Reliable sampling method for analysis of the ecology of the human alimentary tract

An intubation method has been developed that allows removal of a sample of human intestinal fluid within a short period of time, that avoids contamination, and that minimizes exposure of the sample to air. Preliminary results obtained with this method have shown that the stomach and duodenum are essentially sterile and that the bacterial population present in the remainder of the small intestine is similar to that described by previous workers except that Veillonella species were encountered frequently and Haemophilus species were also detected in the lower jejunum and ileum of some individuals.     

Combining counts and incidence data: an efficient approach for estimating the log-normal species abundance distribution and diversity indices

Obtaining accurate estimates of diversity indices is difficult because the number of species encountered in a sample increases with sampling intensity. We introduce a novel method that requires that the presence of species in a sample to be assessed while the counts of the number of individuals per species are only required for just a small part of the sample. To account for species included as incidence data in the species abundance distribution, we modify the likelihood function of the classical Poisson log-normal distribution. Using simulated community assemblages, we contrast diversity estimates based on a community sample, a subsample randomly extracted from the community sample, and a mixture sample where incidence data are added to a subsample. We show that the mixture sampling approach provides more accurate estimates than the subsample and at little extra cost. Diversity indices estimated from a freshwater zooplankton community sampled using the mixture approach show the same pattern of results as the simulation study. Our method efficiently increases the accuracy of diversity estimates and comprehension of the left tail of the species abundance distribution. We show how to choose the scale of sample size needed for a compromise between information gained, accuracy of the estimates and cost expended when assessing biological diversity. The sample size estimates are obtained from key community characteristics, such as the expected number of species in the community, the expected number of individuals in a sample and the evenness of the community.     

Comparative application of the coefficient of variation and range-based statistics for assessing the taxonomic composition of fossil samples

Dental variation has been used commonly to assess taxonomic composition in morphologically homogeneous fossil samples. While the coefficient of variation (CV) has been used traditionally, range-based measures of variation, such as the range as a percentage of the mean (R%) and the maximum/minimum index (I_max/min) have recently become popular alternatives. The current study compares the performance of these statistics when applied to single- and pooled-species dental samples of extant Cercopithecus species. A common methodology for such problems of species discrimination has been to simply compare the maximum value of a variation statistic observed in extant samples with that observed in the fossil sample. However, regardless of what statistic is used, this approach has an unknowable Type I error rate, and usually has low power to detect multiple species. A more appropriate method involves a formal hypothesis test. The null hypothesis is that the level of variation in the fossil sample does not exceed what might be expected in a sample drawn randomly from a reference population, taking into account sampling error and the size of the fossil sample. Previous research using this method with the CV has indicated that it offers considerable power at an acceptable Type I error rate. In the current study, the data of primary interest were posterior dental dimensions for single- and pooled species samples from extant Cercopithecus species. In addition, the study also investigated the relative performance of variation statistics when applied to highly dimorphic canine dimensions, since much recent work has employed sexually dimorphic dental dimensions for assessing single-species hypotheses. The results indicate that the CV consistently out-performed the range-based statistics when using posterior dental dimensions to test a single-species hypothesis. Regardless of which statistic was used, tests on sexually dimorphic dimensions offered minimal power. In consideration of these results and the problem of studywise Type I error rates, we recommend against the use of multiple measures of variation to test for multiple species composition, and advocate the CV as the statistic of choice when using the method of Cope & Lacy (1992). For similar reasons, we argue for careful selection of dental variables for inclusion in such analyses, and in particular recommend against including sexually dimorphic dimensions when testing for multiple species composition.     

A simplified method for the biological assessment of the quality of fresh and slightly brackish water

A simplified method for the biological assessment of the saprobic quality of surface water is described. The method has the advantage that it is unnecessery to determine accurately the species of organisms present in the sample, which must be done when existing saprobic systems are used. The method described here cannot be used when there is a great abundance of a single species and a very strong environmental disturbance must be assumed.     

