Size and visibility of Baltic cod eggs with reference to size-selective and stage dependent predation mortality

Size of Baltic cod eggs from incubation experiments and from field samples was determined by microscopic analysis. Results from plankton samples were compared with corresponding size distributions of cod eggs found in herring stomachs. The influence of fixation on size of different developmental stages was studied. Live eggs from incubation experiments were also sized repeatedly throughout the developmental period with an optical plankton counter (OPC) based on light attenuance measurements as this was assumed to be more closely related to the visibility of the eggs for potential predators than egg diameter as obtained by microscopic analysis.
Preservation in formaldehyde solution caused a small reduction in egg diameter (2.2%) whereby no differences between the developmental stages were detected. Egg size decreased slightly during incubation (6.9%) while the OPC measurements revealed a substantial increase in light attenuance during egg development (42.2%). In the field, a general decrease in egg size with increasing depth was observed while no change between the developmental stages was detectable. The mean size of eggs ingested by herring was slightly lower than in the water column which was most pronounced for the late stages containing a well-developed embryo. The frequency of eggs in an advanced stage of development was considerably higher in the stomachs than in corresponding plankton samples. Therefore, it is suggested that the selection of further developed egg stages by predatory fish in the central Baltic Sea, i.e. herring and sprat, is due to an increase of visibility during egg development in relation to growth and pigmentation of the embryo. Thus it is likely that egg mortality due to predation is stage-dependent rather than strictly size-selective.     

Detection and enumeration of Arcobacter spp. in the coastal environment of the Straits of Messina (Italy)

Seawater and plankton samples from the Straits of Messina, Italy, were analysed to confirm the occurrence of potentially pathogenic Arcobacter butzleri, A. cryaerophilus and A. skirrowii. Both classical cultural methods and molecular techniques were used as confirmative steps of the growth in enrichment broth to enumerate and differentiate these bacteria. Only A. butzleri was isolated from seawater and plankton samples and was more abundant when associated with plankton than free-living. A. cryaerophilus was occasionally detected by PCR assay from environmental samples. The PCR procedure, used in a combined method, was useful in enumerating Arcobacter spp. in marine environment.     

Implications of mechanical deformation and formaldehyde preservation for the identification of stage-specific characteristics of Baltic cod eggs

The identification of developmental stages in fish eggs from plankton samples is often complicated by deformation of the embryos due to mechanical stress during the sampling procedure and by dehydration during formaldehyde fixation. The effects of formaldehyde fixation and mechanical stress on Baltic cod eggs ( Gadus morhua callarias L.) were examined separately by visually comparing the morphological features of treated vs. live eggs of identical ontogenetic age. Microphotographs were made concurrently for documentation. In stage IA eggs, mechanical treatment resulted in scattered blastodiscs surrounded by single cells, while in further advanced stages the yolk membrane collapsed entirely, the yolk coagulated and the embryo extending over the yolk shrank. Formaldehyde fixation caused the yolk and the blastodisc or embryo to darken, and in some cases crystalline enclosures occurred. Eggs mechanically deformed during handling were clearly distinguishable from those that died prior to catching; however, staging was generally less accurate for formaldehyde-preserved eggs when compared with living specimens.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

Finding copepod footprints: a protocol for molecular identification of diapausing eggs in lake sediments

Even though calanoid copepods produce diapausing eggs that stay alive in lake sediments, these eggs have rarely been used paleolimnologically, as they lack diagnostic morphological features. In this study, we developed a method to identify copepod diapausing eggs in Japan as a clue toward reconstructing past plankton populations. We first determined a 28S ribosomal DNA (rDNA) (i.e., nc28S) regional sequence library (240 bp) of various calanoid copepod species using ethanol-fixed plankton samples collected from across the Japanese archipelago. Then we applied the UltraSHOT method to extract DNA from an individual diapausing egg. Finally, the nc28S region of diapausing eggs collected from various lakes was sequenced and compared with the regional library for species identification. In total, 21 haplotypes of the nc28S region were recovered from planktonic samples of 11 Japanese freshwater calanoid copepod species. Despite the short length of this region, no identical haplotypes were shared among the species analyzed, including the Acanthodiaptomus pacificus complex treated as a species. Even different lineages of A. pacificus could be separated. These results indicate that the nc28S region can be used as a barcode in Japan. A total of 112 diapausing eggs collected from various lakes and ponds was processed, and the nc28S region of each was successfully sequenced. All of these egg sequences matched one or the other of the nc28S haplotypes in the regional library mentioned above. The set of protocols we applied (i.e., preparing a comprehensive regional sequence library and sequencing egg DNA) is thus useful for involving copepod diapausing eggs in paleolimnological studies in lakes. The nc28S region treated in this study has a strong potential to uncover the paleodiversity of copepods, at least in Japan.     

