Experimental evidence for trait utility of gill raker number in adaptive radiation of a north temperate fish

North temperate fish in post-glacial lakes are textbook examples for rapid parallel adaptive radiation into multiple trophic specialists within individual lakes. Speciation repeatedly proceeded along the benthic – limnetic habitat axis, and benthic – limnetic sister species diverge in the number of gill rakers. Yet, the utility of different numbers of gill rakers for consuming benthic vs. limnetic food has only very rarely been experimentally demonstrated. We bred and raised families of a benthic – limnetic species pair of whitefish under common garden conditions to test whether these species (i) show heritable differentiation in feeding efficiency on zooplankton, and (ii) whether varia- tion in feeding efficiency is predicted by variation in gill raker numbers. We used zooplankton of three different size classes to investigate prey size dependency of divergence in feeding efficiency and to investigate the effect strength of variation in the number of gill rakers. Our results show strong interspecific differences in feeding efficiency. These differences are largest when fish were tested with the smