Combined Analyses of the ITS Loci and the Corresponding 16S rRNA Genes Reveal High Micro- and Macrodiversity of SAR11 Populations in the Red Sea

Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24°C throughout the year, and a remarkable uniform temperature (∼22°C) and salinity (∼41 psu) from the mixed layer (∼200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea’s water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium.     

A rapid and sensitive intracellular flow cytometric assay to identify Theileria parva infection within target cells

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.     

Some aspects of the biology of the stargazer mountain catfish,Amphilius uranoscopus (pfeffer); (Siluriformes: Amphiliidae) indigenous to Kenya streams

A study on some biological parameters of the mountain catfish, Amphilius uranoscopus Pfeffer 1889 (Silurifomes: Amphiliidae), was carried out in the Thego stream on the slopes of Mount Kenya from February to December 2002. Physical and chemical profiles of the Thego show that the water quality parameters is typical of high altitude streams with temperatures rarely exceeding 18°C, DO ranging from 7.9 to 8.2 mg l^?1 and relatively high conductivity (97–137 μS cm^?1) typical of perturbed lotic environments. A total of 1010 fish were caught by an electro‐fisher, with sizes ranging between 8 and 24 cm fork length. The population structure had a unimodal distribution with maxima at 14–16 cm. The length–weight relationship showed relatively narrow range in the slope ranging from 2.61 in April to 2.98 in February 2002, thereby suggesting isometric growth pattern. The fitted growth pattern of A. uranoscopus showed an asymptotic length (L_∞) of 28.5 cm and a growth curvature (K) of 0.56 year^?1 resulting in an estimated natural mortality coefficient (M) of 0.90 year^?1. The Fulton’s condition factor (K) was also relatively stable with a peak in April (0.92 ± 0.21) and lowest value in June (0.86 ± 0.10). As A. uranoscopus is not under commercial exploitation, the seemingly depressed population is possibly attributed to the introduced exotic rainbow trout that heavily predates on the species and environmental perturbations arising from changes in land use. The implications of such changes on A. uranoscopus are discussed.     

Fine-scale metabolic discontinuity in a stratified prokaryote microbiome of a Red Sea deep halocline

Deep-sea hypersaline anoxic basins are polyextreme environments in the ocean’s interior characterized by the high density of brines that prevents mixing with the overlaying seawater, generating sharp chemoclines and redoxclines up to tens of meters thick that host a high concentration of microbial communities. Yet, a fundamental understanding of how such pycnoclines shape microbial life and the associated biogeochemical processes at a fine scale, remains elusive. Here, we applied high-precision sampling of the brine–seawater transition interface in the Suakin Deep, located at 2770 m in the central Red Sea, to reveal previously undocumented fine-scale community structuring and succession of metabolic groups along a salinity gradient only 1 m thick. Metagenomic profiling at a 10-cm-scale resolution highlighted spatial organization of key metabolic pathways and corresponding microbial functional units, emphasizing the prominent role and significance of salinity and oxygen in shaping their ecology. Nitrogen cycling processes are especially affected by the redoxcline with ammonia oxidation processes being taxa and layers specific, highlighting also the presence of novel microorganisms, such as novel Thaumarchaeota and anammox, adapted to the changing conditions of the chemocline. The findings render the transition zone as a critical niche for nitrogen cycling, with complementary metabolic networks, in turn underscoring the biogeochemical complexity of deep-sea brines.Subject terms: Microbial ecology, Metagenomics     

Construction of a genetic map for Theileria parva: identification of hotspots of recombination

The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome-wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 variable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lymphocytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by assigning individual markers to the four chromosome genome, whose physical length is approximately 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62 Kb (∼93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22 cM Kb⁻¹ per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromosomes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks.     

