Single Fish Research Articles

Mechanistic Insights into Forced Chromatin Looping Mediated Activation of Fetal Globin Gene Expression

The β-globin enhancer, called locus control region (LCR), is required for high level expression of all b-type globin genes. The LCR is in physical proximity with the genes it controls, with contacts shifting from embryonic (ε) to fetal (γ) and finally to adult (δ and β) globin gene promoters during development. In prior studies we showed that forced chromatin contacts between the LCR and the β-globin promoter led to transcriptional activation, suggesting that LCR-promoter looping causally underlies β-globin transcription (Deng et al. Cell 2012). In these studies, the transcription co-factor Ldb1 was tethered to the β-globin promoter using artificial zinc finger (ZF) DNA binding proteins, to trigger the promoter-LCR interaction. We subsequently showed that tethering Ldb1 to the promoters of developmentally silenced embryonic or fetal globin genes reactivated their expression in adult erythroblasts in a manner dependent on looped contacts with the LCR (Deng et al. Cell 2014). This work established a novel strategy to raise fetal globin expression in patients with sickle cell anemia.To examine mechanistically the effects of chromatin looping on gene expression we performed single molecule RNA FISH experiments to precisely measure transcription output at individual alleles before and after enforced LCR-γ-globin looping. The experiments were carried out in primary human erythroblasts, which produce elevated levels of γ-globin when exposed to culture conditions. Preliminary data suggest that the majority of transcripts emerge from the β-globin gene with a smaller fraction of transcripts coming from the γ-globin gene as expected. Among cells producing any type of globin primary transcripts, a significant fraction (25%-35%) of cells co-express γ- and β-globin. Importantly, γ- and β-globin are frequently transcribed from the same allele. Forced juxtaposition of the LCR and the γ-globin promoter increases the number of alleles expressing only γ-globin while reducing the number of alleles expressing only the β-globin gene. This result is consistent with the γ- and β-globin genes competing for LCR activity, and emphasizes the usefulness of this approach in the context of sickle cell anemia in which not only elevated levels of fetal hemoglobin but also reduction of the mutant hemoglobin are desirable. Surprisingly, however, the proportion of alleles co-expressing γ-globin and β-globin remains largely constant. We are testing whether co-expression from the same allele is LCR independent. Finally, our studies suggest that LCR-promoter contacts increase the probability of transcription of a given allele.We will also present work addressing the critical question as to how alteration of chromatin architecture overcomes the action of transcriptional repressive complexes, such as Bcl11a, which normally maintain embryonic and fetal globin genes in a repressed state throughout adulthood.In sum, our studies produce a deeper understanding of the interplay of chromatin architecture and gene expression in a system that holds great potential for therapeutic application in patients with hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Toxic elements in free-living freshwater fish, water and sediments in Poland

Abstract Samples for analysis were collected from 10 areas, including the major Polish rivers and lakes, with different sources of environmental pollution (industrial, municipal, and farming). The materials was taken from the lakes of Mazury, located in a non-industrialised region, from the Brda River, an area impacted by pig farms, from the lakes of Lipczyno Wielkie/Pomerania, from the Wkra River, an area impacted by poultry farms, from the Dunajec River at the Roznowski Reservoir, from the Vistula River at Cracow and Warsaw, from the Odra River at Wroclaw and the Warta River estuary, and also from Rybnik Power Station Reservoir. Concentrations of Pb, Cd, Hg, and As were analysed in 397 fish muscle and 128 sediment samples using an atomic absorption spectrometry technique. The analytical procedures were covered by a quality assurance programme. It was demonstrated that the average concentrations of lead, cadmium, and arsenic in fish were in the low hundredths and thousandths of a mg/kg and never exceeded permitted limits established for food. Higher values of these elements were found in fish from bodies of water located in the zone of influence of large urban agglomerations, especially the Cracow region. High concentrations of lead and cadmium were also found in Vistula River sediments near Cracow, where the maximum values were 134.10 mg/kg and 21.24 mg/kg dry weight for lead and cadmium respectively. The average concentration of mercury in a predatory fish muscle (0.179 mg/kg) was almost twice as high as in the omnivorous fish (0.103 mg/kg). Only a single fish sample exceeded the maximum limit for this metal (0.50 mg/kg) and did not present a risk to consumers’ health.

