Divergent responses of Atlantic cod to ocean acidification and food limitation

In order to understand the effect of global change on marine fishes, it is imperative to quantify the effects on fundamental parameters such as survival and growth. Larval survival and recruitment of the Atlantic cod (Gadus morhua) were found to be heavily impaired by end‐of‐century levels of ocean acidification. Here, we analysed larval growth among 35–36 days old surviving larvae, along with organ development and ossification of the skeleton. We combined CO₂ treatments (ambient: 503 µatm, elevated: 1,179 µatm) with food availability in order to evaluate the effect of energy limitation in addition to the ocean acidification stressor. As expected, larval size (as a proxy for growth) and skeletogenesis were positively affected by high food availability. We found significant interactions between acidification and food availability. Larvae fed ad libitum showed little difference in growth and skeletogenesis due to the CO₂ treatment. Larvae under energy limitation were significantly larger and had further developed skeletal structures in the elevated CO₂ treatment compared to the ambient CO₂ treatment. However, the elevated CO₂ group revealed impairments in critically important organs, such as the liver, and had comparatively smaller functional gills indicating a mismatch between size and function. It is therefore likely that individual larvae that had survived acidification treatments will suffer from impairments later during ontogeny. Our study highlights important allocation trade‐off between growth and organ development, which is critically important to interpret acidification effects on early life stages of fish.     

A readily usable two‐photon fluorescence lifetime microendoscope

A two‐photon fluorescence lifetime (2P‐FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub‐cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table‐top microscope performances are achieved through a comprehensive system including a home‐designed spectro‐temporal pulse shaper and a custom air‐silica double‐clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.      

Phylogeny of spiny frogs Nanorana (Anura: Dicroglossidae) supports a Tibetan origin of a Himalayan species group

Recent advances in the understanding of the evolution of the Asian continent challenge the long‐held belief of a faunal immigration into the Himalaya. Spiny frogs of the genus Nanorana are a characteristic faunal group of the Himalaya–Tibet orogen (HTO). We examine the phylogeny of these frogs to explore alternative biogeographic scenarios for their origin in the Greater Himalaya, namely, immigration, South Tibetan origin, strict vicariance. We sequenced 150 Nanorana samples from 62 localities for three mitochondrial (1,524 bp) and three nuclear markers (2,043 bp) and complemented the data with sequence data available from GenBank. We reconstructed a gene tree, phylogenetic networks, and ancestral areas. Based on the nuDNA, we also generated a time‐calibrated species tree. The results revealed two major clades (Nanorana and Quasipaa), which originated in the Lower Miocene from eastern China and subsequently spread into the HTO (Nanorana). Five well‐supported subclades are found within Nanorana: from the East, Central, and Northwest Himalaya, the Tibetan Plateau, and the southeastern Plateau margin. The latter subclade represents the most basal group (subgenus Chaparana), the Plateau group (Nanorana) represents the sister clade to all species of the Greater Himalaya (Paa). We found no evidence for an east–west range expansion of Paa along the Himalaya, nor clear support for a strict vicariance model. Diversification in each of the three Himalayan subclades has probably occurred in distinct areas. Specimens from the NW Himalaya are placed basally relative to the highly diverse Central Himalayan group, while the lineage from the Tibetan Plateau is placed within a more terminal clade. Our data indicate a Tibetan origin of Himalayan Nanorana and support a previous hypothesis, which implies that a significant part of the Himalayan biodiversity results from primary diversification of the species groups in South Tibet before this part of the HTO was uplifted to its recent heights.     

Calreticulin affects fibronectin-based cell-substratum adhesion via the regulation of c-Src activity

Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.     

The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway

Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.     

Using likelihood-free inference to compare evolutionary dynamics of the protein networks of H. pylori and P. falciparum

Gene duplication with subsequent interaction divergence is one of the primary driving forces in the evolution of genetic systems. Yet little is known about the precise mechanisms and the role of duplication divergence in the evolution of protein networks from the prokaryote and eukaryote domains. We developed a novel, model-based approach for Bayesian inference on biological network data that centres on approximate Bayesian computation, or likelihood-free inference. Instead of computing the intractable likelihood of the protein network topology, our method summarizes key features of the network and, based on these, uses a MCMC algorithm to approximate the posterior distribution of the model parameters. This allowed us to reliably fit a flexible mixture model that captures hallmarks of evolution by gene duplication and subfunctionalization to protein interaction network data of Helicobacter pylori and Plasmodium falciparum. The 80% credible intervals for the duplication–divergence component are [0.64, 0.98] for H. pylori and [0.87, 0.99] for P. falciparum. The remaining parameter estimates are not inconsistent with sequence data. An extensive sensitivity analysis showed that incompleteness of PIN data does not largely affect the analysis of models of protein network evolution, and that the degree sequence alone barely captures the evolutionary footprints of protein networks relative to other statistics. Our likelihood-free inference approach enables a fully Bayesian analysis of a complex and highly stochastic system that is otherwise intractable at present. Modelling the evolutionary history of PIN data, it transpires that only the simultaneous analysis of several global aspects of protein networks enables credible and consistent inference to be made from available datasets. Our results indicate that gene duplication has played a larger part in the network evolution of the eukaryote than in the prokaryote, and suggests that single gene duplications with immediate divergence alone may explain more than 60% of biological network data in both domains.