A family of pneumatically-operated thin layer samplers for replicate sampling of heterogeneous water columns

A series of inexpensive, pneumatic thin layer water samplers is described. They can be operated from small boats, and permit sampling at 2.5 cm intervals with little or no disturbance of stratified systems such as oxyclines or redoxclines of meromictic lakes, or microstratification of flagellates in sheltered epilimnia. Some models permit replicate sampling at closely-spaced intervals in a two-dimensional array. Their performance abilities are illustrated with examples of microstratification.     

Optimal allocation of replicates for measurement evaluation studies

Optimal experimental design is important for the efficient use of modern highthroughput technologies such as microarrays and proteomics. Multiple factors including the reliability of measurement system, which itself must be estimated from prior experimental work, could influence design decisions. In this study, we describe how the optimal number of replicate measures （technical replicates） for each biological sample （biological replicate） can be determined. Different allocations of biological and technical replicates were evaluated by minimizing the variance of the ratio of technical variance （measurement error） to the total variance （sum of sampling error and measurement error）. We demonstrate that if the number of biological replicates and the number of technical replicates per biological sample are variable, while the total number of available measures is fixed, then the optimal allocation of replicates for measurement evaluation experiments requires two technical replicates for each biological replicate. Therefore, it is recommended to use two technical replicates for each biological replicate if the goal is to evaluate the reproducibility of measurements.     

Use of invertebrate traits for the biomonitoring of European large rivers: the effects of sampling effort on genus richness and functional diversity

1. Studies on biodiversity and ecosystem function require considering metrics for accurately describing the functional diversity of communities. The number of taxa (richness) is commonly used to characterise biological diversity. The disadvantage of richness as a measure of biological diversity is that all taxa are taken into account on an equal basis regardless of their abundance, their biological characteristics or their function in the ecosystem. 2. To circumvent this problem, we applied a recently described measure of biological diversity that incorporates dissimilarities among taxa. Dissimilarities were defined from biological traits (e.g. life history, morphology, physiology and behaviour) of stream invertebrate taxa and the resulting biological diversity index was considered as a surrogate for functional diversity. 3. As sampling effort is known to affect the number of taxa collected within a reach, we investigated how change in functional diversity is affected by sampling effort. We used stream invertebrate community data from three large European rivers to model accumulation curves and to assess the number of samples required to estimate (i.e. closeness to the maximal value) functional diversity and genera richness. We further evaluated the precision of estimates (i.e. similarity of temporal or spatial replicates) of the total functional diversity. 4. As expected, richness estimates were strongly dependent on sampling effort, and 10 replicate samples were found to underestimate actual richness. Moreover, richness estimates showed much variation with season and location. In contrast, functional diversity had greater accuracy with less sampling effort and the precision of the estimates was higher than richness both across sampling occasions and sampling reaches. These results are further arguments towards conducting research on the design of a biomonitoring tool based on biological traits.     

Subsampling Variance for 2,4-DNT in Firing Point Soils

At 105-mm howitzer firing points, 2,4-DNT is detectable in the surface soils, but field sampling and laboratory subsampling uncertainty can be large during quantitation. The 2,4-DNT is in particulate form, within fibers or slivers of the nitrocellulose-based propellant. The slender fibers range up to 7.5 mm in length with masses of several 100 μ g. Size fractionation of a firing point soil revealed that most of the 2,4-DNT was in the 0.595- to 2.00-mm size range, although the bulk of the soil was less than 0.6 mm prior to grinding. Machine grinding for five minutes was needed to pulverize the propellant fibers sufficiently so that estimates of 2,4-DNT were reproducible in replicate analytical subsamples. To determine 2,4-DNT, we have adopted the practice of grinding firing point soils for five one-minute intervals, with time for heat dissipation between grinds, prior to obtaining individual or replicate 10-g subsamples.     

