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Microorganisms play a critical role in the decomposition of organic matter, which contributes to energy and nutrient transformation in every ecosystem. Yet, little is known about the functional activity of epinecrotic microbial communities associated with carrion. The objective of this study was to provide a description of the carrion associated microbial community functional activity using differential carbon source use throughout decomposition over seasons, between years and when microbial communities were isolated from eukaryotic colonizers (e.g., necrophagous insects). Additionally, microbial communities were identified at the phyletic level using high throughput sequencing during a single study. We hypothesized that carrion microbial community functional profiles would change over the duration of decomposition, and that this change would depend on season, year and presence of necrophagous insect colonization. Biolog EcoPlates™ were used to measure the variation in epinecrotic microbial community function by the differential use of 29 carbon sources throughout vertebrate carrion decomposition. Pyrosequencing was used to describe the bacterial community composition in one experiment to identify key phyla associated with community functional changes. Overall, microbial functional activity increased throughout decomposition in spring, summer and winter while it decreased in autumn. Additionally, microbial functional activity was higher in 2011 when necrophagous arthropod colonizer effects were tested. There were inconsistent trends in the microbial function of communities isolated from remains colonized by necrophagous insects between 2010 and 2011, suggesting a greater need for a mechanistic understanding of the process. These data indicate that functional analyses can be implemented in carrion studies and will be important in understanding the influence of microbial communities on an essential ecosystem process, carrion decomposition.
Citation: Pechal JL, Crippen TL, Tarone AM, Lewis AJ, Tomberlin JK, Benbow ME (2013) Microbial Community Functional Change during Vertebrate Carrion Decomposition. PLoS ONE 8(11): e79035. https://doi.org/10.1371/journal.pone.0079035
Editor: Chongle Pan, Oak Ridge National Lab, United States of America
Received: August 5, 2013; Accepted: September 27, 2013; Published: November 12, 2013
Copyright: © 2013 Pechal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors (AJL and MEB) would like to thank the University of Dayton Research Council and the Department of Biology for financial support of this project. JLP was funded by the Department of Entomology and the Whole Systems Genomics Initiative for Improved Human, Animal, and Environmental Wellbeing at Texas A & M University. JKT and AMT are funded by the Department of Entomology at Texas A & M University and Texas A&M University AgriLife Research. This project was also funded (TLC, AMT, JKT and MEB), in part, by the National Institute of Justice, Office of Justice Programs, United States Department of Justice through Grant 2010-DN-BX-K243. Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the United States Department of Justice. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Decomposition is an important component of nutrient and organic matter cycling in ecosystems [1]–[3] and forms the base of many food webs [4]. Microbial communities are ubiquitous in the biosphere [5]–[7]. Identifying the functional activity of microbial communities involved in the decomposition process can provide a mechanistic understanding into how detrital community ecology influences ecosystem processes [8]–[10]. Microbes are an important, but often understudied, trophic level in decomposition ecology. For instance, microbes contribute to the decay and recycling of plant organic matter and nutrients [11], which accounts for approximately 99% of the organic matter on Earth [12]. Microbes are also a major component of large carrion resource pulses such as salmon runs [13], [14] and cicada emergences [15], which are important in mediating ecosystem structure and function [16], [17]. However, often in such studies, decay rates or nutrient fluxes provide a measure of the overall decomposition process [10], with little attention given to the discrete function, specifically the carbon source use, of the associated microbial communities and their influence on decomposition rates [18]–[20].
Microbial communities associated with carrion are part of the necrobiome, sensu Benbow et al. [21]. These communities can be from the pre-existing living flora of the organism [22] and the environmental communities existing where carrion falls [23]. Those microbial communities associated with external surfaces of carrion are considered the epinecrotic microbial community, which has been hypothesized to regulate ecological interactions occurring during carrion decomposition through several mechanisms [24].
Prokaryotes can compete with eukaryotes by producing toxic compounds that affect resource "appeal" to consumers and thus reduce competition from eukaryotes [25]. Undisturbed microbial proliferation can deter consumption from higher trophic arthropods such as crustaceans [19] and insects [26]. Additionally, carrion microbial communities influence the physiology and behavior of necrophagous insects through the production of metabolically derived volatile molecules [9], [27]–[29]. Recent studies have documented potential interkingdom interactions with insects eavesdropping on microbial community communication; specifically, in laboratory studies insects have detected and behaviorally responded to quorum sensing and swarming associated molecules produced by bacteria [30], [31]. Yet, there is little understanding of how these interactions may occur in the natural environment, and how they may change during carrion decomposition. If metabolic by-products affect the arrival sequence of necrophagous insects (often the primary consumers of carrion) then microbial functional succession may define consumer community assembly and therefore mediate decomposition and the pathways of nutrient and energy recycling.
There are several factors influencing microbial community assembly and function during vertebrate carrion decomposition: temperature [32]–[34], moisture and humidity [35], tissue type [20], surrounding vegetation [36], [37] and soil pH [38]. However, there are a variety of challenges for researchers studying microbial assemblages during decomposition. Metabolic or physiological profiling is a practical and affordable approach to understand the functional role of microbes within the environment. The study of metabolomics uses a systematic approach to assess metabolic by-products for understanding biological processes [39]. The function of entire microbial communities can be assessed using techniques like Biolog EcoPlates™ [40], [41]. These phenotypic microarrays provide microbial metabolic community profiles (MMCPs) determined by the differential use of carbon sources from environmental samples [42], [43]. Plates similar to Biolog EcoPlates™ were originally developed for the medical field, and have had successful application in many ecological studies [42]–[46]. This technique has been used to study culturable microbial community functional activity in terrestrial [47]–[49] and aquatic habitats [45], [50], [51]. Biolog EcoPlates™ are ideal for evaluating the functional response of entire microbial communities, not individual species or taxa within the community, based on differential utilization of carbon sources (i.e., carbohydrates, polymers, carboxylic and acetic acids, amino acids, and amines/amides). Thus, this approach provides a community-level functional profile (i.e., MMCP) that can be compared between sample types or over time [51]. However, there are limitations to using this technique that have been previously highlighted (see review Preston-Mafham et al. 2011). Some of these limitations for heterogeneous environmental samples are related to the difficulty of quantifying and consistently maintaining the inoculation amount for each carbon source, the potential bias of dominant taxa outcompeting rare taxa present in the community and sensitivity to oxygen concentrations [52].
There are several emerging areas of research focused on a better understanding of microbial community functional ecology importance in carrion decomposition: 1) how does the functional activity of microbial communities change during decomposition; 2) the role of temperature driving this functional succession; 3) how microbial function is mediated by necrophagous insect colonization and succession; and 4) intra- and inter-seasonal variation in these processes [9], [27]. The overall aim of this study was to use Biolog EcoPlate™ MMCPs to characterize microbial community function throughout carrion decomposition, which is the first time the microbial metabolic activity will be characterized in carrion decomposition systems and assess that function with one data set of bacterial metagenomic sequence from matched samples. We tested three hypotheses: 1) microbial functional activity would be dependent on seasonal variation related to local ambient environmental (e.g., temperature) conditions; 2) microbial functional diversity would vary between years depending on temperatures; and 3) that necrophagous insect colonization would have a significant impact on microbial community functional change throughout carrion decomposition. The effects of necrophagous insects on microbial succession were further assessed during one field season by describing the structure of bacterial communities using 454-pyrosequencing with the objective to identify key phyla associated with community functional changes.
Materials and Methods
Seasonal Variation
Seasonal variation in MMCPs was studied within Morris Bean Reserve of Greene County, Ohio, USA (39° 45′53.67″ N, 83° 54′ 41.12″W), a Midwest temperate forest of 12.2 hectares surrounded by agricultural fields with a small tributary stream running adjacent to the reserve that empties into the Little Beavercreek River. The predominant trees were honey locust (Gleditsia triacanthos) and a variety of maples (Acer spp.), while the most common sub-canopy cover was Amur honeysuckle (Lonicera maackii). Sus scrofa (swine) carcasses weighing 14–18 kg were used as models of vertebrate carrion decomposition [53] during four seasonal trials: spring (March until June 2009), summer (July until August 2009), autumn (November 2009 until March 2010) and winter (February until March 2010).
Carcasses purchased from a local farm were double bagged during transport and prior to field placement to prevent insect colonization. Using the same experimental design of Benbow et al. [21], six replicate carcasses were used in the spring and summer trials whereas three were used for the autumn and winter trials; the latter trials only included three replicates because of cost constraints and longer decomposition time expected during the colder months. One carcass was placed randomly along five-1 m2 plots, along each of six transects (50 m) for a total of 30 plots within 10–80 m from each other as previously described [21], [54]. All carcasses were placed under anti-scavenging cages constructed of poultry netting wrapping a wooden frame (0.6×0.9×0.6 m) to prevent disturbance by large vertebrate scavengers (e.g., coyotes, vultures). Temperature was recorded every 0.25 h using NexSens DS1921G micro-T data loggers (Fondriest Environmental, Inc., Alpha, Ohio, USA). Temperature data were converted into accumulated degree hours (ADH), which accounts for temperature variation over decomposition time [55].
Annual Variation and Necrophagous Insect Effects
Annual variation of microbial community activity and effects of necrophagous insect colonization was studied in a Midwestern temperate forest habitat surrounded by agricultural fields in Xenia, Ohio, USA (39° 38′14.83″ N, 83° 1′ 37.82″W). The dominant tree fauna consisted of oak (Quercus spp.) and maple. Carcasses ranging from 5–30 kg were sampled from 5 August 2010 until 14 August 2010 and 26 July 2011 until 2 August 2011. Carcasses purchased from the same farm as described above were double bagged during transport and prior to field placement to prevent insect colonization. In 2010, six male carcasses were randomly placed at a minimum of 20 m apart along three transects. In 2011, using the same methods as described in 2010, six carcasses (three females and three males) were purchased from the same farm, double bagged during transport to the field and randomly placed along three new transects.
During each year, three random carcasses were enclosed in individual 1.8 m3 Lumite® screen (18×14 mesh size) portable field cages (BioQuip Products, Rancho Dominguez, California, USA) to exclude insect colonization. These carcasses were considered the necrophagous insect exclusion treatment (EXC), while the three remaining carcasses were the insect access treatment (ACC) with insects immediately allowed access to the carcasses. As with the previous seasonal study, all carcasses were covered with anti-scavenging cages with NexSens DS1923 micro-T temperatures loggers measuring local ambient temperature every 0.25 h. Temperature data were converted into ADH.
Permission was granted by the Green County Park District for the seasonal trials at Morris Bean Reserve, while no permits were necessary for the annual and necrophagous insect colonization studies, as the landowners granted permission to use the privately owned property. The field studies did not involve endangered or protected species.
Epinecrotic Microbial Community Sampling
During the seasonal trials, sterile cotton applicators were used to sample the microbial communities of the buccal cavity (the top area of the mouth and under the tongue), skin and the interior anal cavity. Epinecrotic microbial communities were sampled from the carcasses every three days, weather and field conditions permitting, until the dry stage of decomposition as defined by Payne [56].
In both the annual and necrophagous insect effect studies, sterile cotton applicators were used to sample microbial communities from two regions on each carcass for 60s: the buccal cavity and the skin, which consisted of combining three areas (approximately 2.54×15.24 cm) along a single transect of a carcass. Care was taken to assure that new areas were swabbed at subsequent samplings. Duplicate samples were collected from the carcasses for 16S rRNA analysis of the bacterial communities using 454-pyrosequencing during the first necrophagous insect effect study (2010). Carcasses were sampled until the dry stage of decomposition as described by Payne [56].
Epinecrotic Microbial Function
Microbial community function was evaluated using phenotype Biolog EcoPlates™ (Biolog Inc., Hayward, California, USA) to provide MMCPs in all experiments [57], [58]. Biolog EcoPlates™ provide quantifiable functional responses of environmental microbial communities using 29 carbon sources (Table S1 in Materials S1); the tweens were used as a positive control to assess microbial growth on the plate and were excluded from analyses [58], [59]. The samples taken for seasonal microbial assessments were stored at −20°C for several months, slowly thawed and then processed using a modified protocol described by Insam and Goberna [60]. All samples from the annual and necrophagous insect effect studies were stored at 4°C and processed within 12 h. Briefly, samples were added individually to 50 ml Falcon tubes containing 40 ml of 25% Ringer solution and 15 sterilized 3 mm glass beads. All samples were homogenized using a Burrell Wrist-Action® shaker (Burrell Scientific, Pittsburg, Pennsylvania, USA) at the power ranking 9 for 10 min. Samples were centrifuged at 500×g for 2 min and the supernatant was retained. The plates were inoculated with 100 µl supernatant aliquots per well and incubated at 25°C in darkness based on preliminary experiments for these types of samples (unpublished data). Absorbance, or overall plate functional activity, was measured at 590 nm every 12 h up to 120 h or until the average plate absorbance reached 0.7 OD using a Wallac 1420 VICTOR2™ with Wallac 1420 Workstation software version 2.0 (Perkin Elmer, Inc., Waltham, Massachusetts, USA) during 2009 and 2010, and a Tecan Sunrise™ with Magellan™ software version 7.0 (Tecan Group Ltd., Männedorf, Switzerland) during 2011. A two-tailed paired t-test was used to test for any differences between the plate reader models, and there was not a significant difference in data between plate readers (t = 0.4620, df = 95, P = 0.645).
Data Analysis
After all seasonal samples were collected, three dates representing three different phases of decomposition (i.e., early, middle, and late) were chosen for analysis. The spring and summer trials followed the same sampling regime, with the first sampling date corresponding with the initial oviposition of Calliphoridae (Insecta: Diptera) signifying the early phase of decomposition (e.g., fresh and bloat). The second sampling date took place when the carcasses reached the middle phase of decomposition (e.g., active decay), while the last sampling date represented the late phase of decomposition (e.g., advanced decay and dry). For the autumn trial, the first sampling date corresponded with initial Calliphoridae oviposition; however, the second sampling date occurred prior to the first snow of the season. It was decided at the beginning of the study that carcasses would not be disturbed once they were covered with snow. The third sampling date took place in January of 2011, which corresponded with increased temperatures and snowmelt. For the winter trial, there were four sampling dates instead of three because of little insect activity during the beginning of the trial. The first sampling date took place immediately after carcass exposure since a delay in initial oviposition was expected during winter temperature conditions. The second date coincided with the next snowmelt. Both a third and fourth sampling date were chosen to represent different decomposition time points, even though there appeared to be negligible visible change to the carcasses and little adult or larval blow fly activity on the carcasses during the entire trial. Sampling for the annual and necrophagous insect colonizer effects studies occurred at initial field placement and on days 1, 3 and 5 during 2010. While in 2011, sampling occurred at initial field placement and then daily until 5 days of decomposition had occurred.
Analyses were performed according to Stefanowicz [44] and Weber and Legge [40]. To generate MMCPs, data were standardized by using the mean metabolic activity that approached or met 0.7 OD or after 10 consecutive readings. This was done to account for possible cell density differences among samples and provided a means to assess overall average microbial activity [40]. First, the average well color development, as determined by the absorbance measurement at 590 nm, was used to calculate the average carbon use of each plate. These data were then normalized using methods modified from Thottathil et al. [45] and Calbrix [46] by subtracting the average water well (background) activity for each plate, then subtracting that value from each carbon source usage, and then dividing by the average of all normalized carbon resources of the plate (Weber et al. 2010). The mean normalized metabolic activity for each carbon source was used for statistical analyses. A minimum base temperature of 0°C was assumed for these microbial community analyses as previously described [24].
454-Pyrosequencing of Communities
Samples collected in 2010 were used to identify microbial communities through 454-pyrosequencing using methods described in Pechal et al. [24]; a modified chloroform-phenol protocol was used for DNA extractions from each sample [24]. Samples were sent to Molecular Research Laboratories (Lubbock, TX) for bacterial tagged encoded FLX amplicon pyrosequencing. PCR amplification of V1–3 regions of 16S rRNA was performed using bacterial primers: Gray28F (5′ TTTGATCNTGGCTCAG) and Gray519r (5′GTNTTAC NGCGGCKGCTG) [61], [62]. The sequences have been deposited in the Sequence Read Archive at the European Bioinformatics Institute (accession number ERP001998). Taxonomic classification of the sequences were conducted using Naïve Bayesian rRNA classifier version 2.2 in the Ribosomal Database Project [63], [64]. The data from the ACC treatment in this study were presented in Pechal et al. [24], with a focus and analysis on family level modeling to estimate decomposition time. Here, we present phyletic data for both the ACC and EXC treatments as a qualitative comparison to functional profile changes over decomposition. For more specific (family) information on the ACC communities see Pechal et al. [24].
Statistical Analyses
Bray-Curtis distance with nonmetric multidimensional scaling (NMDS) and 999 permutations using vegan 2.0–7 library in R [65] was used to visualize MMCP differences among seasons, sample dates, replicate carcasses, sample location (i.e., anal, buccal, skin) and treatment (EXC or ACC). Outliers were identified and removed prior to ordination using Jackknife distances in JMP 9.0.0 (SAS Institute Inc., Cary, NC, USA) as recommended by McCune and Grace [66]. For each of the ordinations, the two axes that explained the most variation with the strongest orthogonality (lowest stress) were used for representing the data in multidimensional space [66].
Differences in microbial function and variation over decomposition were tested using Bray-Curtis dissimilarity values with permutational multivariate analysis of variance (PERMANOVA) with the adonis function using the vegan 2.0–7 library in the R statistical package [65], [67]. PERMANOVA is a nonparametric technique used to differentiate groups of data based on a dissimilarity matrix [67].
Seasonal Variation
Spring overall microbial functional use of the carbon resources was highest of all seasons (Fig. 1) and increased by 6.0% from the first (121 ADH) to the last sampling day (4,426 ADH); the greatest functional activity change occurred during the summer with an increase of 27.1% from the first (345 ADH) to last (3,066 ADH) sampling day; autumn activity decreased by 20.9% over decomposition from 671 to 11,075 ADH; and winter microbial activity increased by 26.2% from 0 to 12,504 ADH.
