Lessons learned from microsatellite development for nonmodel organisms using 454 pyrosequencing.

Microsatellites, also known as simple sequence repeats (SSRs), are among the most commonly used marker types in evolutionary and ecological studies. Next Generation Sequencing techniques such as 454 pyrosequencing allow the rapid development of microsatellite markers in nonmodel organisms. 454 pyrosequencing is a straightforward approach to develop a high number of microsatellite markers. Therefore, developing microsatellites using 454 pyrosequencing has become the method of choice for marker development. Here, we describe a user friendly way of microsatellite development from 454 pyrosequencing data and analyse data sets of 17 nonmodel species (plants, fungi, invertebrates, birds and a mammal) for microsatellite repeats and flanking regions suitable for primer development. We then compare the numbers of successfully lab-tested microsatellite markers for the various species and furthermore describe diverse challenges that might arise in different study species, for example, large genome size or nonpure extraction of genomic DNA. Successful primer identification was feasible for all species. We found that in species for which large repeat numbers are uncommon, such as fungi, polymorphic markers can nevertheless be developed from 454 pyrosequencing reads containing small repeat numbers (five to six repeats). Furthermore, the development of microsatellite markers for species with large genomes was also with Next Generation Sequencing techniques more cost and time-consuming than for species with smaller genomes. In this study, we showed that depending on the species, a different amount of 454 pyrosequencing data might be required for successful identification of a sufficient number of microsatellite markers for ecological genetic studies.

Castrating parasites and colonial hosts.

Trajectories of life-history traits such as growth and reproduction generally level off with age and increasing size. However, colonial animals may exhibit indefinite, exponential growth via modular iteration thus providing a long-lived host source for parasite exploitation. In addition, modular iteration entails a lack of germ line sequestration. Castration of such hosts by parasites may therefore be impermanent or precluded, unlike the general case for unitary animal hosts. Despite these intriguing correlates of coloniality, patterns of colonial host exploitation have not been well studied. We examined these patterns by characterizing the responses of a myxozoan endoparasite, Tetracapsuloides bryosalmonae, and its colonial bryozoan host, Fredericella sultana, to 3 different resource levels. We show that (1) the development of infectious stages nearly always castrates colonies regardless of host condition, (2) castration reduces partial mortality and (3) development of transmission stages is resource-mediated. Unlike familiar castrator-host systems, this system appears to be characterized by periodic rather than permanent castration. Periodic castration may be permitted by 2 key life history traits: developmental cycling of the parasite between quiescent (covert infections) and virulent infectious stages (overt infections) and the absence of germ line sequestration which allows host reproduction in between bouts of castration.

Efficiency of liming in controlling the mobility of lead in shooting range soils as assessed by different experimental approaches.

Shooting range soils contaminated by lead (Pb) are a great environmental risk. Reducing mobility and leaching of Pb by liming, for example, has produced contradictory results. This laboratory study compares the efficiency of two liming agents differing in their reactivity, CaCO(3) and blast furnace slag (BFS), in diminishing the mobility of Pb. In a batch test, contaminated humic soil samples were incubated in closed vessels without and with liming materials added in quantities to correspond additions of 5 t ha(-1). Water soluble Pb (Pb(w)), dissolved organic carbon (DOC), pH and substrate induced respiration (SIR) in soils were monitored for 21 days. In the experiment carried out with freely drained vessels, contaminated humic soil treated without and with liming agents was leached five times during the experimental period of 141 days. Leachates were analyzed for pH, DOC and Pb. At the end of the experiment, soil samples were analyzed for pH, DOC, Pb(w), and SIR. In both systems, CaCO(3) raised pH and DOC more than BFS. The liming agents did not significantly differ in their effect on Pb chemistry. Neither had any effect on SIR: however, liming agents markedly reduced the leaching of Pb in the open system, while in the closed system they increased rather than reduced the extractability of Pb. Incubation in a closed vessel proved not to be a suitable experimental system for Pb mobility estimation, since the ionic strength may be raised to abnormal levels, resulting from accumulated reaction products of liming agents.

Effect of organic amendment and plant roots on the solubility and mobilization of lead in soils at a shooting range.

Lead (Pb) dissolving gradually from spent pellets constitutes a serious environmental risk in and near shooting ranges, and remediation measures are necessary to prevent its movement to deeper soil layers and ground water. In this study, the effectiveness of organic amendment and plant roots in stabilizing Pb was assessed in a microcosm experiment. Planted (Scots pine, Pinus sylvestris L.) and unplanted microcosms consisting of coarse-textured mineral soil covered with Pb-contaminated humic topsoil were coated with uncontaminated peat layers of 1 to 3 cm and incubated for 77 d. In a percolation test, the microcosms were washed with ultra pure water to simulate heavy rain so as to rinse water-soluble lead (Pbw) from the topsoil layer. Although Pbw remained below detection limits in the mineral soils in all test units, acid-soluble lead (Pba) increased. Peat amendment diminished Pba in the mineral soil layer, this effect being more pronounced in planted soils, indicating that Pb was taken up by the plants. The percolation test showed that the effect of Scots pine seedlings on Pb movement was minor when peat was added. A long-term dissolution test revealed that considerably more Pb was released from old pellets into soil extracts than from new ones, whereas only traces of Pb, if any, were dissolved in sterilized pure water.

Seasonality of gestational weight gain and foetal growth in rural Malawi.

