The revised edition of a computerized plankton counter for plankton,periphyton and sediment diatom analyses

A revised computer counting program is presented with faster processing and more efficient features to enhance counting precision. The program is now capable of counting epilithic communities and sediment samples for diatom analysis. This version (2.3) is available for APPLE, IBM, TANDY-RADIO SHACK, and HYPERION systems using standard Basic commands. New improvements include, density and biovolume calculations, a Chi square transect test for randomness, D'Agostino's test for a normal taxa distribution and a three file output with raw and processed data.     

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.     

A cost-effective maize ear phenotyping platform enables rapid categorization and quantification of kernels

High-throughput phenotyping systems are powerful, dramatically changing our ability to document, measure, and detect biological phenomena. Here, we describe a cost-effective combination of a custom-built imaging platform and deep-learning-based computer vision pipeline. A minimal version of the maize (Zea mays) ear scanner was built with low-cost and readily available parts. The scanner rotates a maize ear while a digital camera captures a video of the surface of the ear, which is then digitally flattened into a two-dimensional projection. Segregating GFP and anthocyanin kernel phenotypes are clearly distinguishable in ear projections and can be manually annotated and analyzed using image analysis software. Increased throughput was attained by designing and implementing an automated kernel counting system using transfer learning and a deep learning object detection model. The computer vision model was able to rapidly assess over 390 000 kernels, identifying male-specific transmission defects across a wide range of GFP-marked mutant alleles. This includes a previously undescribed defect putatively associated with mutation of Zm00001d002824, a gene predicted to encode a vacuolar processing enzyme. Thus, by using this system, the quantification of transmission data and other ear and kernel phenotypes can be accelerated and scaled to generate large datasets for robust analyses.     

DIRECT ENUMERATION OF BACTERIA FROM MACROALGAE BY EPIFLUORESCENCE MICROSCOPY AS APPLIED TO THE FLESHY RED ALGAE KAPPAPHYCUS ALVAREZII AND GRACILARIA SPP. (RHODOPHYTA)1

This report describes a simplified method for direct counting of total bacteria associated with the fleshy red algae Kappaphycus alvarezii (Doty) Doty and Gracilaria spp. A Nuclepore® polycarbonate membrane (0.2–μm pore size) fitted to a vacuum filtration apparatus was used to filter algal tissue homogenate after serial dilution and staining with the fluorochrome 4′,6-diamidino-2-phenylindole. Using epifluorescence microscopy, it is possible to count bacteria without preseparating them from the algae. The technique requires homogenized algal tissue diluted with 0.2-μm-filtered, autoclaved seawater to a level appropriate for counting. Dilution reduces the amount of autofluorescent algal debris, which may interfere with Counting. The membrane filtration method yielded a bacterial count two orders of magnitude higher than that of the conventional agarspread plate technique. This method offers a more accurate approach to counting the total number of bacteria on macroalgae.     

AllelicImbalance: an R/ bioconductor package for detecting,managing, and visualizing allele expression imbalance data from RNA sequencing

Background

One aspect in which RNA sequencing is more valuable than microarray-based methods is the ability to examine the allelic imbalance of the expression of a gene. This process is often a complex task that entails quality control, alignment, and the counting of reads over heterozygous single-nucleotide polymorphisms. Allelic imbalance analysis is subject to technical biases, due to differences in the sequences of the measured alleles. Flexible bioinformatics tools are needed to ease the workflow while retaining as much RNA sequencing information as possible throughout the analysis to detect and address the possible biases.

Results

We present AllelicImblance, a software program that is designed to detect, manage, and visualize allelic imbalances comprehensively. The purpose of this software is to allow users to pose genetic questions in any RNA sequencing experiment quickly, enhancing the general utility of RNA sequencing. The visualization features can reveal notable, non-trivial allelic imbalance behavior over specific regions, such as exons.

Conclusions

The software provides a complete framework to perform allelic imbalance analyses of aligned RNA sequencing data, from detection to visualization, within the robust and versatile management class, ASEset.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0620-2) contains supplementary material, which is available to authorized users.     

Intertidal meiofaunal biodiversity with respect to different algal habitats: a test using phytal ostracodes from Southern California

Rocky intertidal algae harbor a diverse invertebrate meiofauna of arthropods, nematodes and other invertebrates. Despite its ecological importance, relatively little is known about the diversity and composition of this important component of intertidal biodiversity. In this study, we quantified species composition, abundance and distribution of ostracodes, an important group of phytal meiofauna, at two different intertidal areas in southern California. In total, we recovered 22 ostracode species from three different orders (16 podocopids, five myodocopids and one platycopid), nearly a quarter of which could not be assigned to existing taxa. The abundance of ostracodes differed significantly among algal types, with structurally complex algae bearing many more ostracodes per gram of algae than simple forms (blade-like algae and the surfgrass Phyllospadix). Although most ostracode species were recovered from multiple kinds of algae, different algae harbored distinct assemblages that could be discriminated statistically on the basis of relative abundances of ostracode species. This segregation of the ostracode fauna according to algal species is evident even over very short spatial scales (<1 m). Finally, ostracode samples from turf-forming algae were more species rich than samples from other kinds of macroalgae. Since turf-forming algae are easily damaged by human trampling, this component of ostracode biodiversity may be particularly vulnerable to anthropogenic impacts on the intertidal habitat. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: K. Martens     

SOME FACTORS IN THE COMPETITION OR ANTAGONISM AMONG BACTERIA, ALGAE, AND AQUATIC WEEDS

Field observations of changes in the populations of aquatic weeds and phytoplankton have confirmed that aquatic weeds have antagonistic activity toward phytoplankton. Nutritional studies in the laboratory indicate that cultures of the aquatic weeds, Myriophyllum sp., Ceratophyllum sp., and duckweed (Lemma minor L.); liquid cultures of barley (Hordeum vulgare L., Dickson variety); and cultures of the filamentous green algae, Cladophora sp. and Pithophora oedogonium (Mont.) Withrock, will remain relatively free of epiphytes or competing phytoplankton if the cultures are nitrogen-limited. Field observations of Cladophora sp. have confirmed that the growth of epiphytes on the Cladophora is related to conditions of surplus available nitrogen compounds. It is proposed that this antagonistic activity may be due to a “nitrogen sink” effect in which the aquatic weeds or filamentous green algae prevent the growth of contaminating algae by competition for the limited nitrogen compounds available. However, the presence of bacteria-sized organisms which have selective toxicity to certain algae indicates that perhaps multiple factors exist. Discussed are the ecological implications of associations of certain algae with bacteria that have selective toxicities for other species of algae under certain environmental conditions such as nitrogen-limited growth.     

Comparative growth and survival of juvenile hard clams,Mercenaria mercenaria,fed commercially available diets

Hobbyist and researchers often use commercially available phytoplankton concentrates to maintain filter feeding organisms held in their ornamental or experimental tanks. This study investigated the nutritional value of 10 products available commercially for juvenile hard clams, Mercenaria mercenaria. Growth and mortality rates in clams fed these products were compared with those found in clams fed fresh cultures of the microalgae Isochrysis galbana, which is considered an industry standard for supporting growth of juvenile bivalves. Our results show a clear difference in feed nutritional value between non‐living and living commercial diets, and among commercial diets advertised as containing live algae. Overall, results showed that juvenile hard clams fed fresh cultures of I. galbana displayed the best growth and lowest mortality rates, followed by those fed the commercial diet DT's Live Marine Phytoplankton. Growth and mortality rates in unfed controls were similar to those found in clams fed commercial non‐living algae mixes or diets advertised as containing live algae (Phyto‐Feast Live product). Results also showed that the nutritional value of fresh algae (I. galbana) cultures is lost rapidly when cultures are maintained at 4°C, suggesting that algae present in some commercial diets may lose their nutritional value during processing or refrigerated storage. The commercial blend, DT's Live Marine Phytoplankton, seems to represent a good substitute to lab grown algae for clams held in ornamental or experimental aquariums. Zoo Biol 0:1–13, 2006. © 2006 Wiley‐Liss, Inc.     

Diet,food preference,and algal availability for fishes and crabs on intertidal reef communities in southern California