The mean microbial functional activity for spring (blue circle), summer (green square), autumn (red triangle), and winter (black inverted triangle) over accumulated degree hours (ADH), which was used as a surrogate of decomposition time.
Using PERMANOVA we found no significant differences in normalized MMCPs among the sample locations (buccal, skin, anus) of each carcass; therefore, data were pooled for the remainder of analyses. A two-dimensional NMDS ordination (stress = 0.198, R2 = 0.88) was sufficient to describe normalized carcass MMCPs among seasons (Fig. S1). There were significant differences (PERMANOVA: P = 0.001) in MMCPs among all seasons (Table 1; Fig. 1). Seasons were then separated to assess MMCP differences among the phases (early, middle and late) of decomposition (Fig. S2). There were significantly different MMCPs among decomposition phases in spring and summer (PERMANOVA: P = 0.001), but no differences during the autumn (PERMANOVA: P = 0.226) or winter studies (PERMANOVA: P = 0.101).
Annual Variation
There were significant differences in carcass MMCPs over decomposition, a significant difference between years, and no significant interaction (Table 2). Overall mean functional activity was significantly higher (PERMANOVA: P<0.001) in 2011 (Fig. S3). A two-dimensional NMDS ordination (stress = 0.199, R2 = 0.87) described the variation in carcass MMCPs between years (Fig. S4).
Annual Variation and Necrophagous Insect Effects
There was a significant difference in carcass MMCPs over decomposition, no difference between treatments (EXC and ACC) and a significant interaction effect (Table 3). Overall, mean microbial functional activity of ACC carcasses increased by 4.2% over decomposition; however, it decreased by 35.7% in EXC carcasses (Fig. 2). There were significant differences in MMCPs over decomposition and between sampling regions (buccal and skin), with no significant interaction effects (Table S2 in Materials S1); buccal community microbial activity initially decreased on sampling day 1 followed by a slight increase in activity until the fifth sampling day with an overall net decrease from the initial to last sampling day (Fig. S5). Skin communities exhibited a similar pattern of decreased overall microbial activity until sampling day 3 followed by increased activity as decomposition progressed with an overall net decrease in microbial activity from the initial to last sampling day (Fig. S5).
The mean microbial functional activity in the A) 2010 field trial for carcasses excluded from insect access (EXC-black circle) and carcasses allowed to have insect access (ACC-green square), and in B) 2011 field trial for carcasses excluded from insect access (EXC-black circle) and carcasses allowed to have insect access (ACC-green square) over accumulated degree hours (ADH), which was used as a surrogate of decomposition time.
A two-dimensional NMDS ordination (stress = 0.177, R2 = 0.91) was sufficient to describe the variation in carcass MMCPs between ACC and EXC carcasses (Fig. S6). There were not significantly different MMCPs between insect exclusion and insect access carcasses, but there was a significant difference in MMCPs over decomposition time and a significant interaction (Table 3); thus data from each treatment were pooled for further analysis. Pair-wise comparisons indicated significantly different MMCPs between the initial day (Day 0) and each subsequent day of decomposition (Days 1, 3 and 5).
454-Pyrosequencing of Communities
A total of 378,904 sequences were obtained throughout carcass decomposition. At initial field placement, Proteobacteria was the predominant phyla for EXC (62%) and ACC (70%) carcasses with Firmicutes being the next predominant phyla at 33% and 20% of EXC and ACC communities, respectively. Proteobacteria remained predominant on the first (35%), third (63%), and fifth (82%) sampling days for EXC carcasses. While Firmicutes decreased as decomposition progressed on the first (43%), third (28%), and fifth (16%) day. However, ACC displayed an inverse trend as Proteobacteria decreased from the first (42%) and third (44%) days to only 3% total abundance on the fifth day, but with some inter-carcass variation as discussed in detail in Pechal et al. [24]. Firmicutes was the predominant phyla as decomposition progressed comprising of 36, 48, and 96% on the first, third and fifth sampling day, respectively. Rare phyla accounted for less than 0.5% of the total relative abundance across decomposition (Fig. 3).
Phyletic level taxonomic relative abundance of the microbial communities based on 454-pyrosequencing between treatments (EXC and ACC) over decomposition days during 2010. Day 0 is the initial field placement followed by subsequent sample collections on days 1, 3 and 5 post-field placement. Rare taxa includes any phyla with <3% of the total relative abundance.
Over decomposition, mean microbial functional activity of ACC carcasses decreased by 46.3% while there was a 45.2% increase on EXC carcasses (Fig. 2). There were no significant differences in carcass MMCPs over decomposition, between treatments nor was there a significant interaction (Table 3). However, there was a significant difference in microbial functional activity between the buccal and skin communities and the composite sample but there was no significant difference over decomposition and no significant interaction. The buccal communities increased on day 1, decreased by day 3, and increased on day 5 with an overall net increase in mean microbial activity from the initial to last sampling day (Fig. S5). Skin communities exhibited an inverse trend of decreased activity on day 1, increased by day 3, and finally decreased on day 5 with an overall net decrease in mean microbial activity from the initial to last sampling day (Fig. S5). However, there were no significant differences in MMCPs between sampling regions (buccal, skin), but a significant interaction (Table S2 in Materials S1).
A two-dimensional NMDS ordination (stress = 0.166, R2 = 0. 89) was sufficient to describe the variation in carcass MMCPs between ACC and EXC carcasses (Fig. S6).
Abiotic Conditions
Mean daily ambient temperature among carcasses was 23.2±2.1°C during 2010 and 25.1±1.0°C in 2011. Mean ADH associated with the carcasses in 2011 was significantly higher (F1,7 = 432.9, P<0.001) by 8–18% than 2010 (data not shown) throughout decomposition, except at initial placement in the field when ADH for all carcasses was zero.
Here we report the use of Biolog EcoPlates™ for describing microbial community metabolic profiles throughout carrion decomposition as a representation of the overall functional activity and differentiated carbon source use of epinecrotic communities. The results demonstrated 1) seasonal variation of microbial community function, with overall higher and more variable activity during spring when compared to the other seasons; 2) annual differences in metabolic profiles; and 3) inconsistent effects of necrophagous insect activity (e.g., blow fly oviposition and subsequent larval development) on microbial functional profiles and overall activity.
Fungi and bacteria have been documented as facilitators of the initial decomposition processes of carrion [19], [68]. The inherent stochastic spatial and temporal nature of microbial communities on ephemeral resources, such as carrion, may contribute to the variation both within and across carcasses [69]. We demonstrated that microbial metabolic community profiles varied by season, and within a season they were highly variable during the carcass decomposition process. We also found that when initial insect access and colonization was delayed the metabolic profile changes that occurred during decomposition depended on the year of study, while overall metabolic activity was more highly variable for microbial communities of insect exclusion carcasses during both years. These results suggest that microbial abundance and activity of epinecrotic communities are patchy and sensitive to environmental factors such as temperature and habitat. This variability in metabolic profile responses both within and between seasons and years may be tied to corresponding shifts in the microbial community composition throughout the decomposition process, but this requires additional study. There are limitations associated with sole-carbon source utilization profiles [52]; however, in this study metabolic profiles were used as a potential surrogate for differentiating microbial community changes on the resource over time (e.g., decomposition).
Seasonal Variation
Seasonal variance in abiotic conditions was hypothesized to significantly impact microbial community function. For the spring and winter trials, microbial functional activity decreased during early decomposition followed by an increase in later phases of decomposition. The increase in functional activity during the winter trial most likely results from a lack of competition with invertebrates (e.g., blow fly larvae) because of the colder temperatures (i.e., <5°C). Summer had an increase in microbial activity throughout decomposition. Autumn was the only season that demonstrated a decreasing trend in overall activity throughout decomposition, which can be due to competition with invertebrates, the depletion of the carrion resource, or both. Competition between microbes and insects for a resource occurs by microbes producing toxins that deter eukaryotic consumers [25], while insects can produce antimicrobial peptides that inhibit microbial proliferation [70].
We found significantly different metabolic profiles over decomposition for carcasses in spring and summer but not autumn and winter. However, why did microbial functional activity increase in the winter trial as temperatures decreased? Carter and Tibbett [33] demonstrated when chicken carcasses were subjected to different temperatures, the efficiency of the microbial metabolic activity (respiration) during decomposition varied. They reported that when carcasses decomposed under 2, 12, and 22°C, the process was most efficient at 2°C. This may explain the increased functional activity between the second and last sampling dates for the winter trial, but this requires additional investigation into the optimal metabolic temperatures of multi-species assemblages of microbes. This could also aide in explaining microbial functional variability in different seasons. For the spring and summer trials, large Calliphoridae larval masses colonized and consumed the carcass throughout the decomposition process. Carter and Tibbett [33] also speculated that higher ambient temperatures might have negative impacts on microbial community catabolic reactions. Dipteran larval masses have the potential to generate increased temperatures up to 50°C higher than the surrounding ambient temperature through collective thermogenesis [71]. The depletion of the carrion resource by an invertebrate competitor, but also increased aggregate temperatures resulting from calliphorid larval masses may be affecting microbial community assembly and overall activity during carrion decomposition. Additional studies are warranted to further understand this process.
Annual Variation
Qualitative phases of decomposition could be differentiated for carcasses based on microbial functional activity, and insects appeared to be reducing the microbial community throughout the decomposition process in 2010. However, these trends were not consistent between years. The lack of similarities between years may result from abiotic (e.g., temperature) and biotic factors (e.g., pre-existing microbial communities), as was demonstrated in our seasonal study. Abiotic factors including temperature may account for microbial community function variation between years and throughout decomposition. Ambient temperatures were up to 18% higher in 2011. Previous studies, however, have reported conflicting microbial species response to changing temperature. For instance, soil bacteria abundance increased with temperature (+3°C) in the presence of elevated carbon dioxide, but decreased under conditions of similar temperature conditions without elevated carbon dioxide [72]. Therefore, during 2011, higher average temperature may have been associated with increased available carbon and overall activity of the carcass microbial communities regardless of treatment. Finally, the variation may be associated with other parameters or interactions (e.g., pH changes occurring as part of the carrion or soil) that were not assessed during this study [73].
Necrophagous Insect Effects
Primary insect colonizers utilize the carrion resource as nutrition, mating or an oviposition site. Subsequent larval development may disrupt established microbial communities through direct or indirect competitive interactions on the carcass [25], [70]. Blow flies may directly impact microbial species through chemical secretions while consuming carrion tissue as changes in the microbial community have been demonstrated when using calliphorid larvae for wound debridement therapy [74], [75]. Insects arriving to colonize carrion could introduce their own exogenous microbial community [76], such as Musca domestica (Diptera: Muscidae), which carries over 100 pathogenic microbes [77], [78]. The introduction of insect associated microbial communities may influence carrion microbial community function through microbially mediated competitive mechanisms, which can alter metacommunity dynamics and biogeographic patterns of microbial communities in the landscape [79], [80]. For example, one of the Biolog EcoPlates™ carbon sources, putrescine, is a well know volatile associated with decomposing remains [81]–[84] and is a blow fly attractant but repellant for male carrion beetles (Coleoptera: Silphidae) [85]. Putrescine is also a molecule required for the swarming behavior of bacteria such as Proteus mirabilis, which is commonly associated with blow flies [30], [31]. Alternatively, specific microbes or functional groups may be outcompeting other species present during later phases of undisturbed decomposition, which has been found in other systems [86], [87]. In this study, we found that overall functional activity was affected by the initial access or exclusion of insect colonizers, but the response depended on the year of study, and presumably associated temperature conditions. These functional responses were also associated with the phyletic differences in communities associated with insect exclusion, suggesting 1) there is variability in the structure and function of carrion microbial communities and 2) that necrophagous flies have important effects on microbial community assembly during decomposition. A similar pattern has been observed for undisturbed vernal rain pools that produced significantly higher species richness of protozoan and metazoan species [88] and higher protozoan and rotifer richness in undisturbed artificial container communities filled by rain than that found in disturbed vernal pools [89], [90]. While the variation of microbial metabolic profiles among years and in the presence of necrophagous insects is not surprising, these insect-microbe interactions are still poorly understood in carrion decomposition systems.
Additionally, we documented four major phyla associated with the carcasses throughout decomposition using 454-pyrosequencing that included Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. These phyla have been reported in one other study where epinecrotic communities were described using 454-pyrosequencing (Pechal et al. [24]). Taxa within Proteobacteria are commonly associated with the spoiling of meat and are found on the hides of slaughtered animals [91]. While taxa in the remaining three phyla are associated with the human microbiome [92] and soil communities [93]. Thus, shifts in functional profiles exhibited throughout the 2010 field trial may have resulted from the changes in the relative abundance of these four main phyla. It is unknown at this time whether or not there is a difference of species composition or functional groups based on taxonomic identification of bacteria.
These results demonstrate for the first time the use of metabolic profiling to assess carrion decomposition, and that there is great potential to use this technique for carrion decomposition research. Current research in this field has been aimed at determining what microorganisms compose communities involved in carrion decomposition [19], [20]. Our results demonstrate that the microbial metabolic profiles describe significant functional changes in the community during decomposition both within and among seasons, similar to other studies in aquatic habitats [19], [20]. This demonstrates community-level functional changes occurring over decomposition; however, more detailed research involving which species are changing over time is needed using high throughput metagenomic sequencing approaches [94], [95]. Specifically, using microbial functional data in conjunction with metagenomic and entomological data is needed to better understand the complexity and importance of the necrobiome to decomposition.
Overall, empirical data are sparse within the microbe-insect-carrion model in terrestrial ecosystems. We have demonstrated that insects may have moderating effects on decomposition by mediating microbial structure and function. It is important to further investigate the role of microbes and their importance in determining underlying mechanisms controlling community assembly, biomass turnover and nutrient cycling of ephemeral resources.
Supporting Information
Figure S1.
Seasonal Bray-Curtis dissimilarity based non-metric multidimensional scaling. Using Bray-Curtis dissimilarity values, MMCPs for seasonal carrion communities were plotted using a two dimensional non-metric multidimensional scaling model (stress = 0.198, R2 = 0.88). The MMCPs were significantly different (PERMANOVA: P = 0.001) among seasons. The circles indicate 95% standard error of each season.
Figure S2.
Decomposition phase within season Bray-Curtis dissimilarity based non-metric multidimensional scaling. Non-metric multidimensional scaling ordinations of MMCPs were plotted on two dimensions for carrion communities of decomposition phases (early, middle and late) during A) spring (stress = 0.122, R2 = 0.94), B) summer (stress = 0.098, R2 = 0. 98), C) autumn (stress = 0.149, R2 = 0.91), and D) winter (stress = 0.158, R2 = 0.91) seasons. There were significant differences among decomposition phases in spring and summer (PERMANOVA: P = 0.001), but no significant difference among decomposition phases in autumn (PERMANOVA: P = 0.226) or winter (PERMANOVA: P = 0.011). The circles indicate 95% standard error of each decomposition phases.
Figure S3.
Mean functional activity over decomposition between years. The mean (SEM) microbial functional activity between the 2010 (gray circle) and 2011 (black square) field trials at initial field placement (Day 0) and subsequent sampling on days 1, 3, and 5.
Figure S4.
Annual Bray-Curtis dissimilarity based non-metric multidimensional scaling. Using Bray-Curtis dissimilarity values, MMCPs from both 2010 and 2011 field seasons were plotted using a two dimensional non-metric multidimensional scaling model (stress = 0.199, R2 = 0.87). There were significantly different MMCPs (PERMANOVA: P = 0.001) between years. The circles indicate 95% standard error of each year.
Figure S5.
Mean functional activity over decomposition between sampling regions in both field seasons. The mean microbial functional activity for the buccal (orange circle) and skin (yellow square) sampling region in the A) 2010 and B) 2011 field trials at initial field placement (Day 0) and subsequent sampling on days 1, 3, and 5.
Figure S6.
Treatment within field season Bray-Curtis dissimilarity based non-metric multidimensional scaling. Non-metric multidimensional scaling ordinations of MMCPs were plotted on two dimensions for ACC and EXC carrion communities during A) 2010 (stress = 0.177, R2 = 0.91), and B) 2011 (stress = 0.166, R2 = 0. 86) field seasons.
Materials S1.
Supplementary Tables S1–S2. Table S1, Carbon sources and their respective groupings (i.e., amines/amides, amino acid, carbohydrate, and carboxylic and acetic acid) in a Biolog EcoPlate™. Table S2, PERMANOVA results testing MMCPs between sampling region (buccal and skin) communities and among carcass decomposition days including the interaction for both 2010 and 2011 field seasons with significant results indicated by an asterisk.
The authors thank T. Blair and J. White for help during the study. The Blair family is gratefully acknowledged for allowing access to their property for this research. We thank B. Singh for assistance in the metagenomic data sequence classification processing. The authors thank P. Barton and two anonymous reviewers for their critical reviews and useful comments to improve this manuscript.
Author Contributions
Conceived and designed the experiments: JLP TLC AMT JKT MEB. Performed the experiments: JLP AJL MEB. Analyzed the data: JLP MEB. Contributed reagents/materials/analysis tools: MEB. Wrote the paper: JLP TLC AMT AJL JKT MEB.
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Varanasi: Soma Modi, the brother of Prime Minister Narendra Modi, today said that the latter has from his childhood accepted tough challenges and carves a way out even in turbulent phase.
"Challenging tough situations is Narendra Modi's character and it has been there since childhood," he said.
"He carves his way even during turbulent times" he added.
Responding on Prime Minister Modi's behavior while he was a child, Soma said: "As a child he always helped his friends, who faced difficulties in their lives."
"Everybody obeyed him and paid heeds to his advice, this was his technique and he is using this method till today," he added.
Soma also said that all those who oppose Prime Minister Modi and his policies must be appreciating the latter in their personal conversations.
Talking to the media in Prime Minister Modi's parliamentary constituency Varanasi, Soma said that he can see the development taking place in the holy city courtesy the effective and people friendly policies of the NDA regime. (ANI)


