Clearance rates of sessile rotifers: in vitro determinations

We measured laboratory clearance rates of 10 rotifer and one unidentified bryozoan species from 3 different lakes using ³²P labeled algae (Chlamydomonas) or yeast (Rhodotorula). Clearance rates for all rotifers fed yeast ranged from < 2.0 to > 260 µl · animal^–1 · h^–1 depending on species. The in vitro clearance rates of two sessile rotifers (Ptygura crystallina and P. pilula) were not significantly different from previously measured in situ rates (Wallace and Starkweather 1983). Clearance rates for 5 rotifers fed algae ranged from < 5.0 to > 90.0 µl · animal^–1 · h^–1. Ptygura beauchampi, P. crystallina, P. pilula, Floscularia conifera, and F. melicerta ingested both cell types but their clearance rates varied substantially among species and between cell types. There was a substantial time-dependent loss of 32P from formalin-fixed animals (Sinantherina socialis) awaiting processing. This loss stabilized at approximately 20 hours and was estimated to be about 40% of the initial ingested label. Clearance rates for the bryozoan fed yeast or algae were highly variable, ranging from < 1.0 to > 3 000 µl · animal^–1 · h^–1.     

In situ localization of PCR-amplified DNA and cDNA

Combining the high sensitivity of PCR with the cell localizing ability ofin situ hybridization allows for the reproducible detection of low copy targets in intact cells. This article describes several key variables that include fixation, protease digestion, the hot start maneuver, stringency, and, for RNA analysis, DNase digestion that are important to successfulin situ PCR. Also stressed is the importance of performing and interpreting controls with each experiment. Important controls include omission of key components, use of samples known either to contain or lack the target of interest and, most importantly, the in-built controls invariably present in the heterogeneous component of any given tissue type.     

In situ nutrient assays of periphyton growth in a lowland Costa Rican stream

Nutrient limitation of primary production was experimentally assessed using an in situ bioassay technique in the Quebrada Salto, a third-order tropical stream draining the northern foothills of the Cordillera Central in Costa Rica. Bioassays employed artificial substrata enriched with nutrients that slowly diffuse through an agar-sand matrix (Pringle & Bowers, 1984). Multiple comparisons of regression coefficients, describing chlorophyll-a accrual through time for different nutrient treatments, revealed positive micronutrient effect(s). Micronutrient treatment combinations (Fe, B, Mn, Zn, Co, Mo, EDTA), supplemented with and without nitrate and phosphate, exhibited significantly greater chlorophyll-a accrual over all other treatments (P < 0.05), supporting over three times that of the control after 14-d of substratum colonization. Neither of the major nutrients (N or P) produced a significant stimulation, although the N treatment displayed 50% more chlorophyll-a than the control after 14-d. Similarly, Si, EDTA, and Si + N + P treatments did not exhibit chlorophyll-a response curves that were significantly different from the control. During the experiment, mean NH₄-N and (NO₂ + NO₃)-N concentrations in the Salto were 2.0 µM (28.6 µg · l^–1) and 7.2 µM (100.2 µg · l ^–1), respectively. High concentrations of PO₄-P ( = 2.0 µM; 60.9 µg · l^–1) and TP ( = 3.0 µM; 94.0 µg · l^–1) were also found, and consequently low molar N:P ratios = 4.7). Despite the potential for N limitation in the system, both N and P appear to be at growth saturating levels. This may be due to micronutrient limitation and/or light limitation of periphyton growth in densely shaded upstream portions of the stream.     

Evaluation of a fluorescent microparticle technique for measuring filtering rates of Daphnia

The paper demonstrates the applicability of fluorescent microparticles (FMP) for the determination of cladoceran feeding rates. For this purpose we examined the relation between filtering rate, ingestion rate, gut passage time and body length of Daphnia pulex at different food concentrations in laboratory experiments, and diurnal changes of filtering rates of Daphnia longispina in in situ experiments, using FMPs as tracer particles. Good agreement of our results with published data based on cell counts and radiotracer methods proves the suitability of the FMP-method. Potentials and limitations of the method are discussed.     

In situ observation of suspended solid aggregates in rivers

Suspended solids are a major pathway of the biogeochemical fate of many contaminants in aquatic systems. Aggregation processes of particles are poorly reported in rivers but they are likely to exist at low flow, mostly because of sticky polysaccharides produced by living organisms. These processes affect suspended particle transport by changing particle sizes and densities and may also limit exchanges of matter between solid and dissolved phases. The main difficulty in floc studies is aggregate fragility, which requires the use of specialized in situ techniques to analyze aggregated suspended solids.We describe two in situ methods for observing suspended particles which do not need heavy field equipment. The first one is based on the filtration of a thin water layer in the natural flow through a membrane which can subsequently be observed by microscope. The second one is based on in situ video snapshots of suspended solids by an endoscope. The video camera linked to the microscope or the endoscope supplies images which are automatically analyzed by image processing to give size distributions.Procedures and validation for both methods are described and results compared with a standard method. The filtration method has been used to trace suspended solids from sewer overflows in the Seine River downstream of Paris. Freshwater flocs are described and a discussion of the fate of aggregates is presented.