Synopsis Herbivory by wide-ranging fishes is common over tropical reefs, but rare in temperate latitudes where the effects of herbivorous fishes are thought to be minimal. Along the west coast of North America, herbivory by fishes on nearshore reefs is largely restricted to a few members of the Kyphosidae, distributed south of Pt. Conception. This paper presents information on natural diets and results from feeding choice experiments for two abundant kyphosids from intertidal habitats in San Diego, California —Girella nigricans andHermosilla azurea, and similar data for the lined shore crab,Pachygrapsus crassipes, which also forages over intertidal reefs. These results are compared with the availability of algae in intertidal habitats measured during summer and winter, on both disturbed and undisturbed habitats. The diets of juveniles ofG. nigricans andH. azurea collected from nearshore habitats were dominated by animal prey (mainly amphipods), but adults of these fishes, andP. crassipes, consumed algae nearly exclusively, with 26, 10, and 14 taxa of algae identified fromG. nigricans, H. azurea, andP. crassipes, respectively. Algae with sheet-like morphologies (e.g.Ulva sp.,Enteromorpha sp., members of the Delesseriaceae) were the principal algae in the diets of the fishes, and calcareous algae (e.g.Corallina sp.,Lithothrix aspergillum) and sheet-like algae (Enteromorpha sp.) comprised the greatest identifiable portion of the shore crab's diet. Feeding choice experiments indicated that the fishes preferred filamentous algae (e.g.Centroceras clavulatum, Polysiphonia sp.,Chondria californica) and sheet-like algae (e.g.Enteromorpha sp.,Ulva sp.,Cryptopleura crispa) over other algal morphologies, whereas the shore crab chose jointed calcareous algae (e.g.Lithothrix aspergillum, Corallina vancouveriensis, Jania sp.) most frequently. The diets and preferences for algae by the fishes were generally most similar to the assemblage of algae available in early successional (disturbed) habitats during summer when sheet-like and filamentous algae are abundant. The shore crab exhibited the opposite trend with a diet more similar to late successional (undisturbed) habitats.     

Algal response to a thermal effluent: study of a power station on the provo river,Utah, USA

The effect of a thermal effluent on the attached algae of the Provo River, Utah, USA, was studied from 1975 to 1977. Data for macroscopic and microscopic algae were collected and analyzed. Diatoms, Cladophora glomerata, and Hydrurus foetidus dominated the flora. The thermal effluent significantly affected the algal flora in a section of river 100 to 135 meters long immediately below the discharge point. Cladophora growth was increased and Hydrurus was absent in this area. In addition, diatom production was often higher and diversity lower than in the rest of the river. Community structure was unique from all other adjacent areas. Small temperature increases which occured as effluent and river waters mixed farther downstream were apparently not as important to the algal flora as other environmental factors.     

New records of benthic marine algae and Cyanobacteria for Costa Rica,and a comparison with other Central American countries

We present the results of an intensive sampling program carried out from 2000 to 2007 along both coasts of Costa Rica, Central America. The presence of 44 species of benthic marine algae is reported for the first time for Costa Rica. Most of the new records are Rhodophyta (27 spp.), followed by Chlorophyta (15 spp.), and Heterokontophyta, Phaeophycea (2 spp.). Overall, the currently known marine flora of Costa Rica is comprised of 446 benthic marine algae and 24 Cyanobacteria. This species number is an under estimation, and will increase when species of benthic marine algae from taxonomic groups where only limited information is available (e.g., microfilamentous benthic marine algae, Cyanobacteria) are included. The Caribbean coast harbors considerably more benthic marine algae (318 spp.) than the Pacific coast (190 spp.); such a trend has been observed in all neighboring countries. Compared to other Central American countries, Costa Rica has the highest number of reported benthic marine algae; however, Panama may have a similarly high diversity after unpublished results from a Rhodophyta survey (Wysor, unpublished) are included. Sixty-two species have been found along both the Pacific and Caribbean coasts of Costa Rica; we discuss this result in relation to the emergence of the Central American Isthmus.     

Monitoring ofgfp-taggedMoraxella sp. under starvation condition

A PNP(p-nitrophenol)-degradingMoraxella sp. was genetically marked bygfp gene for monitoring. Stable chromosomal integration of the introducedgfp gene was confirmed by examining the transformants under epifluorescent microscope. The survival ofgfp-taggedMoraxella sp. cells during long-term storage under starvation condition was examined by viable cell counting and direct fluorescence microscopic counting. The number of green fluorescent cells obtained by direct microscopic counting was approximately 10 times greater than viable cell counts by plating. The number of cells from both counting methods was higher at lower temperature (4°C), although the drop of cell number after 8 weeks of starvation was comparable (approximately 100 fold drop from initial counts). Results obtained by two different methods correlated well with each other indicating that thegfp markedMoraxella sp. can be directly monitored following environmental release using epifluorescence microscopy.     

ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences

A computer program, ARAGORN, identifies tRNA and tmRNA genes. The program employs heuristic algorithms to predict tRNA secondary structure, based on homology with recognized tRNA consensus sequences and ability to form a base-paired cloverleaf. tmRNA genes are identified using a modified version of the BRUCE program. ARAGORN achieves a detection sensitivity of 99% from a set of 1290 eubacterial, eukaryotic and archaeal tRNA genes and detects all complete tmRNA sequences in the tmRNA database, improving on the performance of the BRUCE program. Recently discovered tmRNA genes in the chloroplasts of two species from the ‘green’ algae lineage are detected. The output of the program reports the proposed tRNA secondary structure and, for tmRNA genes, the secondary structure of the tRNA domain, the tmRNA gene sequence, the tag peptide and a list of organisms with matching tmRNA peptide tags.     