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All LSC coaches and managers are required to take the free CDC online training course Concussion Training for Coaches, (link below and takes approximately 45 minutes) before they may coach or officiate. The certificate of completion at the end of the training is proof the course has been taken. The course must be completed every three years. Please submit a copy of the certificate to the Club Administrator and maintain a copy for your own records.
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A ST DAVID'S Day message from First Minister Rhodri Morgan will be read out to Welsh people in 30 different countries tomorrow.
Rhodri, who is hosting a St David's Day reception in Brussels, said: "St David's Day is the day when Welsh people, whether in New Zealand or New York, Vladivostok or Vancouver, will be celebrating Wales and all that is best about being from there."
oThe Prince of Wales will be visiting the 1st Battalion Welsh Guards for a St David's Day parade at their RAF St Athan base.
oPenderyn Whisky, the first single Welsh Malt Whisky to be produced in over 100 years, will be launched on St David's Day.
oPoliticians and civic leaders are expected to join Wales' first national St David's Day Parade in Cardiff tomorrow. It sets off from Sophia Gardens at 2.30pm.
oFly-on-the-wall TV series The Salon will be celebrating St David's Day in style as it welcomes a host of Welsh celebrities through its doors for a make-over.
Ex-One True Voice singer Jamie Shaw and Hollyoaks actor Bernard Latham will be taking advantage of the salon's luxurious treatments.
oBeatrix Potter's magical tales of Tom Kitten, Peter Rabbit and Samuel Whiskers get released for the first time in Welsh on DVD tomorrow.
oA Welsh language group is planning to celebrate St David's Day in style - by taking the day off. Tired of waiting for the day to become a bank holiday, Pontyberem's Menter Cwm Gwendraeth staff in Llanelli have declared it one themselves!


