Single Fish Research Articles

The t(4;14) Is Present in Patients with Early Stage Plasma Cell Proliferative Disorders Including MGUS and Smoldering Multiple Myeloma (SMM).

Background: Clonal cytogenetic aberrations define multiple myeloma (MM) into unique subsets of the disease each one with unique clinical and biologic features, associations with laboratory and pathologic markers and presumed differential influence on disease progression. While it is widely accepted that translocations represent primordial events in the pathogenesis of MM, it is controversial whether all translocations can be associated with disease stability and non-malignant behavior (i.e. in MGUS/SMM). We have found the translocations in cases of MGUS using dual fusion, cIg enhanced, interphase FISH, while others have found this translocation at lower prevalence using single fusion interphase FISH or the RT-PCR for IgH/MMSET. We thus sought to clarify by expanding the study of the t(4;14) among patients with MGUS and SMM.Materials and methods: We studied 210 patients who had the diagnosis of MGUS and SMM and entered into the Mayo Clinic Dysproteinemia database. Patients were excluded if they had active MM, extramedullary disease or plasmacytoma. Patients with isolated lytic bone lesions could be included if they had no overt evidence of progression. All studied patients had provided written informed consent for the collection of a research bone marrow aliquot that was collected at the time of routine bone marrow procurement. All patients had cytospin slides that were studied for a t(4;14) using cIg-FISH as previously described by us and using a double fusion FISH strategy. The importance of this is that the strategy will detect unbalanced t(4;14) with loss of der(14).Results: Twenty patients (9.5%) had evidence of the chromosome translocation. Detailed demographic data was available in 19; 14 were women (74%), 13 of 19 (68%) had SMM and there was no bias for heavy chain IgA (26%). Patients with t(4;14) were slightly younger than those without the abnormality (52 vs. 61 years), had similar hemoglobin concentrations, had slightly higher concentration of the serum M spike (2.0 vs 1.75 g/dl) and had overall slightly higher bone marrow plasmacytosis (median 23% vs 10%). Patients were followed since diagnosis for a median of 33 months (range 2–162 months). Three patients progressed to active MM at 12, 12 and 26 months after original diagnosis (all three originally having indolent/SMM).Conclusion: The t(4;14) can occur in patients with MGUS and SMM and without evidence of progression to MM. The prevalence is similar to that observed in MM, albeit slightly lower (9.5% versus 14%). In most “benign” plasma cell neoplasms the translocation is associated with greater plasmacytosis and thus enriched for in the smoldering MM category. It is also likely that the t(4;14) confers a higher risk of progression to MM given; a) its enrichment in SMM, b) the slightly higher prevalence of the translocation in MM as opposed to MGUS/SMM, and c) the well documented aggressive clinical course of MM with t(4;14).

Gains on Chromosome Arm 9q Represent a Novel and Independent Marker of Adverse Prognosis in Multiple Myeloma Patients Receiving Upfront High-Dose Chemotherapy and Autologous Stem Cell Transplantation.

Introduction: Genomic aberrations represent important prognostic markers in many hematological cancers. In multiple myeloma (MM), chromosome 13q and 17p deletions (13q−, 17p−) have emerged as important outcome predictors that indicate a dismal prognosis. Other chromosomal abnormalities have been discussed as prognostic markers in this disease but came not out as independent variables when they were tested in a multivariable fashion. However, the complexity of genomic rearrangements and the clinical heterogeneity seen in malignant plasma cell disorders argue against 13q− and 17p− as the sole genomic change of prognostic relevance. The significance of chromosome arm 1q, 9q, and 11q extra copies - three frequent genomic imbalances in MM - is undetermined.Material and Methods: 90 patients (pts.) treated with one or two cycles of high-dose chemotherapy (HD-CTX) followed by autologous stem cell transplantation (ASCT) at a single center were analyzed by tri-color FISH and five DNA probes mapping to chromosome bands 1q21, 9q34, 11q25, 13q14, and 17p13. A multivariable analysis (Cox proportional hazards regression model) including genetic and clinical variables was performed.Results: The most frequent aberrations in the present series were (in order of decreasing prevalence): +1q (n=39/78, 50.0%), +9q (n=38/78, 48.7%), 13q− (n=42/90, 46.6%), and +11q (n=39/85, 45.9%). 17p− was identified in only 3 out of 88 patients (3.4%), while +17p were present in 13 out of 88 patients (14.8%). The median follow-up time was 37 months (m) and the median event free survival (EFS) and overall survival (OS) time from first ASCT of the entire cohort was 26 m and 71 m, respectively. Multivariable analysis including genetic aberrations, ß2-microblobulin, albumin, LDH, Salmon/Durie stage, and age at time of diagnosis revealed +9q and 13q− as the only independent predictors of EFS (p=0.003 and p=0.01, respectively). The mEFS in patients with 13q− was 19.0 m and 20.7 m in patients with +9q. In patients with concurrent +9q and 13q−, mEFS was only 12.2 m. In patients lacking these two abnormalities, mEFS was not reached. OS was not significantly influenced by any genetic or clinical variable in our series, most likely due to effective salvage treatment after relapse.Conclusions: +9q represents a novel and independent marker of adverse prognosis in MM. A single FISH experiment applying two DNA probes allows easy and rapid assessment of outcome in patients with malignant plasma cell disorders.