A measure of species diversity related to the lognormal distribution of individuals among species

An investigation has been made of the relation between species diversity and the lognormal distribution of individuals among species, using phytoplankton samples from the Indian Ocean. The area under the truncated lognormal curve representing a sample gives the total number of species, S, while the logarithmic standard deviation, σ, gives a measure of the scatter of distribution of individuals among species, the other factor affecting diversity. Using a method described by Hald, truncated lognormal curves were fitted to the phytoplankton data and estimates obtained for the mean, ξ, the logarithmic standard deviation, σ, the number of species in the modal octave, N_o, and the number of species in the population universe, N. Since one is interested in a sample only in so far as it reflects the properties of the population from whence it came, estimated population parameters were used to define measures of diversity by which means it was hoped to obtain a diversity index independent of sample size, i.e., a diversity value related more to the population than to the sample.     

A Universal Method for Species Identification of Mammals Utilizing Next Generation Sequencing for the Analysis of DNA Mixtures

Species identification can be interesting in a wide range of areas, for example, in forensic applications, food monitoring and in archeology. The vast majority of existing DNA typing methods developed for species determination, mainly focuses on a single species source. There are, however, many instances where all species from mixed sources need to be determined, even when the species in minority constitutes less than 1 % of the sample. The introduction of next generation sequencing opens new possibilities for such challenging samples. In this study we present a universal deep sequencing method using 454 GS Junior sequencing of a target on the mitochondrial gene 16S rRNA. The method was designed through phylogenetic analyses of DNA reference sequences from more than 300 mammal species. Experiments were performed on artificial species-species mixture samples in order to verify the method’s robustness and its ability to detect all species within a mixture. The method was also tested on samples from authentic forensic casework. The results showed to be promising, discriminating over 99.9 % of mammal species and the ability to detect multiple donors within a mixture and also to detect minor components as low as 1 % of a mixed sample.     

Computer-assisted pattern recognition model for the identification of slowly growing mycobacteria including Mycobacterium tuberculosis.

We present a computerized pattern recognition model used to speciate mycobacteria based on their restriction fragment length polymorphism (RFLP) banding patterns. DNA fragment migration distances were normalized to minimize lane-to-lane variability of band location both within and among gels through the inclusion of two internal size standards in each sample. The computer model used a library of normalized RFLP patterns derived from samples of known origin to create a probability matrix which was then used to classify the RFLP patterns from samples of unknown origin. The probability matrix contained the proportion of bands that fell within defined migration distance windows for each species in the library of reference samples. These proportions were then used to compute the likelihood that the banding pattern of an unknown sample corresponded to that of each species represented in the probability matrix. As a test of this process, we developed an automated, computer-assisted model for the identification of Mycobacterium species based on their normalized RFLP banding patterns. The probability matrix contained values for the M. tuberculosis complex, M. avium, M. intracellulare, M. kansasii and M. gordonae species. Thirty-nine independent strains of known origin, not included in the probability matrix, were used to test the accuracy of the method in classifying unknowns: 37 of 39 (94.9%) were classified correctly. An additional set of 16 strains of known origin representing species not included in the model were tested to gauge the robustness of the probability matrix. Every sample was correctly identified as an outlier, i.e. a member of a species not included in the original matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

A model of the species rank-abundance relation for a community in an open habitat

A mathematical model is proposed to describe the relationship between the abundance and the rank of species in order from the most abundant to the least in a community in an open habitat. This model is derived as a corollary of a species-area equation (Kobayashi , 1975) which could be expected in the case where the individuals of each species are uniformly distributed over a habitat area. Numerical simulation reveals that a rank-abundance curve for a universe results in different species-area or species-individual curves according to the spatial distribution of individuals, and that the relative abundance of each species in a sample varies with sample size unless the spatial distribution of individuals is uniform. A species-individual curve obtained bySanders 's (1968) rarefaction method agrees with that observed actually only for the spatially uniform distribution. Change in the pattern of rank-abundance curve with species diversity and with sample size is discussed in relation to the present model.     