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Résumé Les matières en suspension sont déterminantes dans le cycle biogéochimique de nombreux polluants des systèmes aquatiques. Les phénomènes d'agrégation des particules sont encore mal connus en rivière, mais peuvent se produire dans les fleuves urbains du fait de la lenteur des écoulements et vraisemblablement de l'importance des processus bactériens. Ces phénomènes influencent directement le transport des suspensions en modifiant les tailles et les densités des particules. Ils peuvent également limiter les échanges chimiques entre phase dissoute et particulaire.Le problème de la fragilité des flocs et la complexité des équipements habituellement utilisés in situ nous ont conduit à développer deux méthodes d'observation non destructives des flocs. La première consiste à filtrer une mince lame d'eau directement dans l'écoulement naturel puis à observer les suspensions au microscope. La deuxième méthode est l'observation directe des suspensions dans l'écoulement, grâce à un endoscope. Une caméra vidéo reliée au microscope ou à l'endoscope fournit des images que l'on traite automatiquement (dénombrement et mesure des particules) pour obtenir la granulométrie des agrégats naturels.Nous comparons les résultats des deux méthodes à ceux d'une méthode de référence, puis nous comparons les deux méthodes entre elles. La filtration in situ a été utilisée en Seine à l'aval de gros rejets de temps de pluie de l'agglomération parisienne pour étudier les matières en suspension issues des rejets. La caractérisation des flocs de Seine permet de discuter de leur devenir dans le fleuve.

     

人单拷贝及重复DNA序列的原位PCR

在培养的人小肠癌转移腹水细胞系细胞中进行了Y染色体特异的重复序列及单拷贝序列的原位扩增与检测.结果显示原位PCR法的灵敏度比直接的原位杂交法明显提高.     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

In situ predatory behavior ofMysis relicta in Lake Michigan

Selectivity coefficients (W) and predation rates on Lake Michigan zooplankton were determined forMysis relicta during spring through fall using anin situ method. W values indicated the following ranked order of prey preference: Cladocera > copepod copepodites and copepod nauplii > adult diaptomids and cyclopoids. With few exceptions, W values for different prey categories remained fairly constant despite greatly changing relative abundances of prey. Predation rates and prey selectivity were similar in most cases to those determined in laboratory studies. Ingestion rates (percent dry body weight · day^–1) were correlated to total prey biomass (r = 0.38) and to effective prey biomass (r = 0.85), where the weighting factors were overall mean selectivity coefficients for the different prey categories. This result suggested that seasonally varying composition of prey caused much of the variation in ingestion rates among experiments. Feeding trials performed at the same depth with daytime and nighttime assemblages of zooplankton indicated that Cladocera may escape heavyMysis predation at night by migrating from the metalimnetic-hypolimnetic interface into the epilimnion.Contribution 333 from the Great Lakes Research Division, The University of Michigan and contribution 287 from the Great Lakes Environmental Research Laboratory, National Oceanic and Atmospheric Administration.Contribution 333 from the Great Lakes Research Division, The University of Michigan and contribution 287 from the Great Lakes Environmental Research Laboratory, National Oceanic and Atmospheric Administration.     

The components of feeding behavior in rotifers

Feeding behavior of a rotifer can be broken into two classes of activities: the rate of successful search and the handling process. The former consists of the following components: Perceptual field (in planktonic rotifers the area of the corona), swimming rate, and attack rate. The second class consists of capture rate, handling time, rejection rate, ingestion, digestion, and assimilation. All evidence indicates that the perceptual field cannot be varied by the rotifer. Swimming rate is variable and under rotifer control, but does not appear to vary with degree of starvation. Attack rate is also under control of the rotifer, at least in the genus Asplanchna. Capture rates vary with the species of food item from zero to 100%. Handling times are longer than one would expect, as are rejection times. Digestion and assimilation appear to vary inversely with rate of ingestion. There is some suggestion in the literature that feeding behavior on very small particles differs from that on larger ones.     

Measuringin situ predation byMysis relicta and observations on underdispersed microdistributions of zooplankton

Described are a method and apparatus that allowin situ measurement of predation on zooplankton byMysis relicta. The method, which can be generalized to other predators, involves lowering paired large-volume (30-1) plankton traps to the depth of interest, with subsequent trapping of the ambient zooplankton assemblage in each trap and release of predators into one of the traps. The statistical adequacy of the method was shown by error propagation theory to depend on the percentage of available prey consumed, on the number of prey captured by the traps, and on the distribution of zooplankton within the volume of water captured by the traps. Repeated casts of the apparatus showed that, in contrast to other studies of zooplankton distribution, various zooplankton categories were statistically underdispersed (evenly dispersed in space) or at least not more statistically dispersed (clumped) than was a random distribution at a space scale of 1 m. An error analysis of many replicated feeding experiments showed that the errors obtained were reasonably small and that they conformed with or were less than those predicted by error propagation theory that assumed random distribution of zooplankton. Thus, these results supported the practical application of the method and corroborated the conclusion of random dispersion or underdispersion drawn from the experiment of repeated casts of the apparatus.     