Electric organ discharge frequency jamming during social interactions in brown ghost knifefish, Apteronotus leptorhynchus

In most communication systems, animals avoid jamming when similar signals from two individuals might overlap and cause interference. Most species of wave-type electric fish avoid jamming by shifting their electric organ discharge (EOD) frequencies away from those of conspecifics or away from mimics of sine wave EOD frequencies presented in playback experiments. Both male and female brown ghosts, however, appear to intentionally jam rivals during competitive interactions if the rival has a higher EOD frequency. We studied this behaviour in competitive interactions between two fish in a neutral arena, and in playback studies with single fish in resident–intruder and neutral arena models. During competitive interactions, females jammed the rival's EOD signal only when the rival's EOD frequency was within ∼30 Hz of their own EOD frequency, whereas males tried to jam EOD signals of rivals that were 70 Hz higher than their own EOD frequency. In the resident–intruder model experiment, fish tended to raise their EOD frequencies to within potential jamming range in response to a simulated intruder with a higher EOD frequency than their own. In a neutral arena model, fish directed the most aggressive attacks in response to simulated rivals with lower EOD frequencies, yet were less likely to attack the playback device if it played a frequency elevation that mimicked a fish with a lower EOD frequency attempting to jam them. This is the first evidence that electric fish may actively use jamming as an aggressive behaviour.

Abundance of giant sea anemones and patterns of association with anemonefishin the northern red sea

patterns of distribution and abundance of giant sea anemones and anemonefish were compared among coral reefs along the coastline of sinai in the northern red sea. the sea anemones varied widely in abundance between reef areas containing different habitat types. they were rare on steep reef slopes with abundant coral cover (=low-density anemone sites, 0.09–0.68 anemones per 1000 m2 of reef area), but were common at a site containing patch reefs interspersed with sand (=high-density anemone site, 6.00–8.11 anemones per 1000 m2). distributions of the endemic two-band anemonefish (amphiprion bicinctus) varied significantly between the two main host anemone species. at the high-density site, individuals of the sea anemone heteractis crispa either did not contain anemonefish, or were occupied by single juvenile fish as shown in previous studies. at low-density sites h. crispa usually hosted clusters of juvenile anemonefish. in contrast, individuals of the sea anemone entacmaea quadricolor hosted either single adult fish (high-density site) or pairs of breeding adults (low-density sites), frequently in addition to some juvenile fish. mechanisms that prevent anemonefish from reaching adult size and forming breeding pairs in h. crispa may include high fish mortality above a size threshold because this host cannot adequately protect them from predation when they become large, active emigration of fish to e. quadricolor as described in previous reports, and/or environmentally-controlled cessation of fish growth. we conclude that in the northern red sea, individuals of h. crispa potentially serve as nurseries for anemonefish.

Aggregation in juvenile pike (Esox lucius): interactions between habitat and density in early winter

Summary Juvenile pike (Esox lucius), a cannibalistic fish species, aggregates within habitat patches. The advantages to cannibals of aggregating in the absence of other predators and food constraints are not immediately obvious. In this study we explore the basis for this grouping by observing how spatial distributions of juvenile pike are mediated by the presence of conspecifics. Solitary pike preferred shallow‐water (0·17 m depth) habitats. When fish density was increased, the average time spent in alternative deep‐water habitat (0·33 m) increased, consistent with a despotic type of distribution and suggesting that interference was occurring. In pairs of fish, one pike, nominally the dominant individual, showed a habitat use similar to that of single fish. The second individual mostly occupied deep water, again consistent with a despotic distribution and apparently mediated by intimidation interference. However, dominant pike did on occasion enter deep water, at which times the subordinate pike remained with the dominant fish, appearing to aggregate in the pool. We propose that habitat‐specific risk could explain aggregations of pike in deep water. Although remaining in close proximity to dominant individuals in deep water would seemingly put subordinate fish at great risk, the alternative of moving to shallow water may increase risk still further by reducing the capacity to perceive and/or evade attacks.