Improvements in estimating an acoustic target strength–length relationship for hoki (Macruronus novaezeladiae)

Hoki (Macruronus novaezelandiae) is New Zealand's largest fishery. Acoustic surveys of spawning hoki provide abundance indices for informing management of the stocks. For estimating fish abundance using acoustic methods, relationships between the acoustic backscatter from a single fish, commonly known as the acoustic target strength (TS), and fish length are required. Here we present in situ data on TS from individual live hoki, collected using a trawl-mounted acoustic-optical system (AOS). Deriving TS–length relationships from measurements of individual fish is complicated because of the very large stochastic variability in TS from the same fish, and from individual fish of similar length. A new TS–total length relationship of TS=24.5log10(TL)−83.9 was derived based on a weighted non-linear least-squares fit to acoustic backscattering cross-section measurements from 62 New Zealand hoki between 35 and 93cm TL. It was necessary to use a weighted fitting method to appropriately account for variance, as not doing so can introduce a bias to the estimated mean TS. Using the new TS–TL relationship decreases absolute estimates of hoki abundance by 50–60% from those currently used in stock assessment, but has little impact on relative indices. Estimated 90% confidence intervals around the new TS–TL relationship based on bootstrapping suggested uncertainty in mean TS of ±1.5dB at typical New Zealand hoki lengths, with resulting uncertainty in abundance estimates of up to 50%. Further in situ measurements and modelling are required to reduce this uncertainty and to investigate potential differences in TS between AOS-measured and ‘undisturbed’ fish.

Artificial marine habitats favour a single fish species on a long-term scale: the dominance of <em>Boops boops</em> around off-shore fish cages

Off-shore fish cages are new artificial habitats that can affect pelagic fish assemblages and constitute an important food source for wild fish assemblages. This aggregation has noticeable ecological consequences in cage areas in impoverished ecosystems such as those in the Canary archipelago (NE Atlantic Ocean). However, this new habitat could be dominated by a single species, reducing its positive ecological effects. Wild fish assemblages associated with an off-shore fish lease on the northeastern coast of Tenerife (Canary Islands) were sampled for six years. Fish assemblage structure beneath fish cages and at controls ( > 500 m from cages) differed significantly between locations, with 13 times greater abundance at cage locations. These differences were mainly explained by the dominance of bogue ( Boops boops ) around fish cages. This trend was consistent in the long-term throughout the study period (2004-2009), affecting local fisheries. The presence of fish cages significantly altered wild fish assemblages in the study area, enhancing mainly biomass and abundance of one species, bogue, and causing shifts in species composition.

Perfluoroalkyl acids in various edible Baltic, freshwater, and farmed fish in Finland

In this study, the concentration of perfluoroalkyl acids (PFAAs) in various edible Finnish Baltic Sea, freshwater, and farmed fish species were analysed. PFAAs were present in all the Baltic and freshwater species, but were not observed in any farmed fish. The most abundant compound in each species was perfluorooctane sulfonate (PFOS), comprising 41–100% of the total concentration. The total PFAA concentration varied considerably from 0.31 to 46ngg−1 fresh weight. A notable variation in the PFAA concentrations implies that a single fish species alone is not suitable for monitoring PFAA contamination in a certain area. Our results confirm that wild domestic fish is one of the PFAA source in the Finnish diet.

Migration of juvenile and sub-adult Chinese sturgeon Acipenser sinensis Gray, 1835 in the Yangtze River, China below the Gezhouba Dam