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Welcome to Pesky Cat Designs! I create unique handcrafted bags inspired by nature. Featured on FOX 5 News, CBS Sunday Morning News and Time Out New York. Thanks for visiting! Elisa
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Welcome to Pesky Cat Designs! I create unique handcrafted bags inspired by nature. Featured on FOX 5 News, CBS Sunday Morning News and Time Out New York. Thanks for visiting! Elisa
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I just love my new purse. The workmanship is beautiful and I love all of the fabric choices. The fabric is high end too - no cheap cotton here. All the details in this purse make it so unique. Very fast shipping/turnaround time. Purse was well wrapped and protective so it arrived in excellent shape. Will certainly buy from this vendor again. She was very easy to work with. Just one small thing, I wish the purse was just a bit larger. For those of us who wear glasses we have the bulk from them and they take up so much room in their cases.
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The purse is so well made; I think it will last several years. I have received MANY compliments already. I will definitely buy from this seller again.
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This came quickly and is beautifully made! I'm so happy with my purchase!
Handmade Bags Inspired by Nature
I create unique handcrafted bags inspired by nature using exclusive Harris Tweed wool, Liberty of London and fine upholstery fabric. All the bags are made from patterns I draft myself and are created by hand, by me, from start to finish in my studio in beautiful St. Augustine, FL.
In my shop you will find bags adorned with seahorses, birds, leafed branches, flowers and more.
I also have a selection of handbags created in Harris Tweed wool imported from the Isle of Harris, Scotland. This iconic wool is like no other in the world.
Most days you will find me in my studio or walking seaside enjoying the waves and watching pelicans diving for fish.
All Rights Reserved - Pesky Cat Designs © 2017
Shop members
Owner, Maker, Designer, Curator
When she's not relaxing in her beach chair, Anni likes to assist with photo shoots and organizing thread in the studio.
Shop policies
All the bags are made from patterns I design and draft myself and are created by me from start to finish in my St. Augustine, FL studio. Fabrics are purchased in small batches to keep the work fresh and unique. Each bag is interfaced to add structure and durability. See individual items for details. Main seams are double stitched and stress points are extra reinforced. Made with care to be durable and beautiful.
All Rights Reserved - Pesky Cat Designs © 2017
Cleaning instructions for your bag is included with your shipment. Most bags can be spot cleaned or dry cleaned. Contact me if you have any questions before purchasing.
Accepted payment methods
- Accepts Etsy Gift Cards and Etsy Credits
I offer shipping to limited international destinations. Please contact me for shipping options and prices.
PLEASE NOTE: Any Custom Fees, Duty or Taxes incurred at the time of delivery are at the discretion of your country's tax regulations and are the buyers responsibility. Please check your country's policies before purchasing.
I document the full value of your purchase on the customs form and do not mark as "gift".
Refunds and Exchanges
::: All shipping costs for refunded items are buyer's responsibility.
::: Customized orders are not refundable.


