A computerized plankton counter

A portable computerized plankton counting system is presented, which has the potential to enumerate 255 different species per sample. The software is used to count phytoplankton samples and produce summary data. The hardware allows the results to be reproduced in the form of a hardcopy, stored on a cassette tape, or transferred to a larger computer for analysis.     

Rapid and versatile routine measurements of plankton biovolumes with BACCHUS

A semi-automated biovolume measurement application called BACCHUS (Biovolume Analysis Cum Calculus and High Utility Statistics), based on an image-analysis program combined with a database, was developed. With this application, biovolumes of plankton taxa can be determined while counting routine samples. Statistical analyses of the ln-transformed results are performed on-line. The width of the 95% confidence interval is calculated and indicates the precision of the calculated mean. By setting limits to the maximum allowed width of the interval, the extent of desired precision can be defined. For our laboratory, we decided that the limits of the confidence interval must be <0.2 ln units above or below the mean of the ln-transformed results. As soon as this criterion is reached, the user can stop measuring, thereby minimizing the number of measurements necessary to reach a mean biovolume which is sufficiently precise. Other advantages of the BACCHUS application are a very flexible input of master data, output of stored data and a user-friendly operation environment. BACCHUS is very easy to customize because it does not require any expertise in database programming.      

A microcomputer based aid for counting plankton samples

A microcomputer system which eases the task of counting mixedcommunities of phytoplankton and zooplankton using an opticalmicroscope, is described.     

Metagenome-based analysis: A promising direction for plankton ecological studies

The plankton community plays an especially important role in the functioning of aquatic ecosystems and also in biogeochemical cycles. Since the beginning of marine research expeditions in the 1870s, an enormous number of planktonic organisms have been described and studied. Plankton investigation has become one of the most important areas of aquatic ecological study, as well as a crucial component of aquatic environmental evaluation. Nonetheless, traditional investigations have mainly focused on morphospecies composition, abundances and dynamics, which primarily depend on morphological identification and counting under microscopes. However, for many species/groups, with few readily observable characteristics, morphological identification and counting have historically been a difficult task. Over the past decades, microbiologists have endeavored to apply and extend molecular techniques to address questions in microbial ecology. These culture-independent studies have generated new insights into microbial ecology. One such strategy, metagenome-based analysis, has also proved to be a powerful tool for plankton research. This mini-review presents a brief history of plankton research using morphological and metagenome- based approaches and the potential applications and further directions of metagenomic analyses in plankton ecological studies are discussed. The use of metagenome-based approaches for plankton ecological study in aquatic ecosystems is encouraged.     

Computer simulation of error rates of Poisson-based interval estimates of plankton abundance

The routine assignment of error rates (confidence intervals) to Poisson distribution estimates of plankton abundance should be rejected. In addition to the interval estimation procedure being pseudoreplicative, it is not robust to common violations of its assumptions. Because the spatial dispersion of organisms in sampling units from the counting chamber to the field is rarely random and because counting protocols are usually terminated by a count threshold having been equalled or exceeded, Poisson based estimates are usually derived from sampling non-Poisson distributions. Computer simulation was used to investigate the quantitative consequences of such estimates. The expected mean error rate of 95% confidence intervals is inflated from 5% to 15% as contagion increases, as the parametric variance-mean ratio increases from 1 to 2. Also, count threshold termination of the counting protocol effects both a biased estimate of the parametric mean (or total) and alters expected mean error rates, especially if the total count is low (< 100 organisms) and the mean density in the sampling unit is low.     

A comparison of two methods for evaluating drug-induced chromosome alterations.

Evaluating the technique and procedure for mutagenicity testing in mammals is a prerequisite to the development of a broad spectrum mutagenic assessment program. Two techniques, chromosome examination and micronucleus scoring, show promise but their applicability for mass screening is uncertain. We determined the slide observation time for these two techniques in mice treated orally, subcutaneously, intravenously, and intraperitoneally with cyclophosphamide (CY). In each instance, we detected a dose-response in less observation time by counting micronuclei in polychromatophilic erythrocytes. The simplicity of the scoring method, the ease of micronucleus identification and the rapidity of scoring all suggest the micronucleus test may be favorably integrated into a mutagenicity screening program.     