Summary Acoustic telemetry data and recapture information were collected to study the distribution and migration of sub-adult and juvenile Chinese sturgeon Acipenser sinensis. From 2007 to 2012, 187 537 tagged cultured fish (4637 sub-adults and 182 900 juveniles) were released at two sites on the Yangtze River. Subsequently, 482 fish were caught, comprising 198 cultured fish and 284 wild fish, mostly near sandbanks and sandbars with flat topography and slow water flow rates. Acoustic monitoring showed that a single sub-adult fish migrated upstream to the spawning grounds just below the Gezhouba Dam, a distance of 157 km at an upstream ground speed of 0.40–1.40 km h−1 (mean, 0.70 km h−1). Nine acoustically monitored sub-adults made a downstream migration of 45–1521 km at a ground speed of 0.08–3.80 km h−1. The ground migration speed of fish upstream to Wuhan (rkm 1678–1071) was significantly lower than that from Wuhan to the Yangtze estuary (rkm 1071–94). There was no significant difference in the ground migration speed between cultured and wild individuals. Knowledge of the movement and migration patterns of the stocked juvenile and sub-adult Chinese sturgeon will help to locate areas of the downstream channel in the lower reaches of the Yangtze River critical to the sturgeon and to evaluate the potential impact of the Three Gorges Dam on their downstream migration.

GnRH receptors and peptides: Skating backward

Gonadotropin-releasing hormone (GnRH) and its receptor are essential for reproduction in vertebrates. Although there are three major types of GnRH peptides and two major types of receptors in vertebrates, the pattern of distribution is unusual. Evidence is presented from genome mining that type I GnRHRs are not restricted to mammals, but can be found in the lobe-finned and cartilaginous fishes. This implies that this tail-less GnRH receptor emerged early in vertebrate evolution, followed by several independent losses in different lineages. Also, we have identified representatives from the three major GnRH peptide types (mammalian GnRH1, vertebrate GnRH2 and dogfish GnRH3) in a single cartilaginous fish, the little skate. Skate and coelacanth are the only examples of animals with both type I and II GnRH receptors and all three peptide types, suggesting this was the ancestral condition in vertebrates. Our analysis of receptor synteny in combination with phylogeny suggests that there were three GnRH receptor types present before the two rounds of whole genome duplication in early vertebrates. To further understand the origin of the GnRH peptide-receptor system, the relationship of vertebrate and invertebrate homologs was examined. Our evidence supports the hypothesis of a GnRH superfamily with a common ancestor for the vertebrate GnRHs, invertebrate (inv)GnRHs, corazonins and adipokinetic hormones. The invertebrate deuterostomes (echinoderms, hemichordates and amphioxus) have derived GnRH-like peptides, although one amphioxus GnRH with a syntenic relationship to human GnRHs has been shown to be functional. Phylogenetic analysis suggests that gene duplications in the ancestral bilaterian produced two receptor types, one of which became adipokinetic hormone receptor/GnRHR and the other corazonin receptor/invGnRHR. It appears that the ancestral deuterostome had both a GnRHR and invGnRHR, and this is still the case in amphioxus. During the transition to vertebrates both the invertebrate-type peptide and receptor were lost, leaving only the vertebrate-type system that presently exists.

Comprehensive meiotic segregation analysis of a 4-breakpoint t(1;3;6) complex chromosome rearrangement using single sperm array comparative genomic hybridization and FISH

Complex chromosomal rearrangements (CCR) represent rare structural chromosome abnormalities frequently associated with infertility. In this study, meiotic segregation in spermatozoa of an infertile normospermic carrier of a 4-breakpoint t(1;3;6) CCR was analysed. A newly developed array comparative genomic hybridization protocol was used, and all chromosomes in 50 single sperm cells were simultaneously examined. Three-colour FISH was used to analyse chromosome segregation in 1557 other single sperm cells. It was also used to measure an interchromosomal effect; sperm chromatin structure assay was used to measure chromatin integrity. A high-frequency of unbalanced spermatozoa (84%) was observed, mostly arising from the 3:3 symmetrical segregation mode. Array comparative genomic hybridization was used to detect additional aneuploidies in two out of 50 spermatozoa (4%) in chromosomes not involved in the complex chromosome rearrangement. Significantly increased rates of diploidy and XY disomy were found in the CCR carrier compared with the control group (P < 0.001). Defective condensation of sperm chromatin was also found in 22.7% of spermatozoa by sperm chromatin structure assay. The results indicate that the infertility in the man with CCR and normal spermatozoa was caused by a production of chromosomally unbalanced, XY disomic and diploid spermatozoa and spermatozoa with defective chromatin condensation.

