mermithid parasite found in the common pillbug A. VULGARE beyond that of T. COSGROVEI. Current research involves developing a proof-of-concept multiplex strategy for high-throughput sequencing of numerous nematode mitochondrial genomes. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The wide-ranging goal of this research is to determine genetic affinities among mermthid nematodes that parasitize insects representing host taxa separated by large taxonomic distances. One strategy is to exploit comparative mitochondrial genomics and mitochondrial gene order determinations as markers for distantly related taxa. The work completed in this past year has greatly expanded our understanding of the relationships among the Mermithidae and refined the only published phylogeny of this parasitic nematode family. Studies on mitochondrial gene order identified a previously unknown host-parasite relationship, that of AGAMERMIS and its isopod host ARMIDILLIDIUM VULGARE. While this common pillbug host is not considered an insect pest, the ability to identify new host-parasite relationships using comparative mitochondrial genomics will unlikely expose new parasite-host associations that will be useful in biological control strategies. Assembling a comprehensive mermithid mtDNA phylogenetic tree will help identify relationships among these entomophilic nematodes that elaborate two useful characteristics: broad host-range for simultaneous control of multiple insect species and field persistence so that repetitious application of these nematodes to infested field sites is not needed. This knowledge will also be helpful in breeding programs using efficient crosses to combine useful traits, perhaps eventually creating a "full-service" parasitic nematode, and thus lower the cost of employing nematodes as biological control agents. Understanding the phylogenetic affinities among these nematodes may prove useful in choosing specific entomophilic 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 176:1139-1150.
- 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 (Hymenoptera: Formicidae) in Argentina. Systematic Parasitology, 68:115-128.
- Peat, S.M., Hyman, B.C., Adams, B.J. Phylogenetics and Population Genetics of Entomophilic and Entomopathogenic Nematodes. 2009. Molecular Approaches and Techniques for the Study of Insect Pathogens. Editors: P. Stock, N. Boemare, I. Glazer, J. Vandenberg. CABI International. In press.
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Progress 01/01/07 to 12/31/07
Outputs
This year, a more confined taxonomic unit within the nematode family Mermithidae, the genus Romanomermis, was targeted for understanding phylogenetic relationships and genetic variation, with the anticipation that this group might serve as a paradigm for the mermithid nematodes. Romanomermis culicivorax is an obligate parasitic nematode of mosquitoes that historically has been touted, and tested, as a biological control agent for insect pests. The parasite possesses an unusual mitochondrial genome in that it contains a 3 kilobase (kb) region that is duplicated multiple times within individual mitochondrial DNA (mtDNA) molecules, positioned both as direct tandem copies and as inverted copies in non-adjacent sections of the mtDNA. To understand the molecular evolution of this unique mitochondrial genomic architecture with the expectation of gaining insight into the evolution of mosquito parasitism within the genus, the complete mtDNA sequences of R. culicivorax and two
congeners, R. nielseni and R. iyenguri, were determined. While all three mitochondrial genomes contain sequence duplications, it is striking and unexpected that each of the mtDNAs encodes a different gene order despite the close phylogenetic affinities of these congeners, indicating evolution is proceeding rapidly in these representatives of the nematode family Mermithidae. Modeling of the mechanisms sponsoring gene rearrangements revealed that in R. culicivorax and R. iyenguri, mtDNA segments were first amplified, and later inversion events separated the duplicate copies and rearranged the order mitochondrial genes. An independent mechanism, termed the duplication/random-loss model, appeared to change the gene order within the R. nielseni mitochondrial genome. Four shared gene blocks were identified as possible ancestral features. However, the rampant gene rearrangement did not enable the determination of a gene order for the complete ancestral mitochondrial genome; achieving this
goal will require determining the complete mtDNA nucleotide sequences for additional representatives of the genus Romanomermis. When such a progenitor mitochondrial genome can be identified, it will likely be a descriptor of an ancient lineage within which Mermithid parasitism of mosquitoes arose.
Impacts
The wide-ranging goal of this research is to determine genetic affinities among mermthid nematodes that parasitize insects representing host taxa separated by large taxonomic distances. The work completed in this past year was confined to a small defined, taxonomic unit, the genus Romanomermis, which is a small portion of a much larger effort to map mitochondrial genomes throughout the Mermithidae. Assembling a comprehensive mermithid mtDNA phylogenetic tree will help identify relationships among these entomophilic nematodes that elaborate two useful characteristics: broad host-range for simultaneous control of multiple insect species and field persistence so that repetitious application of these nematodes to infested field sites is not needed. This knowledge will also be helpful in breeding programs using efficient crosses to combine useful traits, perhaps eventually creating a "full-service" parasitic nematode, and thus lower the cost of employing nematodes as
biological control agents. Understanding the phylogenetic affinities among these nematodes may prove useful in choosing specific entomophilic nematodes for targeted biological control programs.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
A new species of mermithid nematode, Allomermis solenopsii, (Mermithidae: Nematoda) is described from colonies of fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae) in Argentina. A molecular framework based on nuclear 18S ribosomal DNA sequences for the nematode family Mermithidae was first developed. The identification of A. solenopsii was then achieved with congruent results from independent molecular phylogenetic and morphological analyses. This report is the first record of Allomermis from South America and the first host record for this genus of mermithids. As this mermithid has potential as a biological control agent in an integrated control program of fire ants in the United States, rearing behavior under controlled laboratory conditions are described. Characterization of mitochondrial genomes from individual Thaumamermis cosgrovei nematodes, obligate parasites of the isopod Armadillidium vulgare, revealed that numerous mtDNA haplotypes, ranging in
size from 19 to 34 kb, are maintained in local populations. The magnitude and frequency of conspecific mtDNA size variation is unprecedented among all studied size polymorphic metazoan mitochondrial genomes. To understand the molecular basis of this hypervariation, complete nucleotide sequences of two T. cosgrovei mtDNA haplotypes were determined. A hypervariable segment, residing between the atp6 and rrnL genes, contributes exclusively to T. cosgrovei mtDNA size variation. Within this region, mtDNA coding genes and noncoding sequences have accumulated substitutions, and are duplicated and rearranged to varying extents. Hypervariation at this level has enabled a first insight into the life history of T. cosgrovei. When A. vulgare hosts are infected with multiple nematodes, individuals parasitizing the same isopod host are typically derived from the same maternal lineages, indicating hosts are multiply infected by ingesting a recently hatched egg clutch or become parasitized by
individuals from the same brood prior to dispersal in the soil.
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.
- 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 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, 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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