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D&D Dice by b.freq
When the dice are rolled you hear them on the wood. The results are sketched on your choice of paper. A dice log keeps and displays any rolls you perform down to the individual dice roll with bonuses, totals and a time stamp. D&D Dice by b.freq allows you to always have your dice with you without losing the feeling of rolling the real thing!…
Free • 551


text: Come socialize at Brandon's newest dining hotspot. Located across from the Keystone Centre, Browns Socialhouse combines upscale yet causal dining with the best nightlife as we're open until 2:00am on Friday & Saturday. Kick back and relax on our year-round patio featuring comfy couches and an open fire place. It's the ultimate year round dining experience.


text:
Does everyone deserve a response?
I'm dating a guy and he is not my boyfriend
We got into an argument And he texted me saying "thanks honey"
Honestly he really hurt my feelings today and I do not see a reason why I should pursue anything further with this man
But I do not want to say "let's be friends"
Because I'm am honest person and I don't tell people let's be friends when I really don't want to be friends with them
Do I really need to explain to someone who hurt me why I don't want to date them anymore?
The way I see it we will just be going back And forth
Most Helpful Guy
In this case, I think you should say "something".
It could be as small as you guys got into a big argument already and you don't want to talk to him anymore. Which sounds like the truth. Just say, I don't want to see you anymore. If you two just had a fight, he'll probably be all for that00|00|0
I don't owe him anything really now that I think about it
He hurt my feelings
- Show AllShow Less
5 months
... you owe an explanation.