Identification of a novel protein allergen in Mediterranean silverside fish species

Fish is one of the most common causes of food allergies. The main risk factors for fish allergy are atopy and exposure to fish. As new fish species are introduced into diets, allergic reactions begin to emerge. Parvalbumins are the major fish allergens, with molecular masses of 15 to 20 kDa.1 However, other allergens that could be responsible for allergic responses to a single fish species have been identified.2,3

Behavior of skipjack tuna (Katsuwonus pelamis) associated with a drifting FAD monitored with ultrasonic transmitters in the equatorial central Pacific Ocean

The behavior of skipjack tuna Katsuwonus pelamis released at a drifting fish aggregating device (FAD) in the equatorial central Pacific Ocean was investigated using ultrasonic transmitters. We conducted two types of field observations: one to track a single fish using a continuous transmitter, and the other to simultaneously monitor multiple individuals with coded transmitters. In the first examination, the behavior of one individual (fork length (FL) of 65.2cm) was monitored when the fish was both near and away from the FAD. In the second examination, the behaviors of 14 individuals (FL between 36.0 and 64.0cm) were monitored only when these were associated with the FAD. The behaviors of the fish were monitored 82.0 and 154.5h using continuous and coded transmitters, respectively. The fish monitored with a continuous transmitter made several excursions from the FAD (maximum distance of approximately 9700m) during the monitoring period. The swimming depth during daytime was deeper than that observed during nighttime, and this difference was markedly more obvious when the fish was away from the FAD. Additionally, the daytime swimming depth was deeper when the fish was away from the FAD. The swimming speed of the fish monitored with a continuous transmitter was usually less than 3KT and was higher during daytime compared with nighttime. The fish monitored with coded transmitters occasionally left the FAD, particularly during nighttime, partly demonstrated diurnal vertical movements, and were mainly distributed in the mixed layer or upper part of the thermocline. In addition, the results showed a synchronicity in the vertical movements of two or more fish. This study indicated a clear difference between the on-FAD and the off-FAD vertical movements, occasional excursions from the FAD, and diurnal movements of the fish, especially when away from the FAD.

Universal Marker and Detection Tool for Human Sarcoma Circulating Tumor Cells

To date, no specific marker exists for the detection of circulating tumor cells (CTC) from different types of sarcomas, though tools are available for detection of CTCs in peripheral blood of patients with cancer for epithelial cancers. Here, we report cell-surface vimentin (CSV) as an exclusive marker on sarcoma CTC regardless of the tissue origin of the sarcoma as detected by a novel monoclonal antibody. Utilizing CSV as a probe, we isolated and enumerated sarcoma CTCs with high sensitivity and specificity from the blood of patients bearing different types of sarcoma, validating their phenotype by single cell genomic amplification, mutation detection, and FISH. Our results establish the first universal and specific CTC marker described for enumerating CTCs from different types of sarcoma, thereby providing a key prognosis tool to monitor cancer metastasis and relapse.

Testing the low-density hypothesis for reversed sex change in polygynous fish: experiments in Labroides dimidiatus.

Hermaphroditism is ubiquitous among plants and widespread in the animal kingdom. It is an unsolved problem why reversed sex change has evolved in polygynous and protogynous reef fish. We have previously suggested that facultative monogamy occurs in low-density populations of polygynous species and that males that become single as a result of accidental mate loss may change sex when they meet larger males. In this study, to test this ‘low-density hypothesis', we conducted field experiments with the coral reef fish Labroides dimidiatus in which a portion of females were removed to create a low-density situation. The ‘widowed' males moved to search for a new mate when no male, female or juvenile fish migrated into their territories and paired with nearby single fish, whether male or female. Alternatively, males expanded their territories to take over the nearest pair whose male was much smaller. These results support our low-density hypothesis.

Do stage‐specific functional responses of consumers dampen the effects of subsidies on trophic cascades in streams?