Comprehensive Genomic Analyses of the OM43 Clade,Including a Novel Species from the Red Sea,Indicate Ecotype Differentiation among Marine Methylotrophs

The OM43 clade within the family Methylophilaceae of Betaproteobacteria represents a group of methylotrophs that play important roles in the metabolism of C₁ compounds in marine environments and other aquatic environments around the globe. Using dilution-to-extinction cultivation techniques, we successfully isolated a novel species of this clade (here designated MBRS-H7) from the ultraoligotrophic open ocean waters of the central Red Sea. Phylogenomic analyses indicate that MBRS-H7 is a novel species that forms a distinct cluster together with isolate KB13 from Hawaii (Hawaii-Red Sea [H-RS] cluster) that is separate from the cluster represented by strain HTCC2181 (from the Oregon coast). Phylogenetic analyses using the robust 16S-23S internal transcribed spacer revealed a potential ecotype separation of the marine OM43 clade members, which was further confirmed by metagenomic fragment recruitment analyses that showed trends of higher abundance in low-chlorophyll and/or high-temperature provinces for the H-RS cluster but a preference for colder, highly productive waters for the HTCC2181 cluster. This potential environmentally driven niche differentiation is also reflected in the metabolic gene inventories, which in the case of the H-RS cluster include those conferring resistance to high levels of UV irradiation, temperature, and salinity. Interestingly, we also found different energy conservation modules between these OM43 subclades, namely, the existence of the NADH:quinone oxidoreductase complex I (NUO) system in the H-RS cluster and the nonhomologous NADH:quinone oxidoreductase (NQR) system in the HTCC2181 cluster, which might have implications for their overall energetic yields.     

A standard tissue as a control for histochemical and immunohistochemical staining

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel^? and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.     

Soil moisture dynamics and restoration of self‐sustaining native vegetation ecosystem on an open‐cut coal mine

Post‐mining landscape reconstruction on open‐cut coal mines aims to support restoration of self‐sustaining native vegetation ecosystems that in perpetuity require no extra inputs relative to unmined analogs. Little is known about the soil moisture retention capacity of the limited layer of topsoil replaced (often <30 cm deep), impacts of deep ripping of the profile, and the combined impacts of these on plant available water during the mine restoration process. We examined changes in soil moisture parameters (soil water potential, Ψ, and soil water content, Θ) daily using automated soil sensors installed at 30 and 45–65 cm depths on mine restoration sites aged between 3 and 22 years and on adjacent remnant vegetation sites following heavy rainfall events at Meandu mine, southeast Queensland, Australia. Consistent patterns in soil moisture attributes were observed among rehabilitated sites with generally marked differences from remnant sites. Remnant site soil profiles had generally higher Θ after drying than rehabilitated sites and maintained high Ψ for extended periods after rain events. There was a relatively rapid decline of Ψ on reconstructed soil profiles compared with remnant sites although the times of decline onset varied. This response indicated that vegetation restoration sites released soil moisture more rapidly than remnant sites but the rate of drying decreased with increasing rehabilitation age and increased with increasing tree stem density. The rapid drying of mine rehabilitated sites may threaten the survival of some remnant forest species, limit tree growth, and delay restoration of self‐sustaining native ecosystem.     

Mimicry in plant-parasitic fungi

Mimicry is the close resemblance of one living organism (the mimic) to another (the model), leading to misidentification by a third organism (the operator). Similar to other organism groups, certain species of plant-parasitic fungi are known to engage in mimetic relationships, thereby increasing their fitness. In some cases, fungal infection can lead to the formation of flower mimics (pseudo flowers) that attract insect pollinators via visual and/or olfactory cues; these insects then either transmit fungal gametes to accomplish outcrossing (e.g. in some heterothallic rust fungi belonging to the genera Puccinia and Uromyces) or vector infectious spores to healthy plants, thereby spreading disease (e.g. in the anther smut fungus Microbotryum violaceum and the mummy berry pathogen Monilinia vaccinii-corymbosi). In what is termed aggressive mimicry, some specialized plant-parasitic fungi are able to mimic host structures or host molecules to gain access to resources. An example is M. vaccinii-corymbosi, whose conidia and germ tubes, respectively, mimic host pollen grains and pollen tubes anatomically and physiologically, allowing the pathogen to gain entry into the host's ovary via stigma and style. We review these and other examples of mimicry by plant-parasitic fungi and some of the mechanisms, signals, and evolutionary implications.