Application of a dense genetic map for assessment of genomic responses to selection and inbreeding in Heliothis virescens.

Adaptation of pest species to laboratory conditions and selection for resistance to toxins in the laboratory are expected to cause inbreeding and genetic bottlenecks that reduce genetic variation. Heliothis virescens, a major cotton pest, has been colonized in the laboratory many times, and a few laboratory colonies have been selected for Bacillus thuringiensis (Bt) resistance. We developed 350-bp double-digest restriction-site associated DNA-sequencing (ddRAD-seq) molecular markers to examine and compare changes in genetic variation associated with laboratory adaptation, artificial selection and inbreeding in this nonmodel insect species. We found that allelic and nucleotide diversity declined dramatically in laboratory-reared H. virescens as compared with field-collected populations. The declines were primarily a result of the loss of low frequency alleles present in field-collected H. virescens. A further, albeit modest decline in genetic diversity was observed in a Bt-selected population. The greatest decline was seen in H. virescens that were sib-mated for 10 generations, in which more than 80% of loci were fixed for a single allele. To determine which regions of the genome were resistant to fixation in our sib-mated line, we generated a dense intraspecific linkage map containing three PCR-based and 659 ddRAD-seq markers. Markers that retained polymorphism were observed in small clusters spread over multiple linkage groups, but this clustering was not statistically significant. Overall, we have confirmed and extended the general expectations for reduced genetic diversity in laboratory colonies, provided tools for further genomic analyses and produced highly homozygous genomic DNA for future whole genome sequencing of H. virescens.

Divergent host preferences of above- and below-ground Culex pipiens mosquitoes and their hybrid offspring.

Culex pipiens form pipiens and Cx. pipiens form molestus (Diptera: Culicidae) belong to a cosmopolitan taxonomic group known as the Pipiens Assemblage. Hybridization between these forms is thought to contribute to human transmission of West Nile virus (WNV) in North America. Complementary choice and no-choice landing assays were developed to examine host acceptance by North American Cx. pipiens in the laboratory. Populations collected from above- and below-ground sites in suburban Chicago were identified as forms pipiens and molestus using a polymerase chain reaction-based assay. Avian and human host acceptance was then quantified for the two populations, as well as for their hybrid and backcross offspring. No-choice tests were used to demonstrate that both the pipiens and molestus forms were capable of feeding on human and avian hosts. Choice tests were used to demonstrate that form pipiens females were strongly avian-seeking; an individual's probability of accepting the chick host was 85%. Form molestus females were more likely to accept the human host (87%). Rates of host acceptance by F1 and backcross progeny were intermediate to those of their parents. The results suggest that host preferences in Cx. pipiens are genetically determined, and that ongoing hybridization between above- and below-ground populations is an important contributor to epizootic transmission of WNV in North America.

Application of a reverse dot blot DNA-DNA hydridization method to quantify host-feeding tendencies of two sibling species in the Anopheles gambiae complex.

A DNA-DNA hybridization method, reverse dot blot analysis (RDBA), was used to identify Anopheles gambiae s.s. and Anopheles arabiensis (Diptera: Culicidae) hosts. Of 299 blood-fed and semi-gravid An. gambiae s.l. collected from Kisian, Kenya, 244 individuals were identifiable to species; of these, 69.5% were An. arabiensis and 29.5% were An. gambiae s.s. Host identifications with RDBA were comparable with those of conventional polymerase chain reaction (PCR) followed by direct sequencing of amplicons of the vertebrate mitochondrial cytochrome b gene. Of the 174 amplicon-producing samples used to compare these two methods, 147 were identifiable by direct sequencing and 139 of these were identifiable by RDBA. Anopheles arabiensis bloodmeals were mostly (94.6%) bovine in origin, whereas An. gambiae s.s. fed upon humans more than 91.8% of the time. Tests by RDBA detected that two of 112 An. arabiensis contained blood from more than one host species, whereas PCR and direct sequencing did not. Recent use of insecticide-treated bednets in Kisian is likely to have caused the shift in the dominant vector species from An. gambiae s.s. to An. arabiensis. Reverse dot blot analysis provides an opportunity to study changes in host-feeding by members of the An. gambiae complex in response to the broadening distribution of vector control measures targeting host-selection behaviours.

Toxicity of bloodmeals from ivermectin-treated cattle to Anopheles gambiae s.l.

Two anthelmintic drugs used as cattle dewormers, ivermectin and moxidectin, were tested for their lethal and sublethal effects on the malarial vectors Anopheles gambiae s.s. and An. arabiensis. In the laboratory, direct addition of ivermectin to bovine blood reduced the survivorship and fecundity of mosquitoes fed on the blood. The median lethal concentration (LC(50)) of ivermectin in the bloodmeal, for the laboratory populations of An. gambiae s.l., was 19.8 ppb. In the field, commercially available formulations containing ivermectin or moxidectin were injected into cattle at three times the recommended dose. Most (90%) of the An. gambiae s.s. that fed on the ivermectin-treated cattle within 2 weeks of treatment failed to survive more than 10 days post-bloodmeal. No eggs were deposited by An. gambiae s.s. that fed on ivermectin-treated cattle within 10 days of treatment. In contrast, the survivorship and egg production of the mosquitoes that fed on the moxidectin-treated cattle were no different from those feeding on untreated cattle. These results indicate that treatment of cattle with ivermectin could be used, as part of an integrated control programme, to reduce the zoophilic vector populations that contribute to the transmission of the parasites responsible for human malaria.

Cost-effectiveness of cardiac troponin I in a systematic chest pain evaluation protocol: use of cardiac troponin I lowers length of stay for low-risk cardiac patients.

We evaluated several measures of clinical and fiscal interest to assess the effect of adding an automated cardiac troponin I (c-TnI) assay to our current cardiac panel, which consists of creatine kinase MB (CK-MB), myoglobin, total CK activity, and a calculated CK-MB relative index. Samples were collected on admission and at 3, 6, and 8 hours after admission as part of our diagnostic protocol. Our study was designed to collect data on a control group of patients, implement a change (i.e., c-TnI testing), and then measure the effect of the change on a test population having otherwise equivalent diagnostic and therapeutic pathways. We assessed differences in patient hospital and cardiac care unit length of stay (LOS), time to cardiac catheterization, and hospital and laboratory charges and costs. We found that adding c-TnI to our testing regimen decreased LOS for the large test population. Within this large test population, patients classified as low risk for acute myocardial infarction experienced statistically and clinically significant shorter LOS and lower total and variable hospital costs; for patients with unstable angina, there was an increase (though not statistically significant) in laboratory costs.