Amphioxus muscle transcriptomes reveal vertebrate-like myoblast fusion genes and a highly conserved role of insulin signalling in the metabolism of muscle

Madeleine E. Aase-Remedios and Clara Coll-Lladó were supported by funding from the University of St Andrews, School of Biology and additional support from St Leonards College (MEAR), the CORBEL grant European Research Infrastructure cluster project and European Assemble Plus (H2020-INFRAIA-1-2016-2017; grant no.730984). Transcriptome sequencing was done with an award under the BBSRC TGAC Capacity and Capability Challenge.Background:   The formation and functioning of muscles are fundamental aspects of animal biology, and the evolution of ‘muscle genes’ is central to our understanding of this tissue. Feeding-fasting-refeeding experiments have been widely used to assess muscle cellular and metabolic responses to nutrition. Though these studies have focused on vertebrate models and only a few invertebrate systems, they have found similar processes are involved in muscle degradation and maintenance. Motivation for these studies stems from interest in diseases whose pathologies involve muscle atrophy, a symptom also triggered by fasting, as well as commercial interest in the muscle mass of animals kept for consumption. Experimentally modelling atrophy by manipulating nutritional state causes muscle mass to be depleted during starvation and replenished with refeeding so that the genetic mechanisms controlling muscle growth and degradation can be understood. Results:  Using amphioxus, the earliest branching chordate lineage, we address the gap in previous work stemming from comparisons between distantly related vertebrate and invertebrate models. Our amphioxus feeding-fasting-refeeding muscle transcriptomes reveal a highly conserved myogenic program and that the pro-orthologues of many vertebrate myoblast fusion genes were present in the ancestral chordate, despite these invertebrate chordates having unfused mononucleate myocytes. We found that genes differentially expressed between fed and fasted amphioxus were orthologous to the genes that respond to nutritional state in vertebrates. This response is driven in a large part by the highly conserved IGF/Akt/FOXO pathway, where depleted nutrient levels result in activation of FOXO, a transcription factor with many autophagy-related gene targets. Conclusion:  Reconstruction of these gene networks and pathways in amphioxus muscle provides a key point of comparison between the distantly related groups assessed thus far, significantly refining the reconstruction of the ancestral state for chordate myoblast fusion genes and identifying the extensive role of duplicated genes in the IGF/Akt/FOXO pathway across animals. Our study elucidates the evolutionary trajectory of muscle genes as they relate to the increased complexity of vertebrate muscles and muscle development.Publisher PDFPeer reviewe

More than one-to-four via 2R : evidence of an independent amphioxus expansion and two-gene ancestral vertebrate state for MyoD-related Muscle Regulatory Factors (MRFs)

Funding: Madeleine Aase-Remedios and Dr Clara Coll-Lladówere supported by funding from the University of St Andrews, School of Biology and additional support from the CORBEL grant European Research Infrastructure cluster project.The evolutionary transition from invertebrates to vertebrates involved extensive gene duplication, but understanding precisely how such duplications contributed to this transition requires more detailed knowledge of specific cases of genes and gene families. MyoD (Myogenic differentiation) has long been recognized as a master developmental control gene and member of the MyoD family of bHLH transcription factors (Myogenic regulatory factors, MRFs) that drive myogenesis across the bilaterians. Phylogenetic reconstructions within this gene family are complicated by multiple instances of gene duplication and loss in several lineages. Following two rounds of whole genome duplication (2R WGD) at the origin of the vertebrates the ancestral function of MRFs is thought to have become partitioned amongst the daughter genes, so that MyoD and Myf5 act early in myogenic determination while Myog and Myf6 are expressed later, in differentiating myoblasts. Comparing chordate MRFs, we find an independent expansion of MRFs in the invertebrate chordate amphioxus, with evidence for a parallel instance of subfunctionalisation relative to that of vertebrates. Conserved synteny between chordate MRF loci supports the 2R WGD events as a major force in shaping the evolution of vertebrate MRFs. We also resolve vertebrate MRF complements and organization, finding a new type of vertebrate MRF gene in the process, which allowed us to infer an ancestral two-gene state in the vertebrates corresponding to the early- and late-acting types of MRFs. This necessitates a revision of previous conclusions about the simple one-to-four origin of vertebrate MRFs.Publisher PDFPeer reviewe

Ocean acidification promotes otolith growth and calcite deposition in gilthead sea bream (Sparus aurata) larvae