Estimation of the biomass of plankton

Summary A mathematical function is demonstrated in the numbers of individuals of the various species in a planktonic biocoenosis. The logarithms of the numbers form a Gauss or normal probability curve. A similar probability relation is found in the volumes of the individuals of the various species as well as in the biomass of the various populations.This relationship in the numbers is caused by the effect of the numerous ecological factors influencing the rate of proliferation of the various plankton species. The cause of this relationship concerning the volumes of the various species is not understood. The relationship between the various biomasses is the mathematical product of number and mean volume.An approximate hyperbolic function can be derived from the population volumes and with the aid of a simple equation the plankton biomass is calculated. A modus operandi is given to abbreviate the work necessary to determine the plankton biomass with Lohmann's method. Only ten or twenty of the most dominant populations out of all species present in a plankton sample, have to be counted and measured.The biomasses of the populations in various plankton samples may easily be compared using the hyperbolic or the probability relationship.The biomasses of plankton in various habitats may easily be compared in a graphic way. The logarithms of the biomasses found during the year follow a probability curve and may be plotted and compared on a cumulative logarithmic probability graph.The number of organisms of each species to be counted depends on the degree of accuracy and has to be about a hundred. A chance determined spread is always found in plankton counts.The spatial distribution of most plankters shows a very broad spread. Therefore, sampling at ten places and working with the mean of the ten samples is compulsory.Some gregariously living zooplankters form bunches in the water. A reliable mean may be calculated using the hyperbolic function which seems to describe their densities at the various places.From the existing methods of collection of plankton the rotary, electric pump is chosen. A translucent hose with a special and moving mouth is let down into the water. First the water passes through the plankton-net and after that through the pump and the water-meter. A series of 7 samples of increasing decimal volumes is drawn in this way. From these samples the plankton is concentrated and fractionated by means of two sedimentation chambers, four small plankton sieves and three plankton nets. The sieves and nets have various standardized meshes.Square counting chambers of 10 cm² area are used. These chambers have a thin glass bottom and a broad rim. The sedimentation chambers and the small plankton sieves fit on and into the chambers thus minimizing the loss of organisms.The plankton organisms are enumerated by means of an inverted microscope projecting the image on a ground glass which makes counting easier. Only those organisms seen within a measured square on the ground glass are counted.By standardization of the sample volumes, the magnifications of the microscope and the dimensions of the squares the conversion factors are so simple that only zeros or a decimal point have to be placed in the number counted to obtain the result.International standardization of the method of estimation of the biomass of plankton and the expression of the results is proposed.     

Community structure and function in prokaryotic marine plankton

Molecular biodiversity studies of microbial communities have provided invaluable information on the existence of heretofore unknown organisms and on community composition. Cloning and ‘fingerprinting’ techniques have been used many times to study prokaryote community composition of marine plankton. There are still many opportunities for new discoveries in this area, but the results have also opened new questions about the activities of these organisms and their function, going beyond just listing taxa or counting organisms. Rarely can the broad function be inferred from phylogenetic position alone (e.g. cyanobacteria). The recent discovery of abundant non-cyanobacterial marine phototrophs points to our inability to link phylogenetic position with function in a detailed way. One approach we have found fruitful is to combine fluorescence in situ hybridization with microautoradiography, a technique dubbed STARFISH. A recent application has shown that ubiquitous archaea from the deep sea, phylogenetically related to extreme thermophiles, are active in the uptake of amino acids from ambient (nanomolar) concentrations. This suggests the group is at least partly heterotrophic and able to compete successfully with bacteria for nutrients. Other as-yet uncultivated groups are also amenable to similar studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

根瘤菌生态学研究中两个数学模型的探讨

以自然重壤土和无菌砂为培养基质,栽种大豆,采用MPN法测数,证明了大豆根瘤菌数量的增长符合逻辑斯谛模型并编写了相应的BASIC程序。通过6组试验数据的计算,发现根瘤菌接种量和其占瘤率之间存在一定的数学关系,并编写了相应的BASIC程序。     

Computer-based image analysis for the automated counting and morphological description of microalgae in culture