Characterization of variability in large-scale gene expression data: implications for study design

Large-scale gene expression measurement techniques provide a unique opportunity to gain insight into biological processes under normal and pathological conditions. To interpret the changes in expression profiles for thousands of genes, we face the nontrivial problem of understanding the significance of these changes. In practice, the sources of background variability in expression data can be divided into three categories: technical, physiological, and sampling. To assess the relative importance of these sources of background variation, we generated replicate gene expression profiles on high-density Affymetrix GeneChip oligonucleotide arrays, using either identical RNA samples or RNA samples obtained under similar biological states. We derived a novel measure of dispersion in two-way comparisons, using a linear characteristic function. When comparing expression profiles from replicate tests using the same RNA sample (a test for technical variability), we observed a level of dispersion similar to the pattern obtained with RNA samples from replicate cultures of the same cell line (a test for physiological variability). On the other hand, a higher level of dispersion was observed when tissue samples of different animals were compared (an example of sampling variability). This implies that, in experiments in which samples from different subjects are used, the variation induced by the stimulus may be masked by non-stimuli-related differences in the subjects' biological state. These analyses underscore the need for replica experiments to reliably interpret large-scale expression data sets, even with simple microarray experiments.     

The estimation and applicability of confidence intervals for Stander's Similarity Index (SIMI) in algal assemblage comparisons

Stander's Similarity Index (SIMI) has become a popular measure for comparing algal assemblages. The interpretation of the value produced by this index, however, has been highly variable between studies. Using replicate sampling of natural algal assemblages and computer simulation techniques, a method by which confidence intervals could be applied to SIMI was developed. This paper presents that method and gives an example using hypothetical species counts of two algal assemblages. Since a more realistic range of the actual similarity between two assemblages is produced, the estimation of confidence intervals when using SIMI is recommended.     

Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ)

We assess the reliability of isobaric-tags for relative and absolute quantitation (iTRAQ), based on different types of replicate analyses taking into account technical, experimental, and biological variations. In total, 10 iTRAQ experiments were analyzed across three domains of life involving Saccharomyces cerevisiae KAY446, Sulfolobus solfataricus P2, and Synechocystis sp. PCC 6803. The coverage of protein expression of iTRAQ analysis increases as the variation tolerance increases. In brief, a cutoff point at +/-50% variation (+/-0.50) would yield 88% coverage in quantification based on an analysis of biological replicates. Technical replicate analysis produces a higher coverage level of 95% at a lower cutoff point of +/-30% variation. Experimental or iTRAQ variations exhibit similar behavior as biological variations, which suggest that most of the measurable deviations come from biological variations. These findings underline the importance of replicate analysis as a validation tool and benchmarking technique in protein expression analysis.     

Aspects of statistics in studies of cell proliferation. I. Multistage sampling

In cell kinetic experiments, as in many other branches of biological science, we often sample at various stages of an experiment: we may for example take a sample of animals, then from each study a sample of sites, and from each site take replicate observations. This sampling process can be optimized to give maximum precision to an estimated quantity, but care must be taken in analysing data so gathered because the analysis depends on the precise sampling strategy.     

Aspects of Statistics In Studies of Cell Proliferation

In cell kinetic experiments, as in many other branches of biological science, we often sample at various stages of an experiment: we may for example take a sample of animals, then from each study a sample of sites, and from each site take replicate observations. This sampling process can be optimized to give maximum precision to an estimated quantity, but care must be taken in analysing data so gathered because the analysis depends on the precise sampling strategy.     

A Vessel for the Replicate Culture of Cells in Monolayer

Modification of a commercially available Carrel-type of disposable plastic flask with 10 compartments, by removing the central junctions of the two middle divisions to create 6 intercommunicating chambers, provides a vessel for easy preparation and handling of exact replicate cell cultures on coverglasses. Periodic sampling, fluid changes, attainment of identical inoculation and culture conditions, and microscopic observation are greatly facilitated in radio-isotope, virological and pharmacological experiments.     

Occupancy Models for Monitoring Marine Fish: A Bayesian Hierarchical Approach to Model Imperfect Detection with a Novel Gear Combination

Occupancy models using incidence data collected repeatedly at sites across the range of a population are increasingly employed to infer patterns and processes influencing population distribution and dynamics. While such work is common in terrestrial systems, fewer examples exist in marine applications. This disparity likely exists because the replicate samples required by these models to account for imperfect detection are often impractical to obtain when surveying aquatic organisms, particularly fishes. We employ simultaneous sampling using fish traps and novel underwater camera observations to generate the requisite replicate samples for occupancy models of red snapper, a reef fish species. Since the replicate samples are collected simultaneously by multiple sampling devices, many typical problems encountered when obtaining replicate observations are avoided. Our results suggest that augmenting traditional fish trap sampling with camera observations not only doubled the probability of detecting red snapper in reef habitats off the Southeast coast of the United States, but supplied the necessary observations to infer factors influencing population distribution and abundance while accounting for imperfect detection. We found that detection probabilities tended to be higher for camera traps than traditional fish traps. Furthermore, camera trap detections were influenced by the current direction and turbidity of the water, indicating that collecting data on these variables is important for future monitoring. These models indicate that the distribution and abundance of this species is more heavily influenced by latitude and depth than by micro-scale reef characteristics lending credence to previous characterizations of red snapper as a reef habitat generalist. This study demonstrates the utility of simultaneous sampling devices, including camera traps, in aquatic environments to inform occupancy models and account for imperfect detection when describing factors influencing fish population distribution and dynamics.     

