Analysis options for high-throughput sequencing in miRNA expression profiling

Background: Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods: We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results: High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers. Conclusions: Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.:Background; Methods; Results; Discussion; Conclusion

Comparison of Aerobic Scope for Metabolic Activity in Aquatic Ectotherms With Temperature Related Metabolic Stimulation: A Novel Approach for Aerobic Power Budget

Considering that swim-flume or chasing methods fail in the estimation of maximum metabolic rate and in the estimation of Aerobic Scope (AS) of sedentary or sluggish aquatic ectotherms, we propose a novel conceptual approach in which high metabolic rates can be obtained through stimulation of organism metabolic activity using high and low non-lethal temperatures that induce high (HMR) and low metabolic rates (LMR), This method was defined as TIMR: Temperature Induced Metabolic Rate, designed to obtain an aerobic power budget based on temperature-induced metabolic scope which may mirror thermal metabolic scope (TMS = HMR—LMR). Prior to use, the researcher should know the critical thermal maximum (CT max) and minimum (CT min) of animals, and calculate temperature TIMR max (at temperatures −5–10% below CT max) and TIMR min (at temperatures +5–10% above CT min), or choose a high and low non-lethal temperature that provoke a higher and lower metabolic rate than observed in routine conditions. Two sets of experiments were carried out. The first compared swim-flume open respirometry and the TIMR protocol using Centropomus undecimalis (snook), an endurance swimmer, acclimated at different temperatures. Results showed that independent of the method used and of the magnitude of the metabolic response, a similar relationship between maximum metabolic budget and acclimation temperature was observed, demonstrating that the TIMR method allows the identification of TMS. The second evaluated the effect of acclimation temperature in snook, semi-sedentary yellow tail (Ocyurus chrysurus), and sedentary clownfish (Amphiprion ocellaris), using TIMR and the chasing method. Both methods produced similar maximum metabolic rates in snook and yellowtail fish, but strong differences became visible in clownfish. In clownfish, the TIMR method led to a significantly higher TMS than the chasing method indicating that chasing may not fully exploit the aerobic power budget in sedentary species. Thus, the TIMR method provides an alternative way to estimate the difference between high and low metabolic activity under different acclimation conditions that, although not equivalent to AS may allow the standardized estimation of TMS that is relevant for sedentary species where measurement of AS via maximal swimming is inappropriate

Physiological characterization of Typhlatya cave shrimps: linking habitat with aerobic metabolism

The anchialine environment is characterized by a vertical stratification of water masses with different salinities. Cave shrimps of the genus Typhlatya are widespread inhabitants of the aquifer in fresh, brackish, and marine groundwater. Here we describe physiological aspects of three of the most abundant and widespread Typhlatya species that thrive in the fresh and marine groundwater habitats of the anchialine ecosystem of the Yucatan Peninsula. The aerobic scope (AS) of Typhlatya mitchelli, Typhlatya pearsei and Typhlatya dzilamensis was estimated through induced physical activity, whilst monitoring protein carbonylation and lipid peroxidation (as indicators of cellular damage), lactate accumulation (as an indicator of anaerobic metabolism) and the antioxidant system response. The critical thermal limits (CTL) of all three species as an additional measure of physiological plasticity were also determined. Our results showed that metabolic rates, AS and CTL were similar amongst the two species that inhabit fresh groundwater habitats, and differed markedly from T. dzilamensis, a species typically found in marine groundwater. The antioxidant system response in all three Typhlatya species accompanied the levels of aerobic metabolism following physical activity. However, the large amount of GSH observed in T. dzilamensis may be indicative of an adaptive trait to a more heterogeneous environment. The differences observed among Typhlatya species reflect different physiological adaptations that correspond to the environmental heterogeneity of their natural habitats. Our results suggest that the marine groundwater species, T. dzilamensis, could be better prepared to respond to a naturally more heterogeneous environment, in contrast to Typhlatya mitchelli and Typhlatya pearsei which rarely face environmental clines in the fresh groundwater habitat. Our findings contribute to a better understanding of the consequences of environmental change on ecologically important species that are restricted to live in the aquifer

Comparison of aerobic scope for metabolic activity in aquatic ectotherms with temperature related metabolic stimulation: a novel approach for aerobic power budget

Considering that swim-flume or chasing methods fail in the estimation of maximum metabolic rate and in the estimation of Aerobic Scope (AS) of sedentary or sluggish aquatic ectotherms, we propose a novel conceptual approach in which high metabolic rates can be obtained through stimulation of organism metabolic activity using high and low non-lethal temperatures that induce high (HMR) and low metabolic rates (LMR), This method was defined as TIMR: Temperature Induced Metabolic Rate, designed to obtain an aerobic power budget based on temperature-induced metabolic scope which may mirror thermal metabolic scope (TMS = HMR—LMR). Prior to use, the researcher should know the critical thermal maximum (CT max) and minimum (CT min) of animals, and calculate temperature TIMR max (at temperatures −5–10% below CT max) and TIMR min (at temperatures +5–10% above CT min), or choose a high and low non-lethal temperature that provoke a higher and lower metabolic rate than observed in routine conditions. Two sets of experiments were carried out. The first compared swim-flume open respirometry and the TIMR protocol using Centropomus undecimalis (snook), an endurance swimmer, acclimated at different temperatures. Results showed that independent of the method used and of the magnitude of the metabolic response, a similar relationship between maximum metabolic budget and acclimation temperature was observed, demonstrating that the TIMR method allows the identification of TMS. The second evaluated the effect of acclimation temperature in snook, semi-sedentary yellow tail (Ocyurus chrysurus), and sedentary clownfish (Amphiprion ocellaris), using TIMR and the chasing method. Both methods produced similar maximum metabolic rates in snook and yellowtail fish, but strong differences became visible in clownfish. In clownfish, the TIMR method led to a significantly higher TMS than the chasing method indicating that chasing may not fully exploit the aerobic power budget in sedentary species. Thus, the TIMR method provides an alternative way to estimate the difference between high and low metabolic activity under different acclimation conditions that, although not equivalent to AS may allow the standardized estimation of TMS that is relevant for sedentary species where measurement of AS via maximal swimming is inappropriate

Observations on feeding and biochemical characteristics to improve larviculture of Robsonella fontaniana (Cephalopoda: Octopodidae)

17 p., 3 figures, 1 table and bibliographyAt present, the production of merobenthic octopuses with a post-eclosion planktonic phase is a great challenge for global aquaculture. So far, juveniles from only four species have been reported to have been obtained under controlled rearing conditions: Octopus vulgaris, Octopus joubini, Enteroctopus dofleini and Robsonella fontaniana, the only Chilean species. Robsonella fontaniana (Orbigny, 1834) is a small-sized octopus traded in international markets as “baby octopus”. It is distributed over nearly the entire southern coast of South America, both in the Pacific and Atlantic coasts. This study shows new results regarding the biochemical characteristics in embryonic and paralarval stages, as well as the characteristics of food consumption during the paralarval stage that allow us to discuss a baseline on which to define a strategy for the larviculture of this species.The present study was financed by FONDEF D04 I1401 and by CONICYT-PBCT ACI-034 research funds.Peer reviewe