In situ primary production in young Antarctic sea ice

An in situ incubation technique used successfully to measure the photosynthetic carbon assimilation of internal algal assemblages within thick multiyear Arctic ice was developed and improved to measure the photosynthetic carbon assimilation within young sea ice only 50 cm thick (Eastern Weddell Sea, Antarctica). The light transmission was improved by the construction of a cylindrical frame instead of using a transparent acrylic-glass barrel. The new device enabled some of the first precise measurements of in situ photosynthetic carbon assimilation in newly formed Antarctic sea ice, which is an important component in the sea ice ecosystem of the Antarctic Ocean. The rates of carbon assimilation of the interior algal assemblage (top to 5 cm from bottom) was 0.25 mg C m^–2 d^–1 whereas the bottom algal community (lowest 5 cm) attained only 0.02 mg C m^–2 d^–1. Chl a specific production rates (P^Chl) for bottom algae (0.020 – 0.056 g C g chl a ^–1 h^–1) revealed strong light limitation, whereas the interior algae (P^Chl = 0.7 – 1.2 g C g chl a ^–1 h^–1) were probably more limited by low temperatures (< –5 °C) and high brine salinities.     

The influence of sampling strategy on the apparent population dynamics of planktonic rotifers

Rotifer composition and density, chlorophyll-a content, secchi depth and temperature were investigated with high sampling frequency (every third day in summer, weekly in winter) in the eutrophic lake Bielersee from April 1981 to April 1982. Long intervals between sampling, especially from spring to autumn, lead to contradictory interpretation of abundance dynamics of both total rotifer community and single species. Short intervals between sampling revealed a sequence of mass developments of the different species. As an example, alternating gradation phases are shown for Synchaeta grandis and Synchaeta stylata.     

Chromosomal mapping of T-DNA inserts in transgenicPetunia byin situ hybridization

The chromosomal location of T-DNa inserts in ten independently derived and confirmed transgenic plants ofP. hybrida was detected byin situ hybridization. Nine transgenic plants had the T-DNA inerts at single sites distributed among each of the seven chromosomes; in one plant the T-DNA inserts were detected on two different chromosomes. Although the T-DNA inserts were integrated randomly among the chromosomes, seven of the 11 total inserts were located at or near the telomere. Thus, T-DNA inserts appear to have potential for tagging chromosomes and chromosome fragments.     

In situ recovery of lycopene during biosynthesis with recombinant Escherichia coli

Lycopene is produced by recombinant Escherichia coli expressing genes to encode for the lycopene biosynthesis. However, the productivity of lycopene seemed to be limited by many factors including product toxicity. In the present study, we have investigated physiology of recombinant E. coli during biosynthesis and in situ recovery of lycopene based on an organic/aqueous two-phase system. Lycopene, the 40-carbon molecule product, was little extracted from recombinant E. coli cells to octane or decane phase. However, partial digestion of cell walls with lysozyme promoted extraction of lycopene into the organic phases. Engineering of an organic/aqueous two-phase system allowed recombinant E. coli cells to produce ca. 40% larger amount of lycopene compared to that in a conventional aqueous single-phase system. Optimization of the in situ product recovery process will lead to further increase of product concentration and productivity.     

The effects of posterolateral spine length and body length on feeding rate in the rotifer,Brachionus calyciflorus

The length of the posterolateral spines in the rotifer, Brachionus calyciflorus, is variable. The effect of the length of these spines on the clearance rates of B. calyciflorus was examined under laboratory conditions using radioactively-labelled yeast (Rhodotorula) and bacteria (Aerobacter). The hypothesis that individuals with longer spines have higher clearance rates than those of similarly-sized individuals with shorter spines was rejected. The effect of body length (L) on the clearance rates (C) of short-spined and long-spined animals was also examined. Statistically significant linear regressions of the form log₁₀ C = aL + b, were derived for both types. The slopes of both regressions were similar and suggest that clearance rate in B. calyciflorus increases as a cube function of body length.     

In situ conservation of crop wild relatives: status and trends

Recognized as a priority three decades ago, in situ conservation of crop wild relatives has developed theoretical and methodological focus and achieved significant on-the-ground progress in the last 10 years, most notably under the impetus of the plant genetic resources community. Literature and Internet searches and interviews with experts were undertaken as a basis for reviewing the current status and trends of this effort worldwide. Country-by-country summaries on in situ crop wild relatives conservation activities are presented, and recommendations are made for future action. Principal recommendations include flagging of appropriate taxa as crop wild relatives in botanical and conservation databases, undertaking gap analyses to locate crop wild relatives hotspots, and enhancing cooperation between the plant genetic resources and plant conservation communities.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Primedin situ labeling (PRINS)

PRimedIn Situ labeling (PRINS) is a fast and sensitive alternative to fluorescencein situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.     

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.