Ichthyophonus in Puget Sound Rockfish from the San Juan Islands Archipelago and Puget Sound, Washington, USA

AbstractIn vitro explant cultures identified Ichthyophonus in 10.9% of 302 Puget Sound rockfish Sebastes emphaeus sampled from five sites in the San Juan Islands archipelago and Puget Sound, Washington, in 2003. None of the infected fish exhibited visible lesions and only a single fish was histologically positive. Significantly more females were infected (12.4%) than males (6.8%), and while infected males were only detected at two of the five sites, infected females were identified at all sites, with no significant differences in infection prevalence. Genomic sequences of Ichthyophonus isolates obtained from Puget Sound rockfish, Pacific herring Clupea pallasii, and Yukon River Chinook salmon Oncorhynchus tshawytscha were identical in both the A and B regions of the small subunit 18S ribosomal DNA but were different from Ichthyophonus sequences previously isolated from four different species of rockfish from the northeastern Pacific Ocean. Ich‐thyophonus in Puget Sound rockfish may not have been previously detected because the infection is subclinical in this species and earlier investigators did not utilize in vitro techniques for diagnosis of ichthyophoniasis. However, since clinical ichthyophoniasis has recently been identified in several other species of northeast Pacific rockfishes, it is hypothesized that this either is an emerging disease resulting from changing marine conditions or the result of introduction by infected southern species that appear during periodic El Niño events.

Preimplantation Genetic Diagnosis in Embryos Created From Oocytes Donation

To consider the concept that conception rates could be improved through routine preimplantation genetic diagnosis (PGD) for chromosome aneuploidy, in embryos created from donor eggs. Retrospective case analysis. PGD was performed for aneuploidy on 289 embryos obtained from 26 fresh IVF cycles and 2 FET cycles using 22 egg donors less than 30 years of age. PGD was performed prior to embryo transfer into gestational surrogates, to assure the transfer of chromosomally normal embryos. PGD was accomplished through fluorescence in situ hybridization on blastomeres removed from Day 3 embryos at 4 cell stage or greater. Three different probe combinations were used, depending on indication, including chromosomes 8, 9, 13, 16, 18, 21, 22, X, and Y. Two probe panel of X and Y only was done in 10 cycles on 103 embryos; a 5 probe panel using 13, 18, 21, X, and Y was done in 16 cycles on 163 embryos; and a 10 probe panel using 8, 9, 13, 15, 16, 18, 21, 22, X and Y was done in 2 cycles on 23 embryos. Developing embryos without aneuploidy were transferred on Day 5, with supernumerary embryos being cryopreserved. Egg donors had appropriate cycle day #3 FSH levels. FISH results using the various probes were combined and summarized in the table. Tabled 1 These results are consistent with our previous data based on a small number of embryos, that there appears to be a high rate of chromosomal aneuploidy (42%) involving embryos of young reproductive age women. The proportion of abnormal embryos varies significantly between individuals (29%-83%) and could not be predicted based on embryo morphology. These results may explain why certain egg donors have poor pregnancy conversion rates despite satisfactory ovarian response, oocyte quantity, and embryo morphologic appearance. In that case, PGD assessment of embryos created from donor eggs may improve implantation and conception rates, and minimize high order pregnancy rates through selection and transfer of only chromosomally competent embryos. However, more data on the genetic make up of the embryos of early reproductive age women is necessary in order to fully understand which factors such as spermatozoa, rate of mosaicism in embryos of young women, regime of ovarian stimulation or a consequence of an inherent error associated with the technique of single cell FISH contributes the most to the high rate of aneuploidy.

