Petersen and removal population size estimates: combining methods to adjust and interpret results when assumptions are violated

Synopsis We present ways to test the assumptions of the Petersen and removal methods of population size estimation and ways to adjust the estimates if violations of the assumptions are found. We were motivated by the facts that (1) results of using both methods are commonly reported without any reference to the testing of assumptions, (2) violations of the assumptions are more likely to occur than not to occur in natural populations, and (3) the estimates can be grossly in error if assumptions are violated. We recognize that in many cases two days in the field is the most time fish biologists can spend in obtaining a population estimate, so the use of alternative models of population estimation that require fewer assumptions is precluded. Hence, for biologists operating with these constraints and only these biologists, we describe and recommend a two-day technique that combines aspects of both capture-recapture and removal methods. We indicate how to test: most of the assumptions of both methods and how to adjust the population estimates obtained if violations of the assumptions occur. We also illustrate the use of this combined method with data from a field study. The results of this application further emphasize the importance of testing the assumptions of whatever method is used and making appropriate adjustments to the population size estimates for any violations identified.     

Production and characterization of monoclonal antibodies to rabbit lymphocyte subpopulations. I. Tissue immunofluorescence and flow cytometric analysis

Nine monoclonal antibodies to rabbit T cells and B subpopulations have been generated from three separate fusions of spleen cells from mice immunized with fractionated populations of rabbit lymphocytes. These monoclonal antibodies, as well as a previously described rabbit T cell monoclonal antibody, 9AE10, have been analyzed by immunofluorescence staining on frozen tissue sections of rabbit thymus, spleen, and appendix. This screening method permits rapid identification of the lymphocyte subdomains in each tissue which is not possible by other screening methods. Each monoclonal antibody selected has a unique tissue staining pattern. Flow cytometric analysis of these monoclonal antibodies, using indirect immunofluorescence techniques on thymocytes, splenocytes, and PBL, revealed varying percentages of positive cells and individual mean fluorescence intensities indicating different epitope densities for each antigen. These monoclonal antibodies are now being used to characterize normal lymphocyte function and the role of specific lymphocyte subpopulations in experimental disease models in the rabbit.     

Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter

The enterobactin system for iron transport in Escherichia coli is well characterized with the exception of the mechanism of enterobactin secretion to the extracellular environment. Escherichia coli membrane protein P43, encoded by ybdA in the chromosomal region of genes involved in enterobactin synthesis, shows strong homology to the 12-transmembrane segment major facilitator superfamily of export pumps. A P43-null mutation was created and siderophore nutrition assays, performed with a panel of E. coli strains expressing one or more outer membrane receptors for enterobactin-related compounds, demonstrated that the P43 mutant was unable to secrete enterobactin efficiently. Products released from the mutant strain were identified with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that the P43 mutant secretes little, if any, enterobactin, but elevated levels of enterobactin breakdown products 2,3- dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer. These data establish that P43 is a critical component of the E. coli enterobactin secretion machinery and provides a rationale for the designation of the previous genetic locus ybdA as entS to reflect its relevant biological function.     

Junker: An Intergenic Explorer for Bacterial Genomes

In the past few decades, scientists from all over the world have taken a keen interest in novel functional units such as small regulatory RNAs, small open reading frames, pseudogenes, transposons, integrase binding attB/attP sites, repeat elements within the bacterial intergenic regions (IGRs) and in the analysis of those junk regions for ge- nomic complexity. Here we have developed a web server, named Junker, to facilitate the in-depth analysis of IGRs for examining their length distribution, four-quadrant...     

Evidence for siderophore-dependent iron acquisition in group B streptococcus

Mutagenesis of group B streptococcus (GBS) with TnphoZ, a transposon designed to identify secreted protein genes, identified the gene homologues fhuD and fhuG. The encoded proteins participate in siderophore (hydroxamate)-dependent iron(III) transport in other bacterial species. Sequence analysis of the genome determined that fhuD and fhuG are members of a polycistronic operon comprised of four genes, fhuCDBG, that encode a putative ATPase, cell surface receptor and two transmembrane proteins respectively. We hypothesized that FhuD was a siderophore receptor. Western analysis of cell extracts localized FhuD to the bacterial cell membrane. Fluorescence quenching experiments determined that purified FhuD bound hydroxamate-type siderophores. FhuD displayed highest affinity for iron(III)-desferroxamine, with a K(D) (microM) = 0.05, identical to that described for FhuD2 from Staphylococcus aureus. The role of Fhu in siderophore-iron transport was also characterized. A fhu mutant, ACFhu1, was equally sensitive to the iron-dependent antibiotic streptonigrin as the wild-type strain, suggesting that ACFhu1 was not reduced for intracellular iron concentrations in the absence of exogenous siderophore. However, ACFhu1 transported significantly less siderophore-bound iron in (55)Fe accumulation assays. These data provide the first evidence of siderophore-mediated iron acquisition by GBS.     

Role of CheB and CheR in the complex chemotactic and aerotactic pathway of Azospirillum brasilense

It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.     

Production of viruses during a spring phytoplankton bloom in the South Pacific Ocean near of New Zealand

Lagrangian studies of virus activity in pelagic environments over extended temporal scales are rare. To address this, viruses and bacteria were examined during the course of a natural phytoplankton bloom in the pelagic South Pacific Ocean east of New Zealand. Daily samples were collected in a mesoscale eddy from year days 263-278 (September 19th-October 4th, 2008). The productive bloom transitioned from a diatom to a pico- and nanoplankton-dominated system, resulting in chlorophyll a concentrations up to 2.43?μg?L(-1) . Virus abundances fluctuated c.?10-fold (1.8?×?10(10) -1.3?×?10(11) L(-1) ) over 16?days. The production rates of virus particles were high compared with those reported in other marine systems, ranging from 1.4?×?10(10) to 2.1?×?10(11) L(-1) day(-1) . Our observations suggest viruses contributed significantly to the mortality of bacteria throughout the bloom, with 19-216% of the bacterial standing stock being lysed daily. This mortality released nutrient elements (N, Fe) that likely helped sustain the bloom through the sampling period. Parametric analyses found significant correlations with both biotic (e.g. potential host abundances) and abiotic parameters (e.g. nutrient concentrations, temperature). These observations demonstrate that viruses may be critical in the extended maintenance of regeneration-driven biological production.