Application of real-time quantitative PCR assays for detecting marine Brucella spp. in fish

Brucella ceti and Brucella pinnipedialis have been documented as occurring in marine mammals, and B. ceti has been identified in 3 naturally acquired human cases. Seroconversion and infection patterns in Pacific Northwest harbor seals ( Phoca vitulina richardii) and North Atlantic hooded seals ( Cystophora cristata) indicate post-weaning exposure through prey consumption or lungworm infection, suggesting fish and possibly invertebrates play an epizootiologic role in marine Brucella transmission and possible foodborne risk to humans. We determined if real-time quantitative PCR (qPCR) assays can detect marine Brucella DNA in fish DNA. Insertion sequence (IS) 711 gene and sequence type (ST)27 primer–probe sets were used to detect Brucella associated with marine mammals and human zoonotic infections, respectively. First, DNA extracts from paired-species fish (containing 2 species) samples were tested and determined to be Brucella DNA negative using both IS 711 and ST27 primer–probe sets. A representative paired-species fish DNA sample was spiked with decreasing concentrations of B. pinnipedialis DNA to verify Brucella detection by the IS 711 primer–probe within fish DNA. A standard curve, developed using isolated DNA from B. pinnipedialis, determined the limit of detection. Finally, the IS 711 primer–probe was used to test Atlantic cod ( Gadus morhua) DNA extracts experimentally infected with the B. pinnipedialis hooded seal strain. In culture-positive cod tissue, the IS 711 limit of detection was ~1 genome copy of Brucella. Agreement between culture and PCR results for the 9 positive and 9 negative cod tissues was 100%. Although a larger sample set is required for validation, our study shows that qPCR can detect marine Brucella in fish.

DNA profiles to identify Dillenia species (Dilleniaceae) in Thailand

Surveying of the species throughout Thailand revealed 13 identified and one unidentified species including D. aurea, D. excelsa, D. grandifolia, D. hookeri, D. indica, D. obovata, D. ovata, D. parviflora, D. pentagyna, D. philippinensis, D. pulchella, D. reticulata, D. suffruticosa and Dillenia sp. They were phylogenetically examined based on RAPD profiles of 2,290 discrete characters including a monomorphic and 226 polymorphic characteristics. The phylogenetic relationships calculated from theses banding data show that the intraspecific genetic similarity (S) values ranged from 0.990 to 1.000, and the interspecific S values ranged from 0.520 to 0.790. The RAPD method can be effectively used in Dillenia study. However, its quality of unreproducible method, it cannot be reasonable used in any study on specific genes or areas. In additions, sequences from two molecular regions, rbcL gene and psbA-trnH spacer, were analyzed and determined for genetic distances. The rbcL gene sequences were rather ineffective, as all of the paired species displayed no or low genetic distance values. The psbA-trnH spacer sequences were rather effective, with only one pair (D. reticulata and D. parviflora) showing low genetic distance values. The other species pairs indicated rather far genetic distances, ranging from 0.006 (D. parviflora and D. grandifolia) to 0.376 (Dillenia specie and D. indica). Intraspecific genetic distance values ranged from 0–0.003 and 0–0.013 for rbcL and psbA-trnH spacer regions, respectively. Based on the results from both nucleotide variations and DNA fingerprinting, D. grandifolia and D. ovata were distinct Dillenia species.

Biology, Management, and Conservation of Lampreys in North America

<em>Abstract</em>.—In most lamprey genera, “paired” species exist in which the larvae (which are microphagous filter feeders) are morphologically similar but the adults differ dramatically, becoming parasitic on teleost fishes or nonparasitic (i.e., do not feed at all) following metamorphosis. Parasitic lampreys feed for several months to several years (either in their natal stream or after migrating to larger fresh or marine water bodies) before embarking on a nontrophic upstream migration, sexual maturation, and spawning (followed by death); nonparasitic lampreys eliminate the parasitic phase, begin sexual maturation toward the end of metamorphosis, and spawn and die within 6–10 months of metamorphosis. In each species pair, the reduction in the length of postlarval life in nonparasitic lampreys is generally accompanied by an increase in the length of the larval period (and size at metamorphosis) so that the evolution of nonparasitism appears to have occurred without a change in the overall life span. Rather, nonparasitism appears to have evolved as a result of a change in the timing of metamorphosis relative to the timing of sexual maturation. Conspicuous morphological (e.g., adult body size, relative eye and oral disk size) and histological (e.g., lack of a functional digestive tract) differences distinguish nonparasitic adults from parasitic forms, and most lamprey taxonomists recognize life history type as a species-specific characteristic. However, plasticity of feeding type (e.g., facultative parasitism) has been observed in some lamprey populations, and molecular data on a number of paired species show no genetic differentiation between sympatric species pairs and suggest a polyphyletic origin for several nonparasitic species. This paper reviews the paired species concept, the repeated and independent evolution of nonparasitism in different genera and even within species, the evidence for facultative parasitism or facultative nonparasitism in some lamprey species, and the potential for hybridization between paired species and attempts to answer the question, are brook lampreys “real” species? The tentative answer is that there likely is not a single answer for all lamprey species pairs; different species pairs represent speciation at different stages. Some pairs appear to be distinct species according to both the biological and phylogenetic species concepts (i.e., they are reproductively isolated and show reciprocal monophyly), although each is not necessarily fixed for feeding type. In contrast, other pairs may represent incipient speciation and others yet may be experiencing ongoing gene flow. Parallels are therefore drawn between different lamprey species pairs and the divergent life history types found in other animal taxa (e.g., echinoderms and amphibians) and other temperate fish species (e.g., anadromous and freshwater-resident salmonids).