Target phase: An extra dimension for fish and plankton target identification

The acoustic signal backscattered from a fish in water, Lbs(f), at a frequency, f, differs from the incident signal in both magnitude, /Lbs(f)/, and phase, arg[Lbs(f)], and it has been common practice for many years to use the backscatter magnitude from individual fish as an aid to species identification. However, very little use has been made of the phase of the backscattered signal relative to that of the incident acoustic pulse. If the gross phase changes due to propagation through water are compensated for, the residual phase signature is found to contain useful target-specific information. The phase signature can be characterized by estimating the rate at which the echo phase changes, relative to the transmitted pulse, during the echo from a fish. Clear groups are produced when single fish targets from in situ data are plotted in complex target space (target strength versus target rate of change of phase) and this is explored here by computing the acoustic backscatter, in complex target space, from a series of simple large and small model fish targets, both with and without gas-filled swimbladders, using a Monte Carlo technique. It is shown that all the features found in the in situ data can be explained in terms of the size and attitude of the fish. The modeling was carried out at the frequency of 38 kHz, which pertained to the in situ data, and the specific results only apply to this frequency. However, the complex target approach is generally applicable to fish target strength analysis independently of frequency.

Effects of host density on furunculosis epidemics determined by the simple SIR model

To determine the transmission dynamics of furunculosis, Chinook salmon, Oncorhynchus tshawytscha held at various densities were challenged with a single infectious fish. Data from the in vivo experiments were compared to predicted values from a susceptible–infected–removed (SIR) model. Seven different densities were tested including 9.13, 4.56, 0.72, 0.36, 0.19, 0.06 and 0.03 fish/L and each produced different transmission coefficients ( β) of 0.01, 0.019, 0.0051, 0.0076, 0.0001, 0.0005 and 0.00, respectively. Furunculosis related mortality rates decreased as density of the host decreased. Mortality rates at the highest fish densities where disease specific mortality (DSM) was observed were 0.42, 0.44, 0.18 and 0.02 infected animals per day. The natural mortality rate also followed a similar pattern with 0.005, 0.005, 0.001, 0.005, 0.0001, 0.000 and 0.000 surviving animals per day. The results indicated that the transmission coefficient and the DSM of furunculosis was dependent on host density. It is possible that fish behavioral changes at the lower densities caused the reproductive rate of disease to be persistent even at very low densities. In summary, the simple SIR model suggested that low mortality does not always mean low prevalence of furunculosis in a given population. This is the first study evaluating host density as a risk factor for fish diseases using experimental furunculosis data.

Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch ( Perca fluviatilis) and domesticated rainbow trout ( Oncorhynchus mykiss)

Eurasian perch ( Perca fluviatilis) is a promising aquaculture candidate, but the growth performance of this non-domesticated species may be negatively affected by its stress responsiveness to intensive culture conditions. To evaluate this potential problem, juvenile Eurasian perch were exposed to a standardized handling stressor twice a week for an 8-week period. A similar study was conducted on domesticated rainbow trout ( Oncorhynchus mykiss) for comparison of intra- and inter-specific differences. The stressed fish of both species showed lower body growth than the non-stressed control fish, however, the final mean body mass was 35.4% lower in the stressed Eurasian perch than in the non-stressed controls, compared to 22.8% difference between the two groups in rainbow trout. The stress responsiveness was examined by comparing the post-stress cortisol and glucose levels in repeatedly stressed fish and fish exposed to the stressor only once. The cortisol stress response in both species strongly indicated a habituation to the repeated stressor. Thus, repeatedly stressed Eurasian perch reached maximum cortisol levels of 130 ng/mL after 0.5 h compared to 200 ng/mL in the fish stressed once, while considerably smaller differences in cortisol levels were shown between the repeatedly and single stressed rainbow trout. Rainbow trout also showed lower post-stress glucose levels in the repeatedly stressed fish compared to the single stressed fish. In contrast, the glucose levels in both groups of Eurasian perch increased abruptly after stress treatment and remained elevated at approximately 19 mM for 6 h; levels were three times as high as the peak levels 3 h post-stress in rainbow trout. Together, the habituation of the stress response shown in both species did not eliminate the growth difference found in the repeatedly stressed fish versus the control fish. Further, the lower growth performance of Eurasian perch compared to rainbow trout could partly be due to the increased energy consumption in the more stress responsive Eurasian perch.