Resource subsidies often weaken trophic cascades in recipient communities via consumers' functional response to the subsidies. Consumer populations are commonly stage-structured and may respond to the subsidies differently among the stages yet less is known about how this might impact the subsidy effects on the strength of trophic cascades in recipient systems. We show here, using a large-scale field experiment, that the stage structure of a recipient consumer would dampen the effects of terrestrial invertebrate subsidies on the strength of trophic cascade in streams. When a high input rate of the terrestrial invertebrates was available, both large and small fish stages switched their diet to the terrestrial subsidy, which weakened the trophic cascade in streams. However, when the input rate of the terrestrial invertebrates was at a moderate level, the terrestrial subsidy did not weaken the trophic cascade. This discrepancy was likely due to small fish stages being competitively excluded from feeding on the subsidy by larger stages of fish and primarily foraging on benthic invertebrates under the moderate input level. Although previous studies using single fish stages have clearly demonstrated that the terrestrial invertebrate input equivalent to our moderate input rate weakened the trophic cascade in streams, this subsidy effect might be overestimated given small fish stage may not switch their diet to the subsidy under competition with large fish stage. Given the ubiquity of consumer stage structure and interaction among consumer stages, the effects we saw might be widespread in nature, requiring future studies that explicitly involve consumer's stage structure into community ecology.

Tracking of Native Viral RNA of Enveloped Viruses in Living Host Cells

Visualization of the temporal and spatial organization of viral RNA species in host cells is essential for understanding the viral replication cycle. This project aims at employing various techniques for imaging of viral RNA at single cell/virus level. For tracking in living cells, multiple short modified oligonucleotides carrying specific intercalating fluorophores whose brightness increases 5-20 fold via forced intercalation (FIT) only upon Watson-Crick hybridization with the complementary target sequence are used. Probes are delivered into living host cells via temporarily permeabilization by streptolysin O. Those probes are employed, for example, for in vivo tracking to investigate progress and localization of influenza virus RNA segments assembly during egress. Complementary, in fixed cells we established single molecule fluorescence in situ hybridization (single molecule FISH) for studying RNA segment organization, i.e., for hanta-virus infections, both for cell-free surface immobilized virions as well as for infected host cells. Parallel dual segment labeling via FISH allows measuring the degree of correlation of two segments in a single virion at a time. Employing a highly sensitive EMCCD camera with epifluorescence microscopy, bleaching steps can be recorded for each segment thus statistically determining the number of segments per virion and the degree of colocalization between them. This approach will resolve the packaging mechanism of hanta viruses.

Phycoremediation of Pb+2, Cd+2, Cu+2, and Cr+3 by Spirulina platensis (Gomont) Geitler

Phycoremediation is an application of algae for remediate environmental pollution by reducing or eliminating toxicity of such components in waste water. One of environmental problem in Indonesia is heavy metals pollution. Heavy metals are required by microalgae in a trace concentration, but in a high concentration heavy metals are toxic. Spirulina platensis is a microalgae belong to Cyanobacteria that has been used as a single cell protein, feed fish, and supplement. Many researchs on S. platensis had been done to optimize the product. Research on the use of S. platensis for remediation environmental pollution were also had been conducted. But, research on the use of local S. platensis to reduce the concentration of Pb2+, Cd2+, Cu2+ and Cr2+, had not yet well developed. Therefore, this research was conducted in order to find out the effect of 1, 3, and 5 mg/L concentrations of Pb2+, Cd2+, Cu2+ and Cr2+ on the S. platensis population growth and its accumulation. A laboratory experiment was developed with those different concentrations with 3 replications and control. The initial concentration of heavy metals were measured as well as the 7 and 15 days treatment to determine the bioaccumulation of heavy metals by S.platensis . Based on this research, the concentration of 1 mg/L heavy metals may induced the population growth of S. platensis, due to improvement enzymatic process, the concentration of 3 mg/L heavy metals inhibit population growth, and the concentration of 5 mg/L heavy metals was toxic for S. platensis. Since S. platensis was found high tolerance to Pb2+, Cd2+, Cu2+ and Cr2+at low concentration, it was recommended that for phycoremediation process using S. platensis more appropriate to be applied for low concentration contaminated water. It was suggested that S. platensis can be used as a metal absorbent and can be cultivated from wastewaters after phycoremediation process for other uses such as fertilizer, with the note that heavy metal contamination in waste water should not exceed 1 mg/L. S. platensis will be better if apply 15 days in contaminated wastewater to get the highest removal heavy metals.