Contrasting below-ground views of an ectomycorrhizal fungal community

Ectomycorrhizal fungal communities have been characterized in a number of ways. Here we compare colonized root-tip and mycelia views of an ectomycorrhizal fungal community. Ectomycorrhizal fungi, both as mycelia and colonized root tips, were identified in soil samples taken from a pine plantation. We determined that for some ectomycorrhizal fungal species multiple root tips from a single soil sample were not independent. Therefore in the comparison of root-tip and mycelia views, we considered species to be present or absent from each soil sample irrespective of the number of root tips colonized by the species. We observed 39 ectomycorrhizal fungal species in total, but 12 were observed exclusively as mycelia and 11 exclusively colonizing root tips. The relative frequencies of 10 species occurring as both mycelia and root tips were not independent of the method of observation. Our results suggest that ectomycorrhizal fungal species differ in their spatial distributions on root tips, and that root-tip and mycelia views of the community are different.     

Predicting species distributions from small numbers of occurrence records: a test case using cryptic geckos in Madagascar

Aim Techniques that predict species potential distributions by combining observed occurrence records with environmental variables show much potential for application across a range of biogeographical analyses. Some of the most promising applications relate to species for which occurrence records are scarce, due to cryptic habits, locally restricted distributions or low sampling effort. However, the minimum sample sizes required to yield useful predictions remain difficult to determine. Here we developed and tested a novel jackknife validation approach to assess the ability to predict species occurrence when fewer than 25 occurrence records are available. Location Madagascar. Methods Models were developed and evaluated for 13 species of secretive leaf‐tailed geckos (Uroplatus spp.) that are endemic to Madagascar, for which available sample sizes range from 4 to 23 occurrence localities (at 1 km² grid resolution). Predictions were based on 20 environmental data layers and were generated using two modelling approaches: a method based on the principle of maximum entropy (Maxent) and a genetic algorithm (GARP). Results We found high success rates and statistical significance in jackknife tests with sample sizes as low as five when the Maxent model was applied. Results for GARP at very low sample sizes (less than c. 10) were less good. When sample sizes were experimentally reduced for those species with the most records, variability among predictions using different combinations of localities demonstrated that models were greatly influenced by exactly which observations were included. Main conclusions We emphasize that models developed using this approach with small sample sizes should be interpreted as identifying regions that have similar environmental conditions to where the species is known to occur, and not as predicting actual limits to the range of a species. The jackknife validation approach proposed here enables assessment of the predictive ability of models built using very small sample sizes, although use of this test with larger sample sizes may lead to overoptimistic estimates of predictive power. Our analyses demonstrate that geographical predictions developed from small numbers of occurrence records may be of great value, for example in targeting field surveys to accelerate the discovery of unknown populations and species.     

A Comparison of the Efficacy of Pond-Net and Box Sampling Methods in Turloughs - Irish Ephemeral Aquatic Systems

Two methods were used to sample aquatic macroinvertebrates in three turloughs. Turloughs are systems that flood periodically from groundwater, in response to local rainfall patterns and contain rare aquatic species and assemblages. The first method used a standard pond net that was swept through the water column, while the second involved fixing a rectangular, open-bottomed box to the substrata and removing all organisms with a net. Similar overall sampling effort was applied to each method and individual box samples were found to take longer to gather than pond net samples. The box method, however, gave the maximum yield for a given sampling effort. Significantly more beetle species and individuals were recorded per unit area of bottom at all three turloughs using the box method. Multivariate analysis segregated samples, firstly according to site and secondly, with respect to method. The box method is a viable alternative to sampling with a pond net. It is more quantitative, objective, specific and reliable. This is particularly important in habitats distinguished by rare species and assemblages, and for which monitoring is driven by legislative needs.     