This work received funding from the Marine Alliance for Science and Technology for Scotland (MASTS) Small Grant initiative (SG330 and SG407). MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions. PBW would like to thank the Royal Society for the award of an Industry Fellowship. Financial support to CRB and JG were through the BIOACID (Biological Impacts of Ocean Acidification; Phase II) research programme.The effects of ocean acidification on otolith crystallization and growth rates were investigated in gilthead sea bream (Sparus aurata) larvae. Larvae were exposed to three different pH levels: pH8.2, pH7.7 and pH7.3 for a period of 18 days post-fertilization. For the first time, we demonstrate that pH has a significant impact on the carbonate polymorph composition, showing calcite in a significant percentage of individuals at low pH. Around 21% of the larvae exposed to pH7.3 showed irregular calcitic otoliths rather than commonly found round aragonitic otoliths. Calcitic otoliths showed a moderate level of heritability suggesting an important role of genetic factors. We also observed significantly larger otoliths in larvae reared at pH7.7 and pH7.3 compared to pH8.2 in both sagittae and lapilli. Our results demonstrate that otolith growth rates in gilthead sea bream larvae increase at low pH while a significant proportion of larvae are prone to the formation of calcitic otoliths at pH7.3.Publisher PDFPeer reviewe

Fast-growing growth hormone transgenic coho salmon (Oncorhynchus kisutch) show a lower incidence of vaterite deposition and malformations in sagittal otoliths

DGS is a Serra Húnter Tenure-Track lecturer, and this work has been partially funded by the Ministerio de Ciencia y Tecnología grant number RTI2018-100757-B-100. RHD acknowledges support from the Canadian Regulatory System for Biotechnology (grant number 61740).In fish otoliths, CaCO3 normally precipitates as aragonite, and more rarely as vaterite or calcite. A higher incidence of vaterite deposition in otoliths from aquaculture-reared fish has been reported and it is thought that high growth rates under farming conditions might promote its deposition. To test this hypothesis, otoliths from growth hormone (GH) transgenic coho salmon (TF) and non-transgenic (NT) fish of matching size were compared. Once morphometric parameters were normalized by animal length, we found that TF fish otoliths were smaller (-24%, -19%, -20% and -30%; P<0.001 for length, width, perimeter and area, respectively) and rounder (-12%, +13.5%, +15% and -15.5% in circularity, form factor, roundness and ellipticity; P<0.001) than otoliths from non-transgenic fish of matching size. Interestingly, transgenic fish had smaller eyes (-30% eye diameter) and showed a strong correlation between eye and otolith size. We also found that the percentage of otoliths showing vaterite deposition was significantly smaller in transgenic fish (21-28%) compared to non-transgenic (69%; P&lt;0.001). Likewise, the area affected with vaterite deposition within individual otoliths was reduced in transgenic fish (21-26%) compared to non-transgenic (42.5%; P<0.001). Our results suggest that high growth rates per se are not sufficient to cause vaterite deposition in all cases, and that GH overexpression might have a protective role against vaterite deposition, an hypothesis that needs further investigation.Publisher PDFPeer reviewe

Pilot study to investigate the effect of long-term exposure to high pCO2 on adult cod (Gadus morhua) otolith morphology and calcium carbonate deposition

Funding was provided by the Bonus Baltic Sea research and development programme (Art 185) BIO-C3 project, funded jointly by the EU and the BMBF (Grant No. 03F0682A), BIOACID project (Biological Impacts of Ocean Acidification: Grant No. 03F0655K) funded by the German Ministry for Education and Research (BMBF), and the EU AQUAEXCEL transnational access grant for aquaculture infrastructures. Part of the work was supported by the FINEAQUA-project (Grant Number 12212001) funded by the Program for the Future Economy (financed by the European Regional Development Fund, the Federal Republic of Germany and the state of Schleswig–Holstein), the Centre for Marine Aquaculture, Tromsø, and the Marine Alliance for Science and Technology for Scotland (MASTS) Small Grant initiative (SG330 and SG407), and their support especially by the staff of the centre for Marine Aquaculture is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions. PBW would like to thank the Royal Society for the award of an Industry Fellowship. DGS is a Serra Húnter Tenure-Track lecturer.To date the study of ocean acidification on fish otolith formation has been mainly focused on larval and juvenile stages. In the present pilot study, wild-captured adult Atlantic cod (Gadus morhua) were exposed to two different levels of pCO2, 422µatm (ambient, low pCO2) or 1091µatm (high pCO2), for a period of 30 weeks (from mid-October to early April 2014–2015) in order to study the effects on otolith size, shape and CaCO3 crystallization amongst other biological parameters. We found that otoliths from cod exposed to high pCO2 were slightly smaller (− 3.4% in length; − 3.3% in perimeter), rounder (− 2.9% circularity and + 4% roundness) but heavier (+ 5%) than the low pCO2 group. Interestingly, there were different effects in males and females; for instance, male cods exposed to high pCO2 exhibited significant changes in circularity (− 3%) and roundness (+ 4%) compared to the low pCO2 males, but without significant changes on otolith dimensions, while females exposed to high pCO2 had smaller otoliths as shown for length (− 5.6%), width (− 2%), perimeter (− 3.5%) and area (− 4.8%). Furthermore, while the majority of the otoliths analysed showed normal aragonite deposition, 10% of fish exposed to 1091µatm of pCO2 had an abnormal accretion of calcite, suggesting a shift on calcium carbonate polymorph crystallization in some individuals under high pCO2 conditions. Our preliminary results indicate that high levels of pCO2 in adult Atlantic cod might affect otolith growth in a gender-specific way. Our findings reveal that otoliths from adult cod are affected by ocean acidification, and we believe that the present study will prompt further research into this currently under-explored areaPublisher PDFPeer reviewe