Comparison of sleds versus plankton nets for sampling fish larvae and eggs

Fish larvae and fish eggs were sampled from the inshore waters of eastern Lake Michigan from 1978 through 1980, using a benthic sled and a plankton net towed within 0.5 m of the lake bottom. Differences between estimates of ichthyoplankton abundance based on the benthic sled and those based on the plankton net towed near bottom were examined along with interactions between gear, bottom depth, and time of day. Time of day was determined to be an important factor in comparing these two gear, but data were inconclusive as to the effect of depth on gear differences. Abundance of fish eggs calculated using sled tow data was significantly higher than that for the plankton net. For nighttime collections, density of alewife Alosa pseudoharengus larvae sampled in the plankton net significantly exceeded that for the sled, whereas density of spottail shiner Notropis hudsonius larvae based on sled data was significantly higher than that based on the plankton net for day sampling. Overall, the plankton net appeared to be adequate for sampling abundance of alewife larvae, while the sled was preferred for sampling fish eggs, spottail shiner larvae, and the following less common, but apparently demersal larvae: trout-perch Percopsis omiscomaycus, johnny darter Etheostoma nigrum, ninespine stickleback Pungitius pungitius, and slimy sculpin Cottus cognatus.     

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry

Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 μm³). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm. Received: 21 January 1999; Accepted: 12 April 1999     

Standing out from the crowd: Spotting your targets in a mixed plankton sample

The diversity of marine organisms is staggering, and this fact is readily appreciated by microscopic examination of the contents of a plankton net after a short tow across the ocean surface. Although this diversity is beautiful, it can present a significant problem for those seeking to extract information about a single species of interest. Enumeration of the eggs and larvae of a specific target species can provide a quantitative window into reproductive dynamics that are of great use for fisheries stock assessment and management. But how do you efficiently sort through the mass of plankton and identify target species’ eggs and larvae that may be morphologically indistinguishable from those of a number of other local species? In this issue of Molecular Ecology Resources, Oxley et al. ( 2017 ) describe an innovative in situ hybridization (ISH) approach that successfully solves this important problem and opens an exciting new avenue to ichthyoplankton analysis that may be widely adopted by both fish ecologists and fisheries managers.     

The influence of sampling method on the classification of wetland macroinvertebrate communities

Macroinvertebrate communities sampled by a corer, plankton net and sweep net from five wetlands on the Swan Coastal Plain were compared. The composition of the fauna collected in sweeps and tows was generally similar and differed from that collected in the cores. Cores caught fewer species than tows and sweeps at all wetlands and did not capture fast swimming hemipterans or less abundant taxa. The highest species richness was recorded in sweep samples in four out of the five wetlands. Classification (TWIN-SPAN) and ordination (SSH) of the samples collected in sweeps and tows gave good separation of the wetlands, whereas classification of core samples did not. Coring appeared to be the least suitable sampling method for describing the major components of the macroinvertebrate communities of these wetlands. Plankton tows were useful if the time available for sorting was limited as these samples were free of sediments and generally gave similar results to those obtained with sweeps. Sweeps appeared to be the most useful method for a large classification study as they collected more species and resulted in the best discrimination amongst wetlands.     