Modeling and Simulation of Fish-Like Swimming in a Straight-Line Swimming State Using Immersed Boundary Method

A self-propelled swimming fish model is established, which can reflect the interaction between fish movement, internal force generated by muscle contraction, and the external force provided by fluid. Using finite element immersed boundary method combined with traditional feedback force method, the self-propelled swimming fish is numerically simulated. Firstly, a self-induced vibration of a cantilever beam immersed in a fluid is one of the benchmarks of fluid-structure interaction, which is used to verify the validity of the numerical method. Secondly, start and cruise process of a single swimming fish in a straight-line swimming state is simulated and analysis of the flow characteristics and fish body movement features is done. The results reveal that the fish gain energy from flow field by the conversion of “C” type and “S” type of fish body.

Shake and stew: a non‐destructive PCR‐ready DNA isolation method from a single preserved fish larva

A rapid non-destructive alternative to isolate DNA from an individual fish larva is presented, based on the suspension of epithelial cells through vortex forces, and the release of DNA in a heated alkaline solution. DNA from >6056 fish larvae isolated using this protocol has yielded a high PCR amplification success rate (>93%), suggesting its applicability to other taxonomic groups or sources when tissue amount is the limiting factor.

Trpc2 is expressed in two olfactory subsystems, the main and the vomeronasal system of larval Xenopus laevis.

Complete segregation of the main olfactory epithelium (MOE) and the vomeronasal epithelium is first observed in amphibians. In contrast, teleost fishes possess a single olfactory surface, in which genetic components of the main and vomeronasal olfactory systems are intermingled. The transient receptor potential channel TRPC2, a marker of vomeronasal neurons, is present in the single fish sensory surface, but is already restricted to the vomeronasal epithelium in a terrestrial amphibian, the red-legged salamander (Plethodon shermani). Here we examined the localization of TRPC2 in an aquatic amphibian and cloned the Xenopus laevis trpc2 gene. We show that it is expressed in both the MOE and the vomeronasal epithelium. This is the first description of a broad trpc2 expression in the MOE of a tetrapod. The expression pattern of trpc2 in the MOE is virtually undistinguishable from that of MOE-specific v2rs, indicating that they are co-expressed in the same neuronal subpopulation.

A non-native snakehead fish in British Columbia, Canada: capture, genetics, isotopes, and policy consequences

In June 2012 a single non-native snakehead fish was captured by local officials in a small pond within an urban park in Burnaby, British Columbia. This single snakehead fish garnered significant attention in the local and national media. DNA analysis determined it to be a blotched snakehead (Channa maculata) or possibly a hybrid; a warm water species native to China and Vietnam which is commonly sold in the live food fish trade, and occasionally kept by hobbyists. By collecting prey items from the pond and snakehead specimens from fish markets we used a novel stable isotope approach to estimate how long it had been since the snakehead had been released into the pond. Using a diet-switching tissue turnover model, we estimated that the snakehead was in the pond between 33 and 93 days. Subsequently, provincial legislation was amended to ban all species of snakehead fish, as well as numerous other potentially invasive fish and invertebrate species.

Evidence-based guideline: Treatment of tardive syndromes: Report of the Guideline Development Subcommittee of the American Academy of Neurology

<h3>Abstract</h3> Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. Measuring the timing of splicing relative to the transcription of nascent RNA has yielded conflicting interpretations. Biochemical fractionation suggests that RNA is spliced primarily during the process of transcription, but imaging of nascent RNA suggests that splicing happens after the process of transcription has been completed. We use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the delay between when an intron is transcribed and when it is spliced out of a pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit some post-transcriptional splicing, and that introns can be spliced in any order. We also show that completely synthesized RNA move slowly through a transcription site proximal zone while they undergo additional splicing and potentially other processing after transcription is completed. In addition, upon leaving this zone, some genes’ transcripts localize to speckles during the process of splicing but some appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Taken together, our observations suggest that the regulation of the timing and localization of splicing is specific to individual introns, as opposed to the previously surmised immediate excision of introns after transcription.