Relationship between induced phytoplankton blooms and the structure and dynamics of the free-living heterotrophic bacterial community

15 pages, 6 figures, 4 tablesBacterial community activity and structure are thought to be directly or indirectly related to phytoplankton development and, in particular, to the phytoplankton species dominating specific algal blooms. To test this hypothesis, we performed a mesocosm experiment designed to generate blooms of different types of phytoplankton through the additions of silicate, urea and phosphorus to oligotrophic water from the Blanes Bay Microbial Observatory (NW Mediterranean). Over 10 d of incubation bacterial activity, bacterial abundance, nutrient composition and free-living bacterial community structure were monitored, as well as phytoplankton composition and the fluorescence characteristics of dissolved organic matter (DOM). While we found clear effects of the different nutrient additions on chlorophyll levels, bacterial production and the type of dominant DOM, bacterial abundance followed a similar pattern across different nutrient treatments, which deviated from that observed in the control. While phytoplankton composition in the treatment with added silicate evolved differently with respect to the other treatments, free-living bacterial community structure (as determined with DGGE) did not show conspicuous differences between treatments. Our results reveal that the changes in bacterial community composition were mostly due to the variation in grazing pressure with time, with a small contribution from changes in bottom-up nutrient supply mediated by the shifts in phytoplankton compositionThis work was supported by the NoE Marine Genomics Europe and MARBEF, and by the Spanish projects MODIVUS (CTM2005-04795/MAR) and STORM (CTM2009-09352/MAR). We thank V. Balagué, I. Forn and everyone participating in the Blanes Bay sampling program, particularly M. Sala, F. Not, E. Vázquez-Domínguez, E.L. Sà and C. Rodríguez for various help. The students of the ‘Marine Genomics Europe Summer Course on Plankton Bloom Dynamics’ also provided feedback, and we particularly thank F. Gómez for a first appraisal of phytoplankton diversityPeer reviewe

Fast-growing growth hormone transgenic coho salmon (Oncorhynchus kisutch) show a lower incidence of vaterite deposition and malformations in sagittal otoliths

In fish otoliths, CaCO3 normally precipitates as aragonite, and more rarely as vaterite or calcite. A higher incidence of vaterite deposition in otoliths from aquaculture-reared fish has been reported and it is thought that high growth rates under farming conditions might promote its deposition. To test this hypothesis, otoliths from growth hormone (GH) transgenic coho salmon and non-transgenic fish of matching size were compared. Once morphometric parameters were normalized by animal length, we found that transgenic fish otoliths were smaller (−24%, −19%, −20% and −30% for length, width, perimeter and area, respectively; P<0.001) and rounder (−12%, +13.5%, +15% and −15.5% in circularity, form factor, roundness and ellipticity; P<0.001) than otoliths from non-transgenic fish of matching size. Interestingly, transgenic fish had smaller eyes (−30% eye diameter) and showed a strong correlation between eye and otolith size. We also found that the percentage of otoliths showing vaterite deposition was significantly smaller in transgenic fish (21-28%) than in non-transgenic fish (69%; P<0.001). Likewise, the area affected by vaterite deposition within individual otoliths was reduced in transgenic fish (21-26%) compared with non-transgenic fish (42.5%; P<0.001). Our results suggest that high growth rates per se are not sufficient to cause vaterite deposition in all cases, and that GH overexpression might have a protective role against vaterite deposition, a hypothesis that needs further investigation