In silico identification and expression analysis of 12 novel CC chemokines in catfish

Chemokines, a superfamily of chemotactic cytokines involved in recruitment, activation, and adhesion of a variety of leukocyte types to inflammatory foci, are a crucial component of the immune system of Sarcopterygiian vertebrates. Although all mammalian chemokines are believed to have been found, the status of these molecules in Actinopterygii was unknown until recently. The identification of chemokines in fish species has been complicated by low sequence conservation and confusion over expected numbers. Earlier discoveries of single fish chemokines coupled with rapidly expanding genetic resources in these species have recently provided a foundation for large-scale in silico discoveries of these important immune regulators. We report here the identification and expression analysis of 12 new CC chemokine sequences from catfish. When added to our previous report of 14 catfish CC chemokines, the number of CC chemokines in catfish now stands at 26, two more than known from humans. Establishing orthologous relationships among the majority of catfish CC chemokines, a newly available set of chicken CC chemokines, and their mammalian counterparts remain difficult, suggesting high levels of duplication and divergence within individual species.

Mosquito development and biological control in a macrophyte-based wastewater treatment plant

A one-year study of the proliferation of mosquito in a Pistia stratiotes-based waste stabilization ponds in Cameroon revealed that Mansonia and Culex were the main breeding genera with about 55% and 42% of the total imagoes respectively. Though the ponds represent a favorable breeding ground for mosquitoes, only 0.02% of captured imagoes was Anopheles gambiae, suggesting that this wastewater treatment plant does not significantly contribute to the development of the malaria vector in the area. Gambusia sp. introduced to control mosquito population in the ponds acclimatized relatively well in most of the ponds (B3-B7) and their feeding rate without any diet ranged from 15.0 to 50.2 larvae/day for a single fish.

Highly unsaturated fatty acid synthesis in vertebrates: New insights with the cloning and characterization of a Δ6 desaturase of Atlantic salmon

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a delta5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterized previously. Here we report cloning and functional characterization of a delta6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon delta5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly delta6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the delta6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both delta6 and delta5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both delta6 and delta5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil.

Effect of density on competition between wild and hatchery‐reared Atlantic salmon for shelter in winter

The effect of varying the density of hatchery‐reared Atlantic salmon Salmo salar on the ability of single wild fish to occupy a shelter is assessed. Although there was strong density‐dependence on sheltering overall, the ability of wild Atlantic salmon parr to occupy a shelter was not affected by the presence of hatchery‐reared fish even when outnumbered by four to one. These findings illustrate a competitive asymmetry for shelter in favour of the wild fish at the densities tested.

Establishment of an In Vitro Culture Method for Multiple Myeloma Cells.

In July 2004 we have established an in vitro method for culturing and expansion of human primary myeloma cells in a cytokine free system. This method which was designed to optimize the cell signaling between myeloma cells and their stromal support is easilly reproducible and so far has been applied successfully in 15 of 15 myeloma patients. This progress allows, for the first time, long term maintenance and expansion of the tumor cells with day by day observation of how the myeloma clone propagates, as assessed by morphology, FACS analysis and by the FISH-Bioview system (see figures 1,2 showing a group of myeloma cells after 28 days in culture along with their single FISH signal for known del13q).The protocol is currently under intensive procedure of standardization for general use.Applications of this culture system have already yielded several new findings. One, relating to the peculiar nuclear changes appearing in myeloma cells during culture is being presented in detail in another abstract. An additional issue is the ability to manipulate the culture to detect very small numbers of myeloma cells in the range of MRD. Yet another use is in vitro drug assay. Preliminary results have revealed no apparent influence of pamidronate or erythropoietin on myeloma cell expansion up to 28 days in co-culture (4 cases). In contrast, thalidomide and simvastatin had deleterious effect on the stromal support (3 cases) but the former did not eliminate the myeloma cells whereas no myeloma cells were seen with simvastatin after 3–7 days in culture. [Display omitted] [Display omitted]

Combination Chemotherapy with Pentostatin, Cyclophosphamide and Rituximab Induces High Rate of Remissions Including Complete Responses and Achievement of Minimal Residual Disease in Previously Untreated B-Chronic Lymphocytic Leukemia.

The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., >90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.