Viruses and the microbial loop

The abundance of viral-like particles in marine ecosystems ranges from <104 ml^–1 to >10⁸ ml^–1. Their distribution in time and space parallels that of other biological parameters such as bacterial abundance and chlorophyll a. There is a lack of consensus between methods used to assess viral activity, i.e., rate of change in viral abundance (increase or decrease). The highest rates, 10–100 days^–1, are observed in experiments with short sampling intervals (0.2–2 h), while lower rates, on the order of 1 day^–1, are observed in experiments with longer sampling intervals (days). Few studies have been carried out, but viruses appear, at least in some cases, to have a significant impact on carbon and nutrient flow in microbial food webs. Viruses have also been demonstrated to exert a species specific control of both bacteria and phytoplankton populations in natural waters.     

Description and evaluation of a sampling strategy for macroinvertebrate communities in Basque rivers (Spain)

This paper describes the results of a pilot study on the macroinvertebrate communities occurring in a fast-flowing river in Northern Spain. The objective was to devise a sampling strategy for use on a series of Basque rivers included in a site classification study. Two contrasted sites were chosen for the pilot-study on the River Lea (Bizkaia), at the headwaters and near the mouth. Taxon richness and distribution across the river was examined using two sampling techniques at each site. It was concluded that, ideally, timed sampling units should be collected at intervals across a river transect in order to acquire a representative list of taxa. In addition, adequate sampling in marginal areas was stressed because a number of taxa appeared to be exclusive to marginal areas. Sampling location and total sampling effort were also shown to be a major consideration when taxon lists were acquired for the application of routine biological surveillance techniques.     

Sample pooling obscures diversity patterns in intertidal ciliate community composition and structure

The aim of this study was to assess the effect of sample pooling on the portrayal of ciliate community structure and composition in intertidal sediment samples. Molecular ciliate community profiles were obtained from nine biological replicates distributed in three discrete sampling plots and from samples that were pooled prior to RNA extraction using terminal restriction fragment polymorphism (T-RFLP) analyses of SSU rRNA. Comparing the individual replicates of one sampling plot with each other, we found a differential variability among the individual biological replicates. T-RFLP profiles of pooled samples displayed a significantly different community composition compared with the cumulative individual biological replicate samples. We conclude that sample pooling obscures diversity patterns in ciliate and possibly also other microbial eukaryote studies. However, differences between pooled samples and replicates were less pronounced when community structure was analyzed. We found that the most abundant T-RFLP peaks were generally shared between biological replicates and pooled samples. Assuming that the most abundant taxa in an ecosystem under study are also the ones driving ecosystem processes, sample pooling may still be effective for the analyses of ecological key players.     

Sources of measurement error in assays of EROD activity of fish for biological effects monitoring

The paper describes the technical, procedural and biological aspects of 7-ethoxyresorufin O-deethylase (EROD) activity measurements in dab, Limanda limanda, from the North Sea. Several sources of measurement error in assays of EROD activity measurements for biological effects monitoring exercises could be defined, particularly for assay conditions, sample storage and sampling. In addition to variability caused by technical errors, biological features inherent in the surveyed fish may lead to even more severe errors in EROD activity measurements. This is particularly true of the choice of sampling time. Disregarding the species-specific spawning periods may lead to a loss of activity by a factor of as much as 90 in females and 40 in males, thus making it necessary to design sampling surveys appropriately. Received: 14 April 1999 / Received in revised form: 10 July 1999 / Accepted: 15 July 1999     

Efficient sampling design for larvae of American lobster

For 13 years, lobster larvae were collected from the same 23stations at approximately weekly intervals for the durationof the larval season (Scarratt, 1964). Using the most complete8 years of collections, we produced several data subsets foreach year by reducing the number of sampling dates and the numberof stations per date. Our analyses were confined to the fourthand final larval stage, the most likely stage to be correlatedwith fishery yields. The means and confidence intervals forannual larval abundance were calculated on the data subsetsand compared with the full dataset. We conclude that the usuallobster larval sampling frequency of weekly, or more often,was unnecessary. Tows taken at 10 or 14 stations per week for4 weeks of the 9 week larval season represented well the meanand confidence intervals on the full dataset. These choicesremained the best after the costs of sampling were included.Further cost reduction could be achieved without loss of precisionby removing collections from the net after every second, ratherthan after every tow. Mean annual density was found to be agood index of annual larval production. Thus, the collectionsduring 4 weeks represented annual abundance and annual production.     

