Three pupfish (Cyprinodon) morphotypes (two endemic) occur in some of the young (6000 ypb) saline lakes on the Bahamian island of San Salvador. The 'normal' morph, a detritivore/omnivore, is not different in its general features from Cyprinodon variegatus from other Bahamian islands. 'Bulldog' is a scale-eater/piscivore that preys upon normal pupfish, and 'bozo' is a specialized molluskivore. Reproductive isolation among these morphs is not predicted by the evolutionary biology of congeneric species because sympatry of even morphogically and ecologically quite divergent pupfishes has usually resulted in hybridization/introgression. Survey of variation at eight microsatellite loci reveals that sympatric normal and bulldog populations are genetically distinctive by several criteria, and are therefore likely reproductively isolated. The bulldog morph in Crescent Pond is markedly divergent from those in Little Lake and Osprey Lake, a finding consistent with, although it does not prove, separate parallel origins of this morphotype. The data also suggest that the bulldogs in the latter two lakes did not evolve by intralacustrine speciation from the current sympatric normal populations. Some of the genetic data suggest that the bozo morph may also be reproductively isolated from the other two pupfishes, but only a small, pooled sample of this rare morphotype was available, and the issue is not resolved. Isolating mechanisms between bulldog and normal morphs are of special interest because of the possibility that they arose as a consequence of a predator-prey relationship. A strong correlation between reproductive isolation and predator-prey interactions could provide an important example of ecological speciation via direct selection against heterotypic interactions.

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor-and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.If a biothreat agent was released, hundreds to thousands of environmental samples of diverse types would need to be rapidly processed and analyzed in order to first characterize the contamination of the site and then assess the effectiveness of decontamination activities. Decision-makers also need rapid results for remobilizing disinfection equipment in the case of incomplete decontamination and for reopening facilities and areas based on results from clearance sampling (12)(13)(14).Current methods used by the Centers for Disease Control and Prevention (CDC) to assess the viability of spores on surfaces rely on culturing samples on solid media (5, 6). These methods involve several manual steps, including pipetting to prepare dilution series, plating of numerous replicates for a series of dilutions, and colony counting, which make it labor-, space-, and time-intensive. Typically, only 30 to 40 samples may be processed each day with confirmed results obtained days later (5, 6). Validated rapid-viability test protocols are therefore needed to ensure public safety and to help mitigate impacts due to facility closures following a biothreat agent release. This critical need was highlighted during the response to the 2001 anthrax attacks, in which clearance sampling and analysis required excessive time prior to facilities reopening.Because risk assessment after such an attack is determined on the basis of the presence of viabl...

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