text:
Discussion in 'Fan Zone' started by unionjack8, Aug 27, 2013.
I have no idea but let's say there was little to no hit would you want to cut him?
It's a toss up for me right now. While I am far from convinced Tanney is ready to lead a team, Orton has really shown serious digression recently. His preseason play was absolutely appalling. Now, there is a chance that is merely a vey knowing he has nothing to prove and no way to improve his place on the depth chart, but he was bad. He makes a serious chunk of change for a backup. If that money could be freed up and used to pry one of the veteran guards out of retirement, I might do it.
There's been serious digression in Orton? Right now it's not even close between him and Tanney. Orton's stats in preseason have been better than his career averages so where's the digression? He's completed 76.2% of his passes which leads the team in preseason. He's had a couple of TD throws which ties him with Romo in preseason and he has a passer rating of 88.9. He's clearly outplayed Tanney. If you're prying a veteran guard out of retirement how much help would they be? If they're retired and still want to play they can't be worth a damn. This team would have no chance of staying afloat with Tanney if Romo goes down for a few games.
He played well in 3 of the 4 games. Don't know what games you watched
Same here. IMO, he has more upside than Sterling Moore. And would rather keep both over Frampton and McCray. Heath can grow into a Frampton type. McCray is one dimensional, beast on ST but lacks instincts to play safety.
When people called Pellerin out a cpl games ago I wasn't really sure what they saw. I saw a kid who looked a little lost in the system over versus under, and knowing where safety help was, but he has a great knack for finding the ball in the air and showed some real toughness last week. Im not saying he is a world beater or a future starter, but I would like to keep him and see how he develops. The problem is, I don't know who I let go in order to keep him, especially if they decide to keep Heath.
Pellerin is a former S and it shows in how physical he is against the run. He has a little ballhawk in him too. I like Moore but Pellerin has more size, physicality and playmaking. Kiffen/Marinelli's D scheme is perfect for his style of play and he not only flashes but has improved in every preseason game.


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Cancun (Mexico), Sept. 9: Commerce minister Arun Jaitley has held a series of high-level meetings here with US trade representative Robert Zoellick and European trade commissioner Pascal Lamy where he made it clear that India and other developing nations would not back off on their demand that the rich nations must first roll back their billion-dollar farm subsidies before the latter would even consider starting negotiations on granting greater market access for agriculture products.
The issue has snowballed into a major casus belli two days ahead of the five-day meeting of the worlds trade ministers which begins here on Wednesday.
India is taking the offensive on agriculture, said commerce secretary Dipak Chatterjee. The commerce minister said bluntly that we are determined to prise them open on the subsidy issue both in terms of domestic support to their farmers and export subsidies.
Reforms on the agriculture front will have to start at your end, Jaitley apparently told both Zoellick and Lamy.
Agriculture is scheduled to dominate the talks at the fifth ministerial meeting of the 146 members of the World Trade Organization, with the poor and developing nations in a funk after the US and EU narrowed their differences on agriculture issues just weeks before the Cancun round.
The US-EU agreement was thin on specifics but the prospect of a deal between them has thrown everyone into a tizzy since they control over 40 per cent of the global farm trade.
The talks with Zoellick and Lamy took place within hours of the arrival of the Indian delegation at Cancun, giving some indication of how seriously the two trading giants view Indias attempts to forge an alliance of 20 developing nations that include China, South Africa, Brazil and Mexico to resist any attempt by the rich nations to hijack the trade round and ram unpalatable decisions down their throats.
Jaitley is already working behind the scenes to widen the coalition of like-minded countries by initiating discussions with nine countries today to protect their collective trade interests. These included Guyana, Jamaica, Nigeria, Botswana, Zambia, Bangladesh (which is the co-ordinator for the LDC nations), China, Brazil and Venezuela.
Some of them are already part of the so-called G-20 a broad coalition that Jaitley hopes will not wilt under pressure from the rich nations. The G-20 is broadly in place, he said.
The commerce minister said the discussions with Zoellick and Lamy especially the latter who has been critical of Indias attempts to cobble the coalition of developing nations had provided the opportunity to discuss our respective stands on agriculture, non-agriculture market access and the Singapore issues.
The EU has tried to play down the talk on trade-distorting subsidies by trying to break up the discussion into blue, amber and green boxes a WTO mandated system that lumps the least trade-distorting support measures into the green box and so forth.
We have used FAO (Food and Agriculture Organisation) statistics to show that cereal production in the rich countries have actually risen as a result of the payouts to their farmers exactly what they said would not happen when these support measures were put into the green box, Indian officials told reporters after the meetings.
It stopped Lamy in his tracks. Hes ordered his officials to check the veracity of the FAO statistics, officials said.
Jaitley also conferred with Mexicos foreign minister Luis Ernesto Derbez to decide on the procedural modalities for the Cancun round which is scheduled to review the pace of trade negotiations since the Doha round in November 2001.
One big bone of contention is the rag-bag of Singapore issues multilateral negotiations on trade and investment, competition policy, transparency in government procurement, and trade facilitation. Developing nations, including India, have been opposing the attempts by the US and the EU to widen the agenda for the trade discussions ever since they first came up at the ministerial meeting in Singapore in 1997.
Investment is an issue that none of the developing countries want to hear about since any discussion on this will put pressure on them to open up their markets in sectors like banking and insurance where most have rigid ceilings on foreign investment. India, for instance, allows only 26 per cent in insurance and 49 per cent in banking and has been under pressure from the US to raise those levels to around 74 per cent which it is loathe to grant.
Jaitley will continue with a flurry of bilateral and plurilateral meetings with ministers from several developing countries.


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Length Overall (m/ft): 36.2m (118.77ft)
Beam: 8m (26.25ft)
Draught Max: 2.49m (8.17ft)
Draught Min: 0m (0ft)
Builder: CBI Navi
Hull #: 41
Country of Build: Italy
Date of Completion: 2006
Hull Material: Steel
Superstructure Material: Aluminium
Naval Architecture: Ginton Naval Architects
Exterior Design: Ginton Naval Architects
Interior Design: Ken Freivokh Design
Class: Lloyds Register
MCA: Yes
Guests: 10
Crew: 6
Number of engines: 2
Manufacturer/ Type: MTU - 8V 2000 M60
Total HP: 1070
Total KW: 797
Max Speed: 13.3kn
Cruise Speed: 12.5kn
Range: 4000 nautical miles
Fuel Capacity: 46000 (L) - 12151 (G)
Water Capacity: 10000 (L) - 2641 (G)
Photo credit: Maarten Janssen / SuperYachtPhoto.com
Content pagina voor Layout toevoegen van afbeelding
The CBI Navi superyacht Metsuyan IV in Kotor
36-metre superyacht Metsuyan IV lying in Kotor, Montenegro
Superyacht Metsuyan IV joins CNI charter fleet
Superyacht Metsuyan IV has joined the Camper & Nicholsons International charter fleet. The yacht is the perfect charter yacht, ideal for accommodating families or large parties of guests looking for a charter vacation of a lifetime. With a sleek and timeless exterior she emerged from the CBI Navi shipyard in 2006. readmore
Superyacht Metsuyan IV in Gibraltar last thursday
Metsuyan IV in Gibraltar last thursday
Superyacht Gattopardo VI launched by CBI Navi
Last April, the CBI Navi Shipyard launched a 40 metre motor yacht built by the FIPA Group member and named Gattopardo VI. Exterior styling and naval architecture were done jointly by the Shipyard Naval Architects and by Jaron Ginton based on the successful M/Y Metsuyan IV, built by the FIPA Group Shipyards in 2006. readmore
Superyacht charter review: Turkish delights on board Metsuyan IV
A recent invitation by Edmiston, to join them on a 4 day charter in Turkey, provided the perfect opportunity to explore this beautiful unspoilt area, on board one of the region's finest superyachts, Metsuyan IV. Our itinerary would take us from Göcek to Fethiye, with stops in beautiful tranquil anchorages along the way. readmore
Metsuyan IV anchored in Turkey
Ginton Naval Architects designs a new 40m CBI Navi motor yacht
Ginton Naval Architects in Holland has received an order for the design of a 40 metre Motor Yacht. The design concept is based on the succesful M/Y Metsuyan IV and she will also be built at CBI Navi, part of Fipa Group, in Italy. She has a steel hull, aluminium superstructure, 2 Caterpillar C32 engines, 850 hp each, ZF 4:1 gearboxes and Caterpillar C4.4 generators. readmore
Sophie Blue off Porto Ercole
Sophie Blue off Porto Ercole
Metsuyan IV cruising off Nice
Metsuyan IV cruising off Nice


text:
Pennzoil TV Commercial: Complete Protection 30sec
Watch the latest TV commercial to launch Pennzoil Platinum® Full Synthetic motor oils with PurePlus™ Technology. It's shot on gas making it a first-of-its kind commercial for a first-of-its-kind motor oil made from natural gas. It's motor oil reimagined. Pennzoil Platinum® and Pennzoil Ultra Platinum™ represent the best synthetic motor oils we've formulated, and they're designed for complete protection by maximizing performance in extreme temperatures and protecting your engine's horsepower. See the magic at http://www.motoroilreimagined.com/ Join the conversation: #MotorOilReimagined Twitter: https://twitter.com/pennzoil Facebook: https://www.facebook.com/PennzoilRead the full article


