DNA barcoding distinguishes pest species of the black fly genus Cnephia (Diptera: Simuliidae).

Accurate species identification is essential for cost-effective pest control strategies. We tested the utility of COI barcodes for identifying members of the black fly genus Cnephia Enderlein (Diptera: Simuliidae). Our efforts focus on four Nearctic Cnephia species-Cnephia dacotensis (Dyar & Shannon), Cnephia eremities Shewell, Cnephia ornithophilia (Davies, Peterson & Wood), and Cnephia pecuarum (Riley)--the latter two being current or potential targets of biological control programs. We also analyzed one Palearctic species, Cnephia pallipes (Fries). Although Cnephia adults can be identified anatomically to species, control programs target the larval stage, which is difficult or impossible to distinguish morphologically. By using neighbor-joining, maximum parsimony, and Bayesian methods, we found that COI barcodes successfully identified three Nearctic Cnephia species, but not C. pecuarum. The Palearctic C. pallipes was also successfully identified. Despite nonmonophyly of C. pecuarum, we show that data from COI barcoding, in combination with geographical and ecological information, can be used to distinguish all four Nearctic species. Finally, we discussed 1) possible reasons for paraphyly in C. pecuarum, 2) topological concordance to previously reported chromosomal dendrograms, and 3) evolution of diverse feeding strategies within the genus Cnephia.

Molecular differentiation of two sibling species of the black fly Simulium vittatum (Diptera: Simuliidae) based on random amplified polymorphic DNA.

Larvae of the black fly morphospecies Simulium vittatum from Colorado, Montana, Nebraska, and New Hampshire were cytologically identified as either the IS-7 or the IIIL-1 cytospecies. DNA was PCR amplified from cytotyped larvae using eight 10-mer primers, labeled with 33P, and run on polyacrylamide gels. The entire data set of 96 amplicons produced incomplete separation of the two cytospecies when subjected to neighbor-joining and maximum parsimony analyses. However, when analyzed within geographical regions, separate species status was supported. Bootstrap support for distinctness of the two cytospecies was best in Colorado where they were collected in true sympatry. The IS-7 cytospecies was more polymorphic in the western states, where it differed most from IIIL-1, which was most variable in the eastern states. The frequencies of the 17 most common amplicons in the two cytospecies were inversely correlated. A model of speciation derived from the molecular evidence suggests that IS-7 evolved in the west and spread eastward, whereas IIIL-1 later originated in the east and spread westward.

Utility of the mitochondrial cytochrome oxidase II gene for resolving relationships among black flies (Diptera: simuliidae).

The complete mitochondrial cytochrome oxidase II gene was sequenced from 17 black flies, representing 13 putative species, and used to infer phylogenetic relationships. A midge (Paratanytarsus sp.) and three mosquitoes (Aedes aegypti, Anopheles quadrimaculatus, and Culex quinquefasciatus) were used as outgroup taxa. All outgroup taxa were highly divergent from black flies. Phylogenetic trees based on weighted parsimony (a priori and a posteriori), maximum likelihood, and neighbor-joining (log-determinant distances) differed topologically, with deeper nodes being the least well-supported. All analyses supported current classification into species groups but relationships among those groups were poorly resolved. The majority of phylogenetic signal came from closely related sister taxa. The CO-II gene may be useful for exploring relationships at or below the subgeneric level, but is of questionable value at higher taxonomic levels. The weighting method employed gave phylogenetic results similar to those reported by other authors for other insect CO-II data sets. A best estimate of phylogenetic relationships based on the CO-II gene is presented and discussed in relation to current black fly classification.

Greenbug (Homoptera: Aphididae) biotypes characterized using random amplified polymorphic DNA.

Genomic DNA was extracted from seven greenbug, Schizaphis graminum, biotypes (B, C, E, F, G, H and I) obtained from laboratory colonies maintained by USDA-ARS, Stillwater, Oklahoma. DNA was amplified using single 10-base primers. Of 100 primers tested, four were found which either alone, or in combination, distinguished all biotypes by distinct size differences in amplified fragments. Results were repeatable using aphids obtained from the same colonies 2 years later. These diagnostic primers produced unvarying banding patterns for all biotype E greenbugs collected in the field in Nebraska, Kansas, Oklahoma, and Texas.

