Molecular Diagnostics of Three \u3ci\u3eDiabrotica\u3c/i\u3e (Coleoptera: Chrysomelidae) Pest Species

A 257 bp region of the mitochondrial ND4 gene was analyzed for genetic variation in three species of corn rootworm, southern corn rootworm (Diabrotica undecim punctata howardi Barber, SCR), northern corn rootworm (D. barberi Smith and Lawrence, NCR), and western corn rootworm (D. virgifera virgifera LeConte, WCR). Nucleotide sequencing revealed 26 polymorphic sites. Genetic distances averaged 8% for all pair-wise comparisons among the three species. Restriction maps were constructed from sequence data and compared to potential species specific restriction sites. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) revealed three restriction enzymes (Alu I, Apo I and Sau 3A) which produced diagnostic patterns for both adults and larvae. Only NCR showed intraspecific polymorphism

Molecular Diagnostics of Three \u3ci\u3eDiabrotica\u3c/i\u3e (Coleoptera: Chrysomelidae) Pest Species

A 257 bp region of the mitochondrial ND4 gene was analyzed for genetic variation in three species of corn rootworm, southern corn rootworm (Diabrotica undecim punctata howardi Barber, SCR), northern corn rootworm (D. barberi Smith and Lawrence, NCR), and western corn rootworm (D. virgifera virgifera LeConte, WCR). Nucleotide sequencing revealed 26 polymorphic sites. Genetic distances averaged 8% for all pair-wise comparisons among the three species. Restriction maps were constructed from sequence data and compared to potential species specific restriction sites. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) revealed three restriction enzymes (Alu I, Apo I and Sau 3A) which produced diagnostic patterns for both adults and larvae. Only NCR showed intraspecific polymorphism

An 18S rDNA Perspective on the Classification of Criconematoidea

In the nematode family Criconematidae, a taxonomy primarily based on cuticle characters has created classifications that are notoriously volatile. Molecular characters may lead to their stabilization. A phylogenetic tree of Criconematoidea was constructed using 166 new near full-length 18S rDNA sequences and 58 sequences from GenBank. Bayesian and maximum likelihood (ML) analyses produced trees with similar topologies. Major features include a strongly supported clade that includes Criconematidae and Hemicycliophoridae, excluding Paratylenchidae and Tylenchulidae. Another well-supported clade groups Criconema, Ogma, Crossonema, and Hemicriconemoides plus Xenocriconemella, combining nematodes with cuticular scales with those without scales at any life stage. Mesocriconema, Discocriconemella limitanea, Hemicaloosia, and Lobocriconema are recognized as monophyletic groups, but Criconemoides is paraphyletic. Both trees support an unexpected sister relationship between Bakernema and Hemicycliophora. The 18S rDNA dataset was insufficient for distinguishing genus boundaries between Criconema, Ogma, and Crossonema. The relationships depicted by the 18S rDNA phylogeny suggest that key morphological characters used in the classification of Criconematidae are not homologous

Morphological and Molecular Characterization of \u3ci\u3eGracilacus wuae\u3c/i\u3e n. sp. (Nematoda: Criconematoidea) Associated with Cow Parsnip (\u3ci\u3eHeracleum maximum\u3c/i\u3e) in Ontario, Canada

Gracilacus wuae n. sp. from soil associated with cow parsnip in Ontario, Canada is described and illustrated. Morphologically, females have a long stylet ranging from 80 to 93 mmlong, the lip region not offset from the body contour, without lateral lips but with large and flat submedian lobes, the mouth opening slit-like elongated laterally and surrounded by lateral flaps, the excretory pore is anterior to the knobs of the stylet; males without stylet and the pharynx degenerated. The fourth-stage juveniles lack a stylet, the pharynx degenerated, and can be differentiated into preadult females and males based on the position of the genital primordia. The third-stage juveniles are similar to females but smaller. Phylogenetic studies using the rDNA small subunit 18S, large subunit 28S D2/D3, and internal transcribed spacer (ITS) sequences collectively provide evidence of a grouping with other Gracilacus and some species of Paratylenchus with stylet length of females longer than 41 mm deposited in GenBank

Morphological and Molecular Characterization of \u3ci\u3eGracilacus wuae\u3c/i\u3e n. sp. (Nematoda: Criconematoidea) Associated with Cow Parsnip (\u3ci\u3eHeracleum maximum\u3c/i\u3e) in Ontario, Canada