粪便样品中大肠杆菌多态性分子研究

以粪便样品中分离到的大肠杆菌为研究对象,比较了3种不同方法在分离鉴定大肠杆菌过程中的应用。首先,通过传统方法从粪便样品中分离,筛选和确定了一批大肠杆菌疑似菌株,再用现代分子生物学方法对待鉴定的大肠杆菌疑似菌株,已知大肠杆菌MG1655以及几种其它细菌进行ARDRA(AmplifiedRibosomalDNARestrictionAnalysis)分析,最后利用ERIC-PCR技术在个体水平上分析菌株的多样性。结果表明,所有由传统方法确定的大肠杆菌疑似菌株和MG1655都属于同一ARDRA型,并与其它细菌的ARDRA条码型不同。这说明ARDRA分析得到的结果与传统分析方法的结果吻合,利用ARDRA分析可以区分大肠杆菌和其它肠道细菌。但是在本实验中ARDRA分析不能反映大肠杆菌中不同菌株之间的多样性,ERIC-PCR则可以区分它们。     

The effects of the presence of stored product pests on the microfauna of a flour community

AIMS: To determine the effect that the presence of some beetles have on the species of bacteria found in their flour. METHODS AND RESULTS: Bacteria were isolated from flour that either did not contain beetles, contained Tribolium beetles in different environments, or contained either Stegobium paniceum or Lasioderma serricorne. These bacteria were tentatively identified by both the gas chromatography-fatty acid methyl esters (GC-FAME) method and partial sequencing of the 16S rRNA gene. All samples contained Bacillus species including the controls, but the non-Tribolium beetles and a Tribolium beetle line known to have low benzoquinones also contained Enterococcus and Enterobacter species. Additionally an unidentified bacteria isolate in the Enterobacteriaceae was also found in the L. serricorne sample. Our results also suggest incongruent identifications when using the GC-FAME method vs sequencing. CONCLUSIONS: Certain species of bacteria can be introduced by the presence of insect pests, but the diversity of species is far less in stocks of Tribolium beetles. SIGNIFICANCE AND IMPACT OF THE STUDY: Stored product pests can alter the bacterial community. Isolated species from this study show a strong genetic relationship to each other, suggesting an isolated evolving system. A unique bacteria was also isolated. GC and sequencing methods of identification are compared.     

Sample size and water dynamics on germinating diaspores: the first step for physiological and molecular studies on the germination process

Water dynamics on germinating diaspores is normally used as a chronological marker in molecular biology and physiological studies focusing on germination. However, there is no protocol to analyze this process, which may undermine all results generated. In this context, we determined the sufficient sample size to study water dynamics on germinating diaspores by means of a modified maximum curvature method (MMCM). As a biological model, we used diaspores from agricultural species with different physiological qualities. These diaspores were put in contact with distilled water and were analyzed regarding mass, every hour, until embryo protrusion. Data collected in the last observation were used to measure the sufficient sample size. Here, we show that (i) MMCM is an adequate method to measure the variability of water dynamics on germinating diaspores; (ii) the b coefficient can be used to infer the homogeneity of water dynamics on diaspores of different species as a function of the increase in sample size; (iii) in general, to study this process in an easier, safer and standardized way, seven diaspores are needed and (iv) water dynamics on germinating diaspores estimated by weighted mass demonstrated to be a very stable process which depends more on the species than on the physiological quality. We also discussed why the sample size and water dynamics on germinating diaspores should be the first step for protocols on physiological and molecular aspects of the germination process.     

