Identification of weakfish Cynoscion (Gill) in the Bay of Panama with RFLP markers

A molecular approach, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), was developed to identify the planktonic larvae of Cynoscion species. Species-specific mitochondrial DNA markers were developed using three restriction endonucleases ( Dde I, Hae III and Hin fI). These markers permitted the accurate discrimination of the five Cynoscion species in the Bay of Panama.     

Targeting the rpoB gene using nested PCR-restriction fragment length polymorphism for identification of nontuberculous mycobacteria in hospital tap water

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and can cause nosocomial infections in immunocompromised patients. Recently the presence of NTM in public drinking water and hospital water distribution systems has been reported. Their ability to form biofilms and their resistance to chlorine both contribute to their survival and colonization in water distribution systems. Here we analyzed thirty-two hospital tap water samples that were collected from different locations in three hospitals so as to evaluate the prevalence of NTM species. The water samples were concentrated by membrane filtration and then eluted with sterilized water following sonication. Two-step direct PCR targeting the rpoB gene, restriction fragment length polymorphism (RFLP) using the MspI restriction enzyme, and sequence analysis were performed for identification of NTM to the species level. The sequences of each PCR product were analyzed using BLASTN. Seven samples (7/32, 21.9%) were positive for NTM as determined by nested-PCR. The PCR-RFLP results indicated five different patterns among the seven positive PCR samples. The water-born NTM were identified, including M. peregrinum, M. chelonae (2 cases), M. abscessus, M. gordonae (2 cases), and Mycobacterium sp. JLS. The direct two-step PCR-RFLP method targeting the rpoB gene was effective for the detection and the differentiation of NTM species from hospital tap water.     

Complex patterns of genetic variability in populations of the New World screwworm fly revealed by mitochondrial DNA markers

Abstract.  Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), the New World screwworm fly, is an important agent of traumatic myiasis, which is endemic in the Neotropical region and which has great economic impact on the development of the livestock industry. International efforts have been aimed at designing programmes to control and eradicate this species from endemic areas. Thorough knowledge of the population genetics of an insect pest is a fundamental component to ensuring the success of a pest management strategy because it enables the determination of an appropriate geographic scale for carrying out effective treatments. This study undertook an analysis of mtDNA polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) in 34 populations of C. hominivorax from 10 countries, encompassing almost all the current distribution of the species. Results showed high levels of mitochondrial DNA variability (π= 2.9%) and a complex pattern of population genetic structure for this species. Significant population structure (> st = 0.5234) and low variability were found in Caribbean populations, suggesting that, in general, islands constitute independent evolutionary entities connected by restricted gene flow. By contrast, high variability and low, but significant, differentiation was found among mainland populations (> st = 0.0483), which could not be attributed to geographic distance. Several processes may be acting to maintain the observed patterns, with different implications for establishing control programmes.     

Identification of screwworm species by polymerase chain reaction-restriction fragment length polymorphism

Abstract. Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR-RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera: Calliphoridae). Twenty-seven restriction enzymes were screened on five regions of mtDNA. Eleven restriction fragment length patterns differentiated New World screwworm, Cochliomyia hominivorax (Coquerel), from secondary screwworm, Cochliomyia macellaria (R). Five restriction fragment length patterns were polymorphic in C. hominivorax while all fragment patterns were fixed in C. macellaria. Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains. The PCR-RFLP technique is flexible with regard to developmental stage of the sample and method of preservation. We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults. PCR-RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less than 2.50.     

Grouping of isolates in AG 2 ofRhizoctonia solani by total cellular fatty acid analysis

Total-cellular fatty acid compositions of 34 isolates ofRhizoctonia solani belonging to intraspecific groups (ISGs) of anastomosis group (AG) 2, i.e., AG 2-1, AG 2-2 IIIB (mat rush), AG 2-2 IV (sugar beet), AG 2-2 LP (turfgrass), and AG 2–3 (soybean), were compared. The major fatty acids identified were palmitic, stearic, and oleic acids. Principal component analysis based on the percentage composition of total cellular fatty acids revealed consistently low variability among isolates of a single ISG of AG 2. Average linkage cluster analysis showed that isolates obtained from turfgrass representing a newly proposed group, AG 2-2 LP, were differentiated from other AG 2 ISGs. Isolates of another newly proposed group AG 2–3, from diseased soybean were also closely related to AG 2-1 and AG 2-2 IIIB but distinguishable from the AG 2-1 and AG 2-2 LP isolates by the average linkage cluster analysis. These results suggested that the percentage composition of total-cellular fatty acids is a distinct characteristic for the five ISGs belonging to AG 2, and fatty acid analysis is useful for the differentiation and characterization of these ISGs of AG 2 inR. solani.