Gracilacus wuae n. sp. from soil associated with cow parsnip in Ontario, Canada is described and illustrated. Morphologically, females have a long stylet ranging from 80 to 93 mmlong, the lip region not offset from the body contour, without lateral lips but with large and flat submedian lobes, the mouth opening slit-like elongated laterally and surrounded by lateral flaps, the excretory pore is anterior to the knobs of the stylet; males without stylet and the pharynx degenerated. The fourth-stage juveniles lack a stylet, the pharynx degenerated, and can be differentiated into preadult females and males based on the position of the genital primordia. The third-stage juveniles are similar to females but smaller. Phylogenetic studies using the rDNA small subunit 18S, large subunit 28S D2/D3, and internal transcribed spacer (ITS) sequences collectively provide evidence of a grouping with other Gracilacus and some species of Paratylenchus with stylet length of females longer than 41 mm deposited in GenBank

Morphological and Molecular Characterization of \u3ci\u3eDiscocriconemella inarata\u3c/i\u3e, an Endemic Nematode from North American Native Tallgrass Prairies

Discocriconemella inarata, a plant parasitic nematode species originally discovered in a virgin tallgrass prairie in northwest Iowa, was re-examined by molecular and morphological analyses of topotype material. This species has never been recorded in cultivated fields and could potentially serve as an indicator for high quality prairie habitats. DNA sequence from a conserved 3’ portion of the 18S ribosomal gene exhibited an identical match between D. inarata topotype specimens and topotype specimens of Mesocriconema xenoplax from Fresno, California. Higher resolution sequence analyses using the internal transcribed spacer 1 (ITS1) and a portion of the mitochondrial gene cytochrome b (cytb) allowed discrimination of D. inarata apart from M. xenoplax. This pair of species formed a well-supported clade with other Mesocriconema species exclusive of tropical Discocriconemella species. Scanning electron microscopy confirmed the absence of submedian lobes on D. inarata, suggesting a secondary loss of this defining morphological characteristic for Mesocriconema. Observations and measurements of D. inarata juveniles were added for the first time. Surveys of other prairies within the Great Plains expanded the known distribution of this species

Characterization of Heterorhabditis isolates by PCR amplification of segments of mtDNA and rDNA genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping

Ecological and morphological differentiation among COI haplotype groups in the plant parasitic nematode species \u3ci\u3eMesocriconema xenoplax\u3c/i\u3e

DNA barcoding with the mitochondrial COI gene reveals distinct haplotype subgroups within the monophyletic and parthenogenetic nematode species, Mesocriconema xenoplax. Biological attributes of these haplotype groups (HG) have not been explored. An analysis of M. xenoplax from 40 North American sites representing both native plant communities and agroecosystems was conducted to identify possible subgroup associations with ecological, physiological, or geographic factors. A dataset of 132 M. xenoplax specimens was used to generate sequences of a 712 bp region of the cytochrome oxidase subunit I gene. Maximum-likelihood and Bayesian phylogenies recognized seven COI HG (≥99/0.99 posterior probability/bootstrap value). Species delimitation metrics largely supported the genetic integrity of the HG. Discriminant function analysis of HG morphological traits identified stylet length, total body length, and stylet knob width as the strongest distinguishing features among the seven groups, with stylet length as the strongest single distinguishing morphological feature. Multivariate analysis identified land cover, ecoregion, and maximum temperature as predictors of 53.6% of the total variation (P = 0.001). Within land cover, HG categorized under “herbaceous,” “woody wetlands,” and “deciduous forest” were distinct in DAPC and RDA analyses and were significantly different (analysis of molecular variance P = 0.001). These results provide empirical evidence for molecular, morphological, and ecological differentiation associated with HG within the monophyletic clade that represents the species Mesocriconema xenoplax

Characterization of \u3ci\u3eHeterorhabditis\u3c/i\u3e Isolates by PCR Amplification of Segments of mtDNA and rDNA Genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping

Distribution of Soybean Cyst Nematode in Nebraska

A survey of 552 soybean fields in 20 counties in Nebraska in 1986-88 revealed 35 fields infested with the soybean cyst nematode (SCN), Heterodera glycines. Identification was confirmed with a greenhouse bioassay, using \u27Lee 74\u27 soybean, and by the application of a DNA hybridization probe derived from SCN mitochondrial DNA. Most of the SCN-infested fields were located on the Missouri River floodplain and in the southeastern corner of the state