LINKING FLOW CYTOMETRIC CELL SORTING AND COMPOUND‐SPECIFIC 13C‐ANALYSIS TO DETERMINE POPULATION‐SPECIFIC ISOTOPIC SIGNATURES AND GROWTH RATES IN CYANOBACTERIA‐DOMINATED LAKE PLANKTON1

A novel methodology was applied to determine the δ¹³C signatures of natural cyanobacterial and algal populations by combined compound‐specific isotope ratio mass spectrometry and pyrolytic methylation‐gas chromatography (Py‐GC‐IRMS) of the fatty acids released from phytoplankton fractions collected using fluorescence‐activated cell sorting. Py‐GC‐IRMS provided direct analysis of the very small samples (<200 ng total C) derived from the cell sorting of individual phototrophic populations, while minimizing the chances on contamination and loss in sample handling. Despite trichome lengths exceeding the diameter of the sort droplets, filamentous cyanobacteria were amenable to population‐specific cell sorting. In concert with ¹³C‐CO₂ labeling, the combined use of flow cytometric cell sorting and Py‐GC‐IRMS enabled both the assessment of standing stocks and of population‐specific growth rates of the predominant cyanobacterial and algal taxa in Lake Loosdrecht (The Netherlands). Filamentous prochlorophytes, formerly the dominant cyanobacterial taxon in the lake, appeared less abundant in recent years and exhibited growth rates 30%–40% lower than the rates recorded for oscillatorioid populations. Diatom and green algal populations grew at rates 4‐ to 10‐fold higher than filamentous cyanobacteria and are thus important for the lake's carbon budget. This approach offers new possibilities in studying plankton dynamics at a resolution not feasible in the past.     

Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods.

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.     

Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.     

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.     

A Fast Estimate for the Population Recombination Rate Based on Regression

Recombination is a fundamental evolutionary force. Therefore the population recombination rate ρ plays an important role in the analysis of population genetic data; however, it is notoriously difficult to estimate. This difficulty applies both to the accuracy of commonly used estimates and to the computational efforts required to obtain them. Some particularly popular methods are based on approximations to the likelihood. They require considerably less computational efforts than the full-likelihood method with not much less accuracy. Nevertheless, the computation of these approximate estimates can still be very time consuming, in particular when the sample size is large. Although auxiliary quantities for composite likelihood estimates can be computed in advance and stored in tables, these tables need to be recomputed if either the sample size or the mutation rate θ changes. Here we introduce a new method based on regression combined with boosting as a model selection technique. For large samples, it requires much less computational effort than other approximate methods, while providing similar levels of accuracy. Notably, for a sample of hundreds or thousands of individuals, the estimate of ρ using regression can be obtained on a single personal computer within a couple of minutes while other methods may need a couple of days or months (or even years). When the sample size is smaller (n ≤ 50), our new method remains computational efficient but produces biased estimates. We expect the new estimates to be helpful when analyzing large samples and/or many loci with possibly different mutation rates.     

An iterative procedure to estimate muscle lines of action in vivo

A method is described to estimate the line of action of muscles in the three-dimensional space from serial images of parallel muscle sections obtained in vivo by means of CT or MRI scanning. The external shape of a muscle, reconstructed from the series of parallel sections, is mathematically divided into a series of imaginary slices directed arbitrarily in the three-dimensional space. The line of action is estimated initially as a regression line through the centroids of these mathematical slices. A new series of mathematical slices is constructed perpendicular to the regression line and a new estimate of the line of action is obtained from their centroids. This procedure is repeated until the estimated line of action is perpendicular to the mathematical slices; it can then be considered as a reliable estimate of the line of action. The accuracy of the method has been tested for various reconstruction parameters and muscle shapes. The results of these tests show that the accuracy is relatively independent of the direction in which the sectional images have been made and that, except for relatively short and thick muscles, the estimated lines of action deviated less than about 2 degrees from the theoretical one. The presented method is a relatively simple mathematical technique which can be used easily for muscles reconstructed in vivo from routinely obtained sectional MRI or CT images.     