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Boots Kids Apple Conditioner 300ml
In order to buy non-prescription medicines you must be a registered user of our site as we are obliged to record your transaction history. We also ask that you complete our questionnaire so our pharmacy team can check that this product is suitable for you to buy.
Disabled or chronically sick people can claim VAT relief on purchases for personal or domestic use that are applicable to their disability or sickness.
Stock coming soon
We're sorry, this product is temporarily out of stock. Most products are back in stock within two weeks so please keep checking back.
Collect 4 Boots Advantage Card points with this purchase
Product details
Boots Kids Apple Conditioner 300ml
Boots Kids Apple Conditioner has been carefully formulated to be gentle yet effective, leaving your child's feeling soft and smelling great.
How to use
Squeeze a small amount of conditioner into your palm and smooth evenly onto wet hair. Leave for a few moments and rinse well.
Hazards and Cautions
Supervise children during use.
Keep out of reach of children.
Avoid contact with the eyes.
If product gets into the eyes rinse wel with water immediately.
Important info
Key features
- Hypoallergenic
- dermatologically tested
- Tear free.
Aqua (Water), Cetyl Alcohol, Stearyl Alcohol, Hydroxyethylcellulose, Sodium Benzoate, Cetearyl Alcohol, Glycerin, Parfum (Fragrance), Phenoxyethanol, Cetrimonium Chloride, Dimethicone, Behentrimonium Methosulphate, Citric Acid, Potassium Sorbate, Disodium EDTA, Dipropylene Glycol, Pyrus Malus (Apple) Fruit Extract, Tocopheryl Acetate, Alcohol Denat, Tris (Tetramethylhydroxypiperidinol) Citrate, Panthenol, Denatonium Benzoate, Cl 19140 (Yellow 5), Cl 42090 (Blue 1).


text:
We ship to over 100 countries, including:
Please select the language that you would like to browse the site in.
Please select the currency you would like to shop in. Your order will be transacted in USD.
Style: 35895077 Color Code:
Soft and printed mini skort featuring pleat detailing along the front. Four-pocket style with a hidden side zip.
Free People
Celeste Pleat Mini Skort styled by the FP Me community
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text:
This is a ripped driver for using the USB storage facility on a Nokia 5510 mobile phone without installing the entire software suite. Works with Windows 98/98SE/Me/2000/XP. Easy, single click install/uninstall.
|Discussion Thread
|Nokia 2626 (Windows XP Home) [USB]
|Jun 19, 2008
|Nokia data cable 6102 (Windows Vista) [USB]
|Dec 27, 2007


text:
The Female Coaching Network (FCN) is the leading Global community of female coaches who support, drive and influence real change in sports diversity.
The World's Only International Multi-Sport Online Community for Female Sports Coaches.
For Female Coaches, By Female Coaches
Sport: Netball
Nationality: Australia
Role: Head Coach
Sport: Various
Nationality: British
Role: Sports Therapist
Sport: Ice-Hockey
Nationality: Canadian
Role: Coach, Mentor
Sport: Basketball
Nationality: America
Role: Assistant Video Coordinator
Sport: Track & Field
Nationality: British (Formerly USSR)
Role: Sprints and Hurdles Coach
Sport: Track & Field - Endurance
Nationality: British
Role: Athlete & Coach
Sport: Wheelchair Basketball
Nationality: America
Role: Head Coach of USA Basketball Team at Rio 2016 & HC for University of Illinois
Sport: Gymnastics
Nationality: America
Role: Personal Coach to Simone Biles & Team USA Head Coach at Rio 2016
Sport: American Football
Nationality: America
Role: Head Coach
Sport: Track & Field
Nationality: British
Role: Olympian & Coach
Sport: Archery
Nationality: Canadian
Role: Team Canada's National Recurve Coach
Sport: Soccer
Nationality: America
Role: 'Self-Directed Learning Coach and ASK for Choice Strategist' for CAC.
Sport: Basketball
Nationality: America
Role: Author of: The Final Season


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for me it is twix
I eat the soft side first... then the crunchy side xD
or snickers
Most Helpful Guy
Milky Way. Gently nibbling and sucking the chocolate away, the using your tongue to get inside to the creamy caramel, enjoying the sensuous taste of it all!11|00|0
aham...*cough cough*
that was a fine description lad
MMMMMMMmmmmmmm. So glad you liked it. :-)
Think you for MHGuy! :-)
Can't wait for more chocolate questions.
Most Helpful Girl
I love all chocolate ❤❤❤11|00|0
hahaha I feel ya :P


text: Definition Edit Abandonware is software that is no longer being maintained or supported by the developer or publisher. Retrieved from "http://itlaw.wikia.com/wiki/Abandonware?oldid=163126" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Software Definition Add category Cancel Save


text:
- Description
Accommodates up to a 46" TV weighing 95 lbs. or less. Two adjustable shelves hold audio/video equipment. Two drawers with metal runners and safety stops. Enclosed back with cord access. Quick and easy assembly with patented T-slot drawer system. Cinnamon Cherry finish.
1 TV Stand
Width: 41.625"
Height: 24.125"
Depth: 16.125"
More from this collection: Beginnings
- Additional Information
Additional Information
SKU 14200484 MPN 413045 GTIN UPC 042666111478 Brand Sauder Collection Beginnings Assemble Length No Assemble Width 16.125 Assemble Height 24.125 Assembly Required Yes Material MDF Finish Cherry Styles Traditional Shipping Time 1-2 Weeks Free Shipping Yes
- Reviews
Disclaimer: Pictures displayed may include optional products not included in the price. Computer monitors often display color variations. Color swatches are shown if alternative colors are available. Please call us or email us if you have any questions!


text: Sorry if this should go in a different spot. I attempted to root my phone, and as usual, the Sw updater couldn't find the drivers. Now, once I put the battery back in my phone, it goes into recovery mode. I'm worried that I broke it. If I select either the reboot or update options it goes to the screen with the "!" and the green android character and stays that way. Any help would be greatly appreciated.


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|Half Baths
|Property type
|Minimum Stay
|7 nights
Enjoy Oceanfront luxury and amenities at Semi-Oceanfront prices! Dream of Jeanne is spacious, beautifully decorated and very well maintained by "hands- on" owners who take pride in making sure you will have a wonderful vacation. Only 100 feet away from the beach access and beautiful views from the home's crows nest, the magical beaches of the Outer Banks are right at your doorstep. Spend your time away from the beach enjoying the heated pool (20 x10) and large Hot Tub (seats 6). Challenge the family to a game of volleyball, corn hole or horseshoes in the yard, or you could test your skill with the giant chess and checkers on the middle front deck.
If you are in need of a little down time you can relax by the fireplace (off season only) and watch a movie on the 52" HDTV with surround sound or take an afternoon nap on the deluxe queen mattresses. In the large recreation room your family will have a blast playing pool, ping pong and the ever fast moving air hockey.
Dream of Jeanne is located in the center of the main OBX shopping and restaurant areas, that is if you even need to venture out from this great home! You may want to spend time fishing at the nearby pier or a walk over to nearby Jockeys Ridge to fly a kite. No matter what your interests are there is something for everyone in this fantastic vacation home with all the extras.
Jeanne and Randolph welcome you to their beach home and hope you come back again and again to "Dream of Jeanne"
Remember cool season rentals receive FREE pool heat! (4/1 - 5/25 & 9/22- 11/1)
Level 1: Recreation room, half bath and pool access
Level 2: Hot tub on deck, laundry area, Queen master bedroom and bath, Queen master bedroom with bath, hall bath, Bunk Room, and an additional Queen bedroom, including a Den with a Full Sleeper Sofa and bath.
Level 3: Living area, dining area, kitchen, ocean view deck, hall bath and Queen bedroom
Our week at Dream of Jeanne was everything we hoped for and more! It is obvious the owners take great pride in their property as everything was in good order and well taken care of. Two days into our rental, the owner called us to ask if we had any questions and to touch base. There was nothing we could think of that was missing or should have been changed. Plenty of space, lots of seating in the great room and for meals at the table and bar. Lots of activities available for the kids, both in the game room and outside sports. The beach access was right across the street. We rented through Elan Vacations and their check-in and check-out procedures couldn't have been more convenient. We will be looking at this property again next year.
This is a very pretty, convenient, well stocked home. There are lots of games, pool and beach toys, dishes, etc. All the appliances you could expect. There are a lot of stairs, like most OBX homes. My family really enjoyed our stay. The Crowe's nest is a great place to interact with a great view and good seating. Wrap around decks are very nice with places to sit or lay everywhere you need.