Effects of preservation methods, parasites, and gut contents of black flies (Diptera: Simuliidae) on polymerase chain reaction products.

Molecular analysis of biological specimens usually requires extraction of high-molecular weight DNA free of foreign DNA contaminants. DNA was extracted from black flies at different life stages that had been preserved by 4 methods: larvae and adults in ethanol, larvae in Carnoy's solution, adults on card-points, and adults hand-swatted and sun-dried. Using specific primers for the mitochondrial ND4 gene, a 257-bp amplicon was obtained from specimens preserved by ethanol, card-point mounting, and sun-drying. Successful amplification often required DNA dilutions > or = 1:20 (< 1-10 ng). DNA from specimens preserved in Carnoy's solution (ethanol: acetic acid, 3:1) yielded degraded DNA, resulting in fewer successful amplications. Parasitic nematodes and, to a lesser extent, gut contents resulted in extra products when amplified with randomly amplified polymorphic DNA (RAPD) primers. Sufficient DNA was extracted from the head of a larva for a successful polymerase chain reaction (PCR), eliminating the need to remove the contaminating gut and parasites.

Molecular phylogeny and typing of blackflies (Diptera: Simuliidae) that serve as vectors of human or bovine onchocerciasis.

A subregion of the mitochondrial large subunit (16s) rRNA gene was amplified by polymerase chain reaction (PCR) from nine species of blackflies (Diptera: Simuliidae) which serve as natural or experimental vectors of human or bovine Onchocerca parasites. PCR products from each species of blackfly were tested by directed heteroduplex analysis (DHDA), and their genotypes established according to diagnostic banding patterns of the heteroduplex products. Three alleles of mitochondrial 16s rRNA were found to exist in members of the Simulium (Ewardsellum) damnosum sensu lato complex from West Africa, and two alleles were found in the Neotropical Simulium (Psilopelmia) ochraceum Walker complex and the Simulium (Simulium) metallicum Bellardi complex. Different single alleles were detected in Austrosimulium bancrofti, in English S.(S)noelleri and in two North American laboratory vectors: Simulium (Psilozia) vittatum Zetterstedt and S.(S.)decorum Walker. Phylogenetic analysis of 16s sequences indicated that blackflies from West Africa and the Americas formed distinct clades. Neotropical onchocerciasis vectors were found to be more closely related to Nearctic and Palaearctic non-vector Simulium species than to the African vectors of onchocerciasis.

Serious falls in hospitalized patients: correlates and resource utilization.

PURPOSE: To evaluate the correlates of serious falls in hospitalized patients and the resource utilization associated with such falls. PATIENTS AND METHODS: This retrospective case-control study was performed in an urban tertiary care hospital. The 62 cases included all inpatients with available charts who were reported as having fallen with a resulting fracture (n = 22), dislocation (n = 1), or laceration or hematoma (n = 39) after being admitted between January 1987 and March 1991. The 62 controls were matched by date of hospitalization (within 3 months), age (within 5 years), gender, and length of stay up to the time of the fall. RESULTS: Univariate correlates of falls (P < 0.05) included severity of underlying disease, Charlson comorbidity score, and Confusion Assessment Method (CAM) score. In multivariate conditional logistic regression analyses, only the Charlson index (P < 0.006) and the CAM score (P < 0.03) were independent correlates of a fall. Exposure to any of a number of drugs did not predict falls, but the power to detect drug effects was limited. A combination of the Charlson comorbidity and CAM scores identified a population at substantially increased risk of fall, including 50% (31/62) of fallers, versus 16% (10/62) of controls (odds ratio 5.2; 95% confidence interval, 2.4 to 12). In multivariate analyses, falls were also independently correlated with increases in length of stay (P < 0.004) and total charges (P < 0.008). Fallers stayed 12 days longer and had charges $4,233 higher than controls, after adjustment for potential clinical and nonclinical confounders. CONCLUSION: Falls during hospitalization are commoner in confused patients and those with greater comorbidity. This profile differs from that of fallers in the community, probably because hospitalized patients are sicker. Injurious falls are associated with substantially increased resource utilization.