OBJECTIVE: To study seasonal variation of maternal anthropometry and newborn weights. DESIGN: A retrospective descriptive study from 1995 to 1998. SETTING: Lungwena, a rural health centre in Malawi, southeast Africa. SUBJECTS: One thousand and thirty two women with singleton pregnancy, a minimum of eight weeks antenatal follow-up and a baby who was weighed in the first month of life. Maternal data were sought from a health centre antenatal register and linked with infant information collected from a newborn clinic. RESULTS: The mean (SD) gestational weight and fundal height gains among the pregnant women were 240 (200) grams and 0.9 (0.2) cm/week and the mean (SD) newborn weight 3360 (530) grams. Gestational weight gains followed a seasonal pattern and they were typically highest among women delivering in the third quarter of the year (mean gain 250-300 g/week) and lowest among those delivering in January-May (mean gain 100-200 g/week) (p < 0.001 for seasonality, ANOVA). For maternal fundal height gains and newborn weights, seasonality was less obvious (but statistically significant, p < 0.05, ANOVA) and its pattern was influenced by year of study. On average, newborn weights peaked in the last quarter (mean 3350-3400 grams) and nadired during the second quarter (mean 3200-3300 grams). There was only a modest correlation between maternal weight gain in pregnancy and the weight of her newborn (Pearson's correlation coefficient 0.13). CONCLUSION: In rural Malawi, maternal weight gains during pregnancy are more strongly associated with season than fundal height gains or newborn weights. In adverse environmental conditions, foetal growth ismaintained at least partially at the expense of mother's nutritional status.

Assessment of the risk of phosphorus loading due to resuspended sediment.

Resuspension is a multiphase phenomenon where suspended solids encounter water layers differing in physico-chemical properties that affect the reactions of phosphorus (P). The role of resuspended sediment as a sink or source of dissolved P was determined in a laboratory study of P desorption-sorption equilibria. Gradual mixing was simulated using decreasing solid concentrations and varying environmental conditions (pH, redox, ionic strength). To describe the P exchange when the particles encounter dissimilar water layers, the extent of P sorption to or desorption from solids was expressed as a function of P concentration in the bath solutions. The equilibrium phosphorus concentration (EPC), at which there is no net P release from or retention to the particles, proved to be a suitable parameter for assessment of P load risk. Under oxic conditions at pH 7, commonly prevailing in lakes, the EPC values ranged from 11 to 27 microg P L(-1). The larger the water volume the suspended material was mixed with, the higher the P concentration, allowing desorption to occur. As for chemical factors affecting P mobilization, EPC followed the order: pH 7 < pH 7 anoxic < pH 9. A separate extraction experiment revealed that elevated pH enhanced P mobilization more as the concentration of solids decresed. The results demonstrate that high pH (a common characteristic in eutrophic lakes during summer), when linked with intensive resuspension, may markedly increase the internal P loading risk. As for the risk assessment, the quantification of the internal P loading would be improved by isotherm studies combined with field observations.

Phosphorus mobilization from various sediment pools in response to increased pH and silicate concentration.

Phosphorus (P) release from sediment particles to the interstitial water has been studied extensively, but the contribution of different inorganic P pools in sediment under differing environmental conditions is not fully understood. This study was undertaken to get more detailed information about the chemical mobilization mechanisms. Phosphorus mobilization from reserves bound by Al, Fe, and Ca compounds in response to increased pH and to inorganic silicon (Si) enrichments was investigated using a sequential fractionation analysis and an isotope-labeling technique. The aerobic sediment of Lake Vesijärvi had a high P retention capacity, and Fe-bound P was the largest inorganic P pool as well as the main source of released P. High Si addition (47 mg Si L-1 sediment) released more P to the interstitial water than did the elevation of pH from 6.6 to 9.5, since Si lowered the resorption of released P onto hydrated Al oxides. This finding reveals that P equilibrium between Fe-bound and Al-bound P in sediments regulates P net mobilization to the interstitial water under aerobic conditions. Furthermore, elevated pH combined with high Si enrichment had a positive synergistic effect, resulting in the most substantial P mobilization. This synergism may cause a self-fueled increase in the internal loading of P. It accentuates the effect of diatom sedimentation on P fluxes in eutrophic lakes with high pH and may favor the appearance of bloom-forming cyanobacteria.

The role of bacteria in the nutrient exchange between sediment and water in a flow-through system.

The contribution of bacteria to phosphorus (P) and nitrogen (N ) release from, or retention in, sediment was studied in a flow-through system. "Live" and formaldehyde-"killed" sediment communities were incubated in 25-liter bottles with a continuous flow of P- or P + N-enriched water. Sediment bacteria in the killed communities were inhibited by adding formaldehyde (final concentration 0.04% v/v) to the sediment before the start of the experiment. Bacterial activity in the live sediments measured with [(3)H]thymidine and [(14)C]leucine incorporation techniques did not change essentially during the experiment period (7-8 days). Chemical mechanisms were found to be of principal importance in PO4-P retention in the sediment. In the live samples, the net retention of PO4-P was lower than in the killed samples, which was likely due to the reduced O2 conditions in the sediment as a consequence of bacterial mineralization. In total P exchange, however, bacteria increased the retention rate by recycling dissolved organic P in the sediment. In the live communities the retention of N was very efficient, and all the introduced NH4 -N and NO3-N was immobilized by sediment bacteria. Nitrogen enrichment, however, did not alter the P exchange rates. The gradual emergence of bacterial activity (and grazing) in the killed communities, subsequent to the dilution of formaldehyde concentration, enhanced the release of PO4-P and NH4-N from sediment.

Comparison of methods for inhibiting bacterial activity in sediment.

Dilute formaldehyde was the most suitable treatment to inhibit sediment bacteria, since bacterial activity remained low during long-term incubations and the chemical changes in the sediment were minimal. The inhibiting effects of HgCl(2), autoclaving, and gamma radiation were diminished during longer incubations; these treatments also caused increases in dissolved nutrients.