Unraveling the commercial market for medicinal plants and plant parts on the witwatersrand, South Africa

To unravel the market for commercial medicinal plants on the Witwatersrand in South Africa, a semiquantitative approach was taken. A stratified random sample of 50 herb-traders was surveyed, and an inventory of all plants and parts sold was compiled. Research participants were questioned on the scarcity and popularity of the plants traded, as well as suppliers and origins. The rarefaction method established that the sample size was adequate. The diversity of the sample was determined using ecological indices of diversity, and found to be comparatively high. In addition, Spearman rank correlations, chi-squared and Fisher’s exact probability tests were used to assess the probability of certain taxa being used. At least 46% of the taxa traded showed a higher than expected probability of being utilized, and taxa tended to be harvested from the largest families proximate to the markets. About 511 species are traded in the region, and there is a low dominance in the use of species. Ethnic and floristic diversity are influential in deciding the trading patterns that have emerged.     

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

Efficiency of sampling methods for accurate estimation of species richness of corticolous microlichens in the Atlantic rainforest of northeastern Brazil

Three different sampling methods were compared with regard to accurate estimation of species richness of crustose and microfoliose lichens in Atlantic rainforest remnants of northeastern Brazil. Quantitative transect sampling yielded three times as many species as non-quantitative opportunistic sampling, even if the number of sampled phorophyte trees was higher on average for the latter method. Repetitive non-quantitative opportunistic sampling of a single site resulted in twice the number of species than one-time sampling, but only two thirds of the number recovered by means of quantitative transect sampling, even of the number of sampled phorophyte trees was highest for this method. The analysis showed that opportunistic sampling fails to detect rare, inconspicuous, sterile, and/or cryptic species, usually neglected or overlooked even by the experienced collector upon visual inspection in the field. On the other hand, quantitative sampling forces even the inexperienced collector to sample lichen thalli or pieces of bark which only after detailed morphological, anatomical, and chemical study in the laboratory reveal themselves as distinct species. Accordingly, of the 456 lichen species collected and identified across the 22 studied localities, no less than 76 were unique to the quantitatively sampled transect, and the lichen species sampled with this method had a significantly higher proportion of rare, inconspicuous, sterile, and/or cryptic species. It is also apparent that it is not the higher number of specimens collected via quantitative sampling that results in a higher number of species, but the method of selection of the specimens, which is subjective and biased towards abundant, conspicuous, fertile and/or distinctive species in opportunistic sampling, but objective and unbiased in quantitative sampling.     

Factors influencing the distribution of nests of the Argentine ant Linepithema humile Mayr (hymenoptera: formicidae), in a foothills ecosystem of the central zone of Chile

The purpose of this research was to demonstrate the dependant relationship between the Argentine ant Linepithema humile Mayr, plant coverage, and human settlements. A method was designed in order to sample the presence of the Argentine ant and other ant species that may had influenced L. humile distribution, under different types of vegetation and percentages of plant coverage, taking in consideration the distance from human settlements. Eight sample stations were established. Four of these stations were found on a dwelling home and the other four were found on similar areas, but far from human settlements. Three transects were established from the center of each sampling station (Tt1, shrublike vegetation, Tt2, herblike vegetation and Tt3, no vegetation). Transects were compound by 10 m side quadrants. A negative correlation was found between nest number of L. humile and human settlement distance, therefore nests were not found at distances higher than 80 m, in all studied areas. There was a positive and significant correlation between nest number of L. humile and plant coverage percentage, regardless the type of vegetation found. The same trend was found for other ant species studied in the area. Finally, it was proved that the distribution of L. humile nests is not influenced by other ant species.     

两种珍稀植物群落物种多度分布的核方法研究

首次提出物种多度分布的非参数核密度估计方法,介绍了此方法的构造和主要性质。珍稀濒危植物观光木群落和长苞铁杉群落的乔木层、灌木层、所有木本植物物种多度分布实例拟合结果表明,核方法能很好地描述群落物种多度分布。非参数核估计方法是群落物种多度分布模拟的一种有效方法,它丰富了物种多度分布拟合方法,为珍稀濒危植物的管理与保护提供了理论参考。     

A sensitive multidimensional method for the detection,characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows.