Heritability of cortisol response to confinement stress in European sea bass dicentrarchus labrax

Background

In fish, the most studied production traits in terms of heritability are body weight or growth, stress or disease resistance, while heritability of cortisol levels, widely used as a measure of response to stress, is less studied. In this study, we have estimated heritabilities of two growth traits (body weight and length) and of cortisol response to confinement stress in the European sea bass.

Findings

The F1 progeny analysed (n = 922) belonged to a small effective breeding population with contributions from an unbalanced family structure of just 10 males and 2 females. Heritability values ranged from 0.54 (±0.21) for body weight to 0.65 (±0.22) for standard body length and were low for cortisol response i.e. 0.08 (±0.06). Genetic correlations were positive (0.94) between standard body length and body weight and negative between cortisol and body weight and between cortisol and standard body length (−0.60 and −0.55, respectively).

Conclusion

This study confirms that in European sea bass, heritability of growth-related traits is high and that selection on such traits has potential. However, heritability of cortisol response to stress is low in European sea bass and since it is known to vary greatly among species, further studies are necessary to understand the reasons for these differences.     

GBSX: a toolkit for experimental design and demultiplexing genotyping by sequencing experiments

Background

Massive parallel sequencing is a powerful tool for variant discovery and genotyping. To reduce costs, sequencing of restriction enzyme based reduced representation libraries can be utilized. This technology is generally referred to as Genotyping By Sequencing (GBS). To deal with GBS experimental design and initial processing specific bioinformatic tools are needed.

Results

GBSX is a package that assists in selecting the appropriate enzyme and the design of compatible in-line barcodes. Post sequencing, it performs optimized demultiplexing using these barcodes to create fastq files per barcode which can easily be plugged into existing variant analysis pipelines. Here we demonstrate the usability of the GBSX toolkit and demonstrate improved in-line barcode demultiplexing and trimming performance compared to existing tools.

Conclusions

GBSX provides an easy to use suite of tools for designing and demultiplexing of GBS experiments.     

Structure and dynamics in DNA looped domains: CAG triplet repeat sequence dynamics probed by 2-aminopurine fluorescence

The triplet repeat sequence (CAG)n and related triplet repeats are associated with dynamic DNA mutations implicated in a number of debilitating human diseases. To gain insight into the dynamics of the (CAG)n repeat, we have substituted a single 2-aminopurine (2AP) fluorescent base for adenine at select positions within the 18 base looped domain of a (GC)3(CAG)6(GC)3 hairpin oligonucleotide. Using temperature-dependent steady-state fluorescence measurements in combination with time correlated photon counting spectroscopy, we show the conformation and dynamics of the C2APG domains to be strongly dependent on the position of the probe in the looped region. In other words, rather than being a uniform, single stranded loop, the (CAG)6 triplet repeat looped domain exhibits order and dynamics that are position dependent. The 2AP fluorescence dynamics within the C2APG repeat are well described by a 4 component exponential decay model, with lifetimes ranging from 5 ps to 4 ns. Differences in global DNA conformation (duplex, hairpin, single strand), as well as the local position of the probe within the loop of a given hairpin, predominantly are reflected in the relative amplitude rather than the lifetime of the probe. The time dependent 2AP anisotropy in the hairpin (CAG)n loops is sensitive to the position of the fluorescent base, with the fluorescence depolarization of a centrally located 2AP probe within the loop proceeding significantly more slowly than 2AP positioned at the 5'- or 3'-end of the repeat sequence near the loop-stem junction. These results are consistent with segmental motions of the CAG repeat, while also suggesting that the 2AP probe is significantly stacked, possibly even hydrogen bonded, within the partially structured CAG looped domain. Our results characterize the position-dependent and conformation-dependent dynamics and order within (CAG)n triplet repeat DNAs, properties of relevance to the biological mechanisms by which such domains can lead to disease states.     

Preliminary evaluation of non-native rainbow trout (Oncorhynchus mykiss) impact on the Cederberg ghost frog (Heleophryne depressa) in South Africa’s Cape Fold Ecoregion

We evaluated the impact of non-native rainbow trout Oncorhynchus mykiss on a population of endemic Cedarberg ghost frog Heleophryne depressa in the upper Krom River (Olifants-Doring River Catchment, Cape Fold Ecoregion). We compared H. depressa abundance (using kick-sampling and underwater video analysis) and environmental conditions between sites above and below a waterfall that marks the upper distribution limit of O. mykiss. Heleophryne depressa abundance was significantly greater above the waterfall than that below it, and, because there was no significant difference in measured environmental variables, O. mykiss presence is identified as the most likely explanation for the observed decrease in H. depressa abundance.     

Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.