Evaluation of mosaic frequency in transgenesis by sperm mediated gene transfer on the cultured prepubertal mouse testicular germ cells

Exogeneous DNA can reproducibly be delivered by co-injected spermatozoa and this transgenesis method is known to be an efficient protocol. But, mosaic patterns of transgenic embryos and offspring were showed frequently. Whole blastomeres integration is very important in transgenic animal economics. This study was performed to evaluate the integration of exogeneous DNA in each blastomere and to improve the transgenic efficiency for sperm mediated gene transfer technique. Prospective animal study was executed. BDF1 mouse oocytes were piezo-pulse assisted microinjected with different sperm source for the production of exogenous eGFP DNA harbouring embryos. Three groups were divided by sperm source. As a control, fresh sperm were used to compare developmental rates in each groups. First group was used by frozen-thawed sperm head co-incubated with linear eGFP DNA for binding. Second group was used in vitro cultured sperm from 14–16 day old ICR mouse testicular germ cells for 2 weeks. For the efficient introduction of eGFP DNA into testicular cells, electroporation was executed at the beginning of in vitro culture. Spermatogenic cell culture was performed with DMEM/F12 basal medium containing rFSH, testosterone, GM-CSF, SCF, ITS, retinoic acid. eGFP protein expressions were confirmed under epifluorescence microscopy. At 8-cell stage, eGFP expressing blastomere number were evaluated. For evaluate mRNA transcript expression and chromosomal integration in each blastomere, single blastomere RT-PCR and FISH were executed in each blastomere at 8-cell stage embryos. PN formation, cleavage and blastocyst formation rates were similar in each group, and the eGFP expression rates were not different between two groups. But, in each blastomere evaluation at 8-cell stage embryos, eGFP expression features were very different. None of embryos derived by co-incubation group were shown whole blastomere expression patterns. To the contrary, embryos by in vitro cultured sperm were showed whole expression patterns (RT-PCR 4/6, FISH 2/5). Development of mouse embryos by in vitro cultured sperm injection Introduction of exogeneous DNA into pre-meiotic spermatogenic cells may improves to integrate DNA on the mouse whole embryos. Therefore, our results suggest that in vitro spermatogenic cell culture would be efficient in spermatogenic cell mediated gene transfer for transgenic animal production.

Fish density and size alter Pacific halibut feeding: implications for stock assessment

Laboratory experiments were conducted with Pacific halibut Hippoglossus stenolepis to test the hypothesis that responsiveness to food cues is density‐ and size‐ dependent. Tanks holding one, two and four fish were video‐taped continuously before and after introduction of an olfactory cue and whole baits (squid). Pre‐cue indices of activity did not vary with fish density. After olfactory stimulation, per capita activity was directly related to density, with no significant increase in activity by single fish. Numbers of baits located, attacked and consumed were directly related to fish density, and average times to first attack varied from 12 s in groups of four fish to >15 min in single fish. The latency period between location and attack decreased significantly with fish density. When large (43–55 cm, total length, LT) and small (31–38 cm LT) Pacific halibut were tested together in pairs and groups of four fish, small individuals located baits first in 61% of the trials. Large fish, however, consumed all of the baits (except one) in trials where both large and small fish responded. Social facilitation in Pacific halibut feeding will result in a non‐linear relationship between catch per unit effort and population density, and the size composition of fish captured may be influenced by interference competition.

Goezia bangladeshi n. sp. (Nematoda: Anisakidae) from an anadromous fish Tenualosa ilisha (Clupeidae).

A total of 1432 hilsa shad, Tenualosa ilisha (=Hilsa ilisha) from 11 fresh water, brackish water and marine localities in Bangladesh yielded 13 adult Goezia bangladeshi n. sp., all specimens being found in the intestine of a single fish host caught in the lower Ganges. A total of 2372 Goezia juveniles were recovered from 490 infected fish (prevalence 34.2%). This is the first Goezia species recorded from Bangladesh, and it differs from other valid species by the number of caudal papillae (pre 7-9, para 3, post 6); encircled by tiny spines, the position of double papillae, the arrangement of body spines, and the length ratio of the intestinal caecum and the ventricular appendix (1: 3.33-4.72). Juveniles were free in the gut, embedded in the gut wall and encapsulated in mesenteries. Tenualosa ilisha serves as the definitive host, but the predominance of juveniles may indicate that piscivorous hosts may also serve as such. A list of the nominal Goezia species with important characters is provided.

Pregnancies by PGD for translocation carriers using single interphase cell fish with chromosome-specific breakpoint spanning probes

Pregnancies by PGD for translocation carriers using single interphase cell fish with chromosome-specific breakpoint spanning probes