Gene Unscrambler for detangling scrambled genes in ciliates

Scrambled genes are surprisingly common in some species of ciliates. Until now there was no software available to analyze automatically these genes. We present here a program that can automatically align the macronuclear and micronuclear forms of a gene, outputting the location of the macronuclear destined segments and pointer sequences. AVAILABILITY: A web version of the program is available free of charge and can be accessed at http://oxytricha.princeton.edu/GeneUnscrambler.htm     

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.     

The inverted microscope method of estimating algal numbers and the statistical basis of estimations by counting

Summary Various methods for the estimation of populations of algae and other small freshwater organisms are described. A method of counting is described in detail. It is basically that of Utermöhl and uses an inverted microscope.If the organisms are randomly distributed, a single count is sufficient to obtain an estimate of their abundance and confidence limits for this estimate, even if pipetting, dilution or concentration are involved.The errors in the actual counting and in converting colony counts to cell numbers are considered and found to be small relative to the random sampling error.Data are also given for a variant of Utermöhl's method using a normal microscope and for a method of using a haemocytometer for the larger plankton algae.     

Chloroplast genome characterization in the red alga Griffithsia pacifica

Summary It has been suggested that cyanobacteria served as the ancestors for rhodophytic algae whose chloroplasts contain chlorophyll a and phycobilins, and that a rhodophyte served as the plastid source for chromophytic plants that contain chlorophylls a and c. Although organellar DNA has been used to assess phylogenetic relatedness among terrestrial plants and green algae whose chloroplasts contain chlorophylls a and b, few data are presently available on the molecular profile of plastid DNA in chromophytes or rhodophytes.In this study the chloroplast genome of the rhodophytic, filamentous alga Griffithsia pacifica has been characterized. DNA was purified from isolated chloroplasts using protease k treatment and sodium dodecyl sulfate lysis followed by density centrifugation in Hoescht-33258 dye-CsCl gradients. Single and double restriction enzyme digests demonstrate that the DNA prepared from purified chloroplasts has a genome size of about 178 kilobase pairs (kb). A restriction map of this chloroplast genome demonstrates that it is circular and, unlike the chloroplast DNA (cpDNA) in most other plants, contains only a single ribosomal DNA operon. DNA was also purified from the mitochondria that co-isolated with chloroplasts. Mitochondrial DNA consists of molecules that range in size from 27 to 350 kb based on restriction endonuclease digestion and electron microscopic analysis.     

Zooplankton grazing methods using radioactive tracers: Technical problems

Some of the technical problems specific to the application of radiotracer techniques for measuring grazing of zooplankton are highlighted and, in some cases, the corrective measures suggested.The major drawbacks of measuring zooplankton grazing in the laboratory, for studying the role of zooplankton in nature, namely, changes in water temperature and light and heavy mortality of some cladocerans, are overcome by the use ofin situ grazing chambers. However, at present there are no suitable techniques available to measure short-term assimilation rates which usually suffer from the respiratory loss during the experiment of the assimilated radiotracer.In the ecosystem research in which zooplankton grazing is an integral part of the studies, the radiotracer food manufactured fromin situ lake seston is preferred to labelled monoculture of algae, bacteria or yeast. The specific activities of the different tracer components and size fractions of the seston comprising the tracer may considerably differ in short-term labelling. On the other hand, long labelling periods,i.e. exceeding a day for example, may lead to shifts in size spectra of and composition of the different phytoplankton species. Changes during the incubation in the size spectra of the tracerfood are probably of greater importance than those in species composition, especially because a size-selective feeding may often exist.Simultaneous use of different radiotracers for labelling phytoplankton and bacteria or for mixtures of algae that differ in sizes offers some promising applications. More work in this field is needed, particularly because of the overlapping energy spectra of the radiotracers, and differential loss rates of these tracers from the preserved animals before the radioactivity counting. Such losses of the tracer from the experimental animals may lead to significant errors, especially in the short-term grazing measurements. This problem can be, nevertheless, prevented to a large extent by a rapid freeze-drying of the animals immediately on concluding the experiment.Self absorption may be a problem in radioactivity counting, more so if the animals are large. Use of sample oxidizers offers a good solution if the tracer can be trapped in its oxidized, gaseous form (e.g. ¹⁴CO₂) before further processing for counting. Modern solubilizing agents, like Soluene 350, with their relatively high water-holding capacity are quite effective in dissolving the different tissues, except the exoskeletal material.dedicated to Prof. H.F. Linskens, chairman of the Managing Committee of the Limnological Institute, in honour of his 65th birthday.