A specific and sensitive method for the determination of the anticoagulant phenprocoumon in plasma

A specific thin-layer chromatographic assay for phenprocoumon has been developed with a sensitivity of 5 ng/ml of plasma, using only 0.2 ml. This sensitivity is more than 20 times higher than that of the published methods. The drug is extracted from acidified plasma, an aliquot of the extract is applied to a silica-gel thin-layer plate and separated from interfering substances. The quantity of phenprocoumon is determined by fluorescence densitometry in situ. The standard deviation of the whole procedure is less than ± 3%. The new procedure permits pharmacokinetic studies withlow doses of phenprocoumon to be performed on volunteers. Furthermore, due to the high sensitivity of the method, it is possible to determine the free drug fraction of this highly protein-bound substance in the plasma of patients. It was shown that, in the therapeutic concentration range, phenprocoumon is bound by about 99.5% to the plasma proteins. Since the assay is simple and quick to perform, a large series of plasma samples can be analysed without any problems.     

Distribution of potentially pathogenic bacteria as free living and plankton associated in a marine coastal zone

AIMS: To determine the abundance of faecal and nonfaecal bacteria related to human and animal health, as free living or associated with small (>64 microm) and large (>200 microm) plankton, samples were collected monthly from the coastal zone at Messina (Italy). METHODS AND RESULTS: Different enrichment and selective cultural methods were used to determine the abundance of bacteria in sea water and plankton. The bacteria were more frequently isolated from water and large plankton than from small plankton. Vibrio and Aeromonas spp. showed different distribution patterns in water and plankton. Faecal indicators were always present in water and the large size class plankton samples. Enterococci associated with large plankton were more abundant than E. coli in the winter. Vibrio species distributions were different in water and plankton samples. Among arcobacters only A. butzleri was isolated from water and plankton samples. Campylobacter spp. was always absent in small plankton and more frequent in large plankton than in water. CONCLUSIONS: The colonization of zooplankton by potentially pathogenic bacteria is a widespread phenomenon. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of potentially pathogenic bacteria in sea water and associated with plankton can have ecological and epidemiological implications.     

Flow Cytometric Analysis and Sorting of Heterodera glycines Eggs

A nondestructive technique was developed to characterize and separate eggs of soybean cyst nematode, Heterodera glycines, by developmental stage using flow cytometry. Eggs from cysts cultured on susceptible soybean roots were suspended in 0.1% xanthan gum or 59% sucrose and loaded into either a Coulter EPICS 752 or EPICS 753 flow cytometer. Eggs were analyzed and sorted according to forward angle and 90° light scatter, flow cytometric parameters that are relative measures of object size and granularity, respectively. Mature eggs containing vermiform juveniles were less granular and slightly larger than eggs in earlier stages of embryogeny, allowing for separation of mature eggs from immature eggs. The effectiveness of flow cytometric sorting was evaluated by comparing the developmental stages of subpopulations of unsorted and sorted eggs. Of a subpopulation of unsorted eggs, 62% contained vermiform juveniles, whereas 85 to 95% of sorted subpopulations of larger, less granular eggs contained vermiform juveniles. Suspending H. glycines eggs in 0.1% xanthan gum or 59% sucrose for flow cytometric analysis had no effect on subsequent egg hatch in vitro. This technique is an efficient and effective means to collect large, relatively homogeneous quantities of H. glycines eggs in early or late embryogeny, and would likely be useful for analyzing and sorting eggs of other nematode species for use in developmental, genetic, or physiological research, or for identification and collection of parasitized eggs.     

A rapid micromethod for apolipoprotein E phenotyping directly in serum

A new method for the apolipoprotein E phenotyping has been developed. The method is based on isoelectric focusing of either delipidated or guanidine-HC1-treated serum or plasma in a horizontal slab gel system followed by immunoblotting using either polyclonal or monoclonal anti-apolipoprotein E antibodies as first antibody. Apolipoprotein E phenotyping with this method in 200 serum samples that had been stored at -20 degrees C for more than one year gave exactly the same results as obtained with the conventional method based on isoelectric focusing of delipidated very low density lipoproteins isolated from fresh serum followed by protein staining. Compared with the conventional method, the present method is less laborious because ultracentrifugation to isolate VLDL is not needed; it is suitable for large scale screening purposes; it needs only a few microliters of serum or plasma, and can easily be performed with samples with low concentrations of apolipoprotein E.