The Simulium damnosum species complex: phylogenetic analysis and molecular identification based upon mitochondrially encoded gene sequences.

The DNA sequence of portions of the 16s rRNA and the NADH dehydrogenase subunit 4 (ND4) genes were used to determine phylogenetic relationships in the Simulium damnosum s.l. species complex. Results suggested that at least two major clades existed in the S. damnosum species complex, and that members of the S. damnosum s.l. species complex were not closely related to North American Simulium species. The sequence variability of the ND4 gene was exploited to develop a method to distinguish the sibling species of the S. damnosum s.l. species complex, based on directed heteroduplex analysis of PCR products derived from the ND4 gene. This method was capable of classifying the six sibling species into at least five groups.

How bad are bacteremia and sepsis? Outcomes in a cohort with suspected bacteremia.

BACKGROUND: To evaluate the short-term and long-term outcomes of patients with suspected bacteremia, we performed a prospective cohort study. METHODS: Clinical data were collected within 24 hours of initial culture from a random sample of 1516 episodes in which blood cultures were performed in an urban tertiary care hospital. One hundred forty-two patients with bacteremia were compared with two comparison groups: (1) 142 randomly selected patients with negative cultures, matched in age within 5 years, gender, severity of underlying disease, and presence of major comorbidity, and (2) all 155 patients with contaminant cultures. The main outcome measures were death, death secondary to bacteremia, and major complications. RESULTS: In the 439 patients, there were 142 deaths (32%), 114 at 1 year (26%) and 46 within 30 days (11%). Mortality at 30 days was most highly correlated with predicted fatality of underlying disease: 48% for the 65 patients with a rapidly fatal disease, 9% for the 156 patients with an eventually fatal disease, and 0.5% for the 217 patients with no fatal disease. In a Cox survival analysis, the risk ratio associated with bacteremia was 1.6 (95% confidence interval, 1.0 to 2.4) vs the comparison groups. When we performed time-dependent Cox analyses in which the hazard ratio was allowed to change at 30 days, we found that the risk ratios associated with bacteremia were 2.3 (95% confidence interval, 1.2 to 4.4) for the first 30 days, and 1.3 (95% confidence interval, 0.76 to 2.1) after 30 days. CONCLUSIONS: We conclude that this population has a high mortality, which is strongly correlated with severity of underlying disease. Short-term mortality was higher in patients with bacteremia even after controlling for severity of illness, but the increase in risk was present only during the first month and most deaths occurred in patients with a rapidly fatal disease.

Mitochondrial transfer RNA genes in a black fly, Simulium vittatum (Diptera: Simuliidae), indicate long divergence from mosquito (Diptera: Culicidae) and fruit fly (Diptera: Drosophilidae).

Sequences are given for nine complete genes and one partial mitochondrial tRNA gene of the black fly, Simulium vittatum (Zetterstedt). Sequenced tRNA genes were for alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, leucine(CUN), lysine, serine(AGN), and valine. Nucleotides were aligned with the same previously sequenced genes in Aedes albopictus Skuse and Drosophila yakuba Burla. A cluster of six tRNA genes, which differ in arrangement in Ae. albopictus and D. yakuba, was amplified by PCR and found to have the same position and orientation in S. vittatum as in D. yakuba. Overall, similarity with either D. yakuba or Ae. albopictus was 86%. Sequences that were common to the three insects suggest that black flies and mosquitoes are as divergent from each other as either is from Drosophila. Sequences for nine species of black flies were obtained for tRNA leucine(CUN) from DNA amplified with another primer set. Little variation occurred within the tRNA gene but, by including the flanking regions to provide 175 base pairs, a phylogeny of the nine species was obtained that was largely consistent with current classification.