text:
Accepting Guests
- Last login 11 months ago
Join Couchsurfing to see Robyn's full profile. It's free!
About Me
Travel the world or my own country and find the elusive Narnia, trek through my own Middle Earth, and experience the world through other cultures and stories from everyone I meet along the way.
22 year old born in Canada looking for epic lifetime adventures. Doesn't matter where, all that matters is that they happen in any form...
Little words are to be said here, lets meet and you´ll know for real.
Be who you want to be, don´t compare yourself to others.
Follow your passion and be open to others' stories and ideas, and then the best people will come into your life.
Why I'm on Couchsurfing
Surfed through Portugal and Barcelona, and hosted in my home town!
Open for meeting whoever is open to meet me.
Never enough experience can be placed under this section.
Anything and everything invloving outdoor activities ranging from walking to rock climbing. Anyone up for adventures?
- walking
- traveling
- music
- outdoor activities
- rock climbing
Music, Movies, and Books
Lord of the Rings\The Hobbit
The Celestine Prophecy
Zen - The art of motorcycle maintanence
Cool Runnings
Into the Wild
No screamo, except for the metal cover of the Gypsy Kings - Bambaleo, of course!
One Amazing Thing I've Done
Build world wide friends... as well as living in Canada
Teach, Learn, Share
Love to teach and learn music on whatever instrument presents itself.
Share stories, most importantly share my passion of life, and learn others'.
What I Can Share with Hosts
The sweet sweet sound of Canadian back bacon sizzle on the grill early in the morning.
Countries I've Visited
Cambodia, Jamaica, Laos, Mexico, Philippines, Portugal, Spain, United States


text:
Many of us will find ourselves in need of some financial advice at some point in our lives. With the ability to answer question online, it's far easier to find answers now than years earlier. Still, you need to know where to look for those answers.
Does your business have a marketing strategy? Life today is extremely busy and can be overwhelming at times. In fact, more people are struggling with life and work balance than ever before.
Many of us who were transplanted from the North witnessed in the past the change of seasons, summer into fall and fall into winter. Just about this time, the leaves on the trees would turn to colors that would be determined by the temperature and the amount of rainfall in the previous months.
Just before I sat down to write this column, I came across a quote from comedian Denis Leary, who once said, "Racism isn't born, folks, it's taught. I have a 2-year-old son. You know what he hates? Naps! End of list."
I am afraid if my church found out, they would dislike me for joining, as well as question where I really stand as a Christian. I feel conflicted about how to address both sides of my beliefs. Can you help? — CAUGHT IN THE MIDDLE IN KENTUCKY
Mindfulness is a key to gaining control over our distracted world. It helps us be aware of our situation, develop empathy for others, and strengthen our ability to listen. Teachers know the challenges of educating a roomful of children, each of whom operates at a different level of readiness with varying states of attentiveness. We adults are no less guilty of distraction. With multiple responsibilities, technological frustrations and frequent interruptions, we are often not as attentive as we should be.
Beginners to the duplicate world are devastated when the director is called on them. It is a necessity, nothing to be embarrassed by. It's not a reflection on your character to have a director called to the table when someone leads out of turn or drops a card face up where it can be seen by others.
Dear Abby, I'm not even positive you still answer these things. The last time I saw one of your columns, I was in college. One was always hanging on my mom and dad's refrigerator. Every time I'd visit, mom would release it from her "I lost my ass in Vegas" magnet and read it to me.
Consumers have long been used to experiencing the dynamic discounts of doubling coupons. However, those days are long gone, and chances are they won't return to supermarkets again.
Dear Dave, My wife just learned she'll be losing her job at the end of the year. She's a project manager for a software company in Seattle, and she will be receiving a severance package if she works until year's end. We were curious as to how you think we should handle the next few months.
The biggest house in a nearby town is a huge white Victorian that looks like a wedding cake. It has a wide front porch that runs the length of the house, three-story turrets with curved glass windows topped with witches' hat roofs. It has gables and wings and filigree and shutters and shingles. It's as if the architect thought they gave a prize for putting the most frippery on one single house. It looks as if it might be the starring attraction in Stephen King's next novel. It is bigger than Perkins' Funeral Home and the Falling Leaves Home for Seniors combined, the other two large houses on Main Street.
Thumbs up to all the people who will make the 2017 Wilson County Fair likely break records for attendance spearheaded by the total solar eclipse and the new Wilson County Expo Center.
There's a certain eloquence to riding the train, both in the ease of commute and the kindness of the people. People wait their turn to board and often yield to those coming down from the second floor. I never heard one angry or loud conversation during the four days, eight trips, that I rode on the train.
DEAR ABBY: I have been in two relationships. The first was with a girl a couple of years older than I am. We were together for several years before she cheated on me and dumped me. I was crushed. The next girl was a few years younger. She did the same thing after we were together a year.
First of all, the Bible does not specifically mention any of the world wars. The Bible does talk about massive death coming in the future through war, pestilence, natural disaster and other things to include civil disorder. Other than what Jesus said in general about "wars and rumors of wars" (Matthew 24:6; Mark 13:7) the current situation between America and North Korea is not in the Bible. Whether current American events will end up a war or only a rumor of war, only time will tell.


text:
Search By Vehicle
Bilstein Shock Absorber 24-186988 DODGE RAM 1500 4WD 5100 SERIES F 06-08 5100 SERIES (RIDE HEIGHT ADJUSTABLE)
Dodge Ram 1500 2008-2006
B8 5100 (Ride Height Adjustable) - Shock Absorber
46mm Monotube Shock Absorber
Bilstein B8 5100 (Ride Height Adjustable) shock absorbers are designed for trucks and SUV's with a front coilover style suspension. The monotube shocks feature a 46mm digressive piston with self-adjusting deflective disc valving. Multiple snap-ring grooves in the shock body allow for different spring seat positions, providing 0"-2.5" of front end lift, depending on application. The Bilstein B8 5100 (Ride Height Adjustable) shock absorber provides increased wheel travel and improved handling and feature a zinc-plated steel body for resistance to harsh off-road elements.
Vehicle Fitment
Extended Information
- 24-186988
- 24-186988
- 651860594767
- Bilstein
- Available
- In Stock: Ships Same Day to 24 Hours
- New
Shipping Information
- Item Requires Shipping
- 4.6 lbs.
- W3.5500" x H3.5500" x L26.8000"
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text:
Paragon 6309 Motla Snow Cone Syrup Red Raspberry, One Gallon
Ships in 2-3 days
Availability: In Stock
✔ Ready to use ✔ For use on shaved ice or snow cones ✔ Made with the finest ingredients ✔ No mixing, no messSee more product details
Earn $0.11 Loyalty Reward Points! More Info
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Item Details
Ready to use with no mixing required, the Paragon 6309 Motla Snow Cone Syrup Red Raspberry, One Gallon is a terrific purchase and a high-profit money maker. Perfect for concession stands, grocery stores, food courts, country fairs and more, Paragon syrup is made of delicious natural ingredients and pure cane sugar.
This one gallon dispenser can yield up to 100 red rasberry flavored ice drinks that will be a welcome treat on a hot day and a thirst quencher always.
- Snow Cone Red Rasberry Syrup
- Ready to use
- For use on shaved ice or snow cones
- Made with the finest ingredients
- No mixing, no mess
- Brand:
- Paragon
- Model #:
- 6309
- Ship weight:
- 10 lbs
- Sold As:
- 1 ea
- Item Number:
- 0475-6309
- TigerChef ID:
- 126164