Protocol for efficient plasma sampling for low density lipoprotein turnover studies

Radiolabeled low density lipoprotein (LDL) is commonly used to study the turnover of LDL apolipoprotein B (apoB), the major protein component of LDL. Following an intravenous injection of radioiodinated LDL, typical sampling schedules have including 20-25 samples over a 14-day period with frequent sampling during the first 12 hr and daily samples thereafter. This is a burdensome task for subjects and investigators. To improve acceptance of the procedure, we have examined the effects of reduced sampling schedules upon the estimation of the fractional catabolic rate (FCR) for LDL apoB. Data from 36 different sets of LDL decay curves obtained from investigations of subjects with a variety of lipoprotein phenotypes have been used to test these schedules. Our results indicate that by choosing specific intervals over a 14-day period only 10 samples are sufficient to accurately determine the fractional catabolic rate for LDL in plasma. This reduced sampling schedule should facilitate the study of LDL turnover in large groups of subjects as outpatients.     

Robust regression for periodicity detection in non-uniformly sampled time-course gene expression data

Background  

In practice many biological time series measurements, including gene microarrays, are conducted at time points that seem to be interesting in the biologist's opinion and not necessarily at fixed time intervals. In many circumstances we are interested in finding targets that are expressed periodically. To tackle the problems of uneven sampling and unknown type of noise in periodicity detection, we propose to use robust regression.     

Effect of sampling interval and temperature on the accuracy of food consumption estimates from stomach contents

The effect of temperature and sampling interval on the accuracy of food consumption estimates based on stomach contents was studied using simulation. Three temporal patterns of feeding were considered (scattered throughout the day, one 5 h period or two 5 h periods) and gastric evacuation was modelled according to published values. Sampling intervals of 3 h gave reasonable food consumption estimates (2 to 19% error) at all temperatures. Comparably, sampling intervals as large as 12 h gave reasonable estimates of food consumption (1 to 20% error) when temperature was set to ≤10° C. At temperatures <5° C, even 24 h intervals (equivalent to one daily sampling) provided reasonable estimates of daily food consumption (2 to 19% error) for all but the highest gastric evacuation rate combined with one daily feeding period (47% error). The temperature effect on estimation error resulted from diminishing temporal fluctuations in stomach contents with slower gastric evacuation rates. It follows that sampling effort may be considerably minimized when estimating food consumption from stomach contents during periods with low temperatures such as the winter time experienced by temperate fishes.     

Effect of sampling interval on serum concentrations of luteinizing hormone, follicle-stimulating hormone, and prolactin in prepubertal, ovariectomized, and cycling gilts.

Three experiments were conducted to determine the effect of sampling interval on serum concentrations of LH, FSH, and prolactin (PRL) in prepubertal, ovariectomized, and cycling gilts. In all experiments, blood samples were drawn at 2-min intervals for 4 h from indwelling jugular catheters. Mean serum hormone concentrations, mean number of peaks, and mean and maximum peak heights of LH, FSH, and PRL were calculated using values reflecting 2-, 6-, 10-, 20-, 30-, and 60-min sampling intervals. For LH, FSH, and PRL, mean serum concentrations can be obtained through blood samples drawn at hourly intervals. Since LH peaks are very distinct in pigs, the number of secretory peaks and mean peak height can be obtained via samples drawn at 20-min intervals. Since FSH and PRL peaks are less well defined, a more frequent sampling interval (10 min) is needed to determine number of peaks and mean peak height. To obtain the maximum peak height or the number of minutes for LH, FSH, or PRL to rise from its nadir to zenith, blood samples need to be drawn at 2-min intervals. Regardless of reproductive state, these data indicate that the sampling interval needed to characterize serum concentrations of LH, FSH, and PRL in the gilt is dependent upon the parameter in question.