A largely unexplored area is the application of digital image processing to counting and sizing of microalgal cells from culture. Commercial systems are available, but have not been tested, nor necessarily optimized for high speed counting and sizing of phytoplankton. The present work describes the design, construction, specifications and comparative performance of an inexpensive system optimized for counting and sizing microalgal cells. This system has been tested with cells of the picoplankton to nanoplankton size ranges (1–20 μm). The hardware was a widely available standard microcomputer, an inexpensive video camera and monitor, and a video digitization board (frame grabber). A modifiable menu-driven program (PHYCOUNT) was written and provisions made to make this program available to other workers. The program is constructed such that it can be adapted to a variety of hardware setups Video digitization boards). Comparison of growth curves for microagae revealed there were no significant differences in division rate and cell yield as assessed by the image analysis method compared to manual counts with a hemacytometer. Several hundred cells were counted routinely within 10–15 s, far exceeding the counting rate achieved by hand tally. A variable transect feature allowed sampling every nth pixel and provided a substantial increase in execution speed. More than 1000 counts can be done per day. A protocol for the use of 96-well plates of polyvinyl chloride as counting chambers contributed to the processing of large numbers of samples rapidly. Other routines developed provided subtended area, defined the coordinates of cell perimeter, and derived cell length and width. The calculation of the latter two parameters was usually done off-line as data output is in standard numerical form accessible by other programs. Experience with daily use of the PHYCOUNT program and imaging hardware reveal that the system is reliable for cell counting and sizing. The presence of bacteria in the algal cultures does not affect cell counting or sizing.     

Algamica: revisions to a key-based computerized counting program for free-living,attached, and benthic algae

A computerized counting program for algae and other microscopic bodies, named Algamica, is presented here. This program is a revised version of the original computer counting program of Hamilton published in 1990. This DOS-based software can enumerate all types of microscopic algae (i.e. phytoplankton, periphyton, diatoms), for which adequate expression of results are provided. Automated calculations of densities, biovolumes, surface areas and carbon biomass are available at the termination of each sample count. A simple counter for other micro-organisms is also available. A comprehensive guide manual file has been added to allow for a friendly first contact with the program and its options. This software conforms to current enumeration methodology. This version is available for PC computers, from website Algamica.ibelgique.com. Minimum required memory is 200 KB.     

A sequential approach to the counting of plankton organisms

A counting strategy is described by means of which it is possible to decide, after carrying out a few counts, whether a sampled plankton population can be considered homogeneously distributed. The time saving of the proposed strategy lies in the fact that if the underlying population is homogeneously distributed only one count, i.e., the one of the mixture of aliquots taken from all individual samples collected, would be necessary to characterize the population sufficiently. The count of this mixture may be considered as a ‘mean value’ equivalent to the mean obtained from the counts of all individual samples.With some experience in predicting where homogeneously and where heterogeneously distributed populations are sampled, it is possible to mix large numbers of samples and thus save a great amount of effort in the analysis of plankton populations. The samples may be collected during a survey in any way desired, e.g., in normal hydrographie casts, with pumps, with the air bubble lift etc. A numerical example is given in the Appendix illustrating the method.     

MUSCLE: multiple sequence alignment with high accuracy and high throughput

We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.     

Dynamic analysis of multivariate failure time data

We present an approach for analyzing internal dependencies in counting processes. This covers the case with repeated events on each of a number of individuals, and more generally, the situation where several processes are observed for each individual. We define dynamic covariates, i.e., covariates depending on the past of the processes. The statistical analysis is performed mainly by the nonparametric additive approach. This yields a method for analyzing multivariate survival data, which is an alternative to the frailty approach. We present cumulative regression plots, statistical tests, residual plots, and a hat matrix plot for studying outliers. A program in R and S-PLUS for analyzing survival data with the additive regression model is available on the web site http://www.med.uio.no/imb/stat/addreg. The program has been developed to fit the counting process framework.     

Time series are critical to understand microbial plankton diversity and ecology

How diverse are marine planktonic protist communities? How much seasonality do they exhibit? For a very long time, these two old and challenging questions in the field of plankton ecology could be addressed only for large‐size protist species, based on cell counting under the microscope. The recent application of molecular techniques, notably massive marker‐gene amplicon sequencing approaches (metabarcoding), has allowed investigating with unprecedented level of resolution the small‐sized (<20 µm) planktonic eukaryotes too. An amazing diversity of these tiny organisms has been unveiled but details about their temporal dynamics remain much more elusive. In a From the Cover article in this issue of Molecular Ecology, Giner et al. (2019) introduce a new Recurrence Index (RI) to specifically look for seasonality in time‐series metabarcoding data. They inspected the temporal dynamics of all operational taxonomic units (OTUs) in a rich sequence data set of pico‐ and nanoplanktonic eukaryotes in samples collected monthly during 10 years. Although most OTUs did not show seasonality, some abundant ones did, which explains why some averaging methods can find seasonality at the less detailed level of whole planktonic communities. Not surprisingly, the very complex small‐sized eukaryotic plankton communities are composed of organisms with miscellaneous temporal dynamics.     

Comparing models and observations of shelf plankton

In a previous paper (Solow and Steele, J. Plankton Res., 17,1995), the differences between variability in zooplankton biomassand in copepod stage structure were demonstrated using datafrom the northern North Sea. Here, a model is used to describethe underlying demographic processes and the effects of interannualphysical variability. Comparison of output with observationscan test the theory and so help to reconcile the apparent contradictionbetween great variability and persistence in these populations.     

Use of the FlowCAM for semi-automated recognition and enumeration of red tide cells (Karenia brevis) in natural plankton samples

Early detection is the most effective way to mitigate the effects of harmful algal blooms (HAB). Cell counts based on examination of microplankton samples using settling chambers and visual inspection with an inverted microscope are tedious and time consuming, and counting precision is generally poor at low cell densities. The FlowCAM is a continuous imaging flow cytometer designed to characterize particles in the microplankton size range (20–200 μm diameter). In this study we examined the ability of the FlowCAM to improve routine monitoring protocols for HAB species by automatically recording information on size and fluorescence per cell. This will eliminate the need to examine cells outside the ranges of these measurements for our target species, Karenia brevis. We also tested the ability of image comparison software to match images of cells in mixed assemblages to images of the target species. For simple mixtures of cultured dinoflagellates, the ability of the image matching software to discriminate target cells varied greatly depending on how similar the two species were in size and shape. When target cells were added to natural plankton samples, the image recognition software correctly identified 80–90% of the target cells, but misidentified 20–50% of non-target cells in the size range of the target species. We conclude that the FlowCAM is less tedious and time-consuming than microscopy, allowing for examination of more cells for greater counting precision. The cell recognition software helps reduce the numbers of cells that must be screened, but images must still be examined by a trained operator to identify the HAB species of interest.     

Computer-aided quantification of RNA levels detected by in situ hybridization of tissue sections.

A semi-automatic program, designed for non-computer scientists, was developed for quantification of RNA levels detected by in situ hybridization in heterogeneous tissues. A video camera was used to acquire microscopic images of autoradiographed tissue sections which are then digitized on a video monitor for semi-automated quantification of silver grains. We describe a data entry and analysis procedure for systematic quantification of RNA levels in which about 300 cells per tissue sample can be analysed within 10 min. When compared with visual counting, computer-aided quantification was found to be more objective and reliable, with the highest variation coefficient between individuals being 7.5% using computer-aided quantification, compared to 24% with manual counting of the same section areas. A comparative study of c-myc oncogene expression in 11 mammary adenocarcinomas from 3 independent experiments showed the good reproducibility of results using the computer-aided method, with an 18% maximum variation between experiments. The program, with its simple user-interface, reliability and rapidity, is convenient for measuring specific genetic expression levels in clinical studies requiring large numbers of specimens.     

A new method for the automated age-at-death evaluation by tooth-cementum annulation (TCA)

A valid age at death estimation is required in historical and also forensic anthropology. Tooth cementum annulation (TCA) is a method for age at death estimation of adult individuals. The method is based on light microscope images taken from tooth-root cross sections. The age is then estimated by manually counting the cementum incremental lines and adding this to the chronological age at the assumed point of tooth eruption. Manual line counting, however, is time consuming, potentially subjective and the number of individual counts is insufficient for statistical evaluations. Software developed for the automated evaluation of TCA images, that uses Fourier analysis and algorithms for image analysis and pattern recognition is presented here. It involves "line-by-line" scanning and the counting of gray scale peaks within a selected region-of-interest (ROI). Each scanning process of a particular ROI yields up to 400 counts that are subsequently statistically evaluated. This simple and time saving program seeks to substitute manual counting and supply consistent and reproducible results as well as reduce the demand of human error by eliminating unavoidable factors such as subjectivity and fatigue.     

Genetic similarity of polyploids: a new version of the computer program POPDIST (version 1.2.0) considers intraspecific genetic differentiation

For evolutionary studies of polyploid species estimates of the genetic identity between species with different degrees of ploidy are particularly required because gene counting in samples of polyploid individuals often cannot be done, e.g., in triploids the phenotype AB can be genotypically either ABB or AAB. We recently suggested a genetic distance measure that is based on phenotype counting and made available the computer program POPDIST. The program provides maximum-likelihood estimates of the genetic identities and distances between polyploid populations, but this approach is not informative for populations within species that only differ in their allele frequencies. We now close this gap by applying the frequencies of shared 'bands' in both populations to Nei's identity measure. Our simulation study demonstrates the close correlation between the band-sharing identity and the genetic identity calculated on the basis of gene frequencies for any degree of ploidy. The new extended version of POPDIST (version 1.2.0) provides the option of choosing either the maximum-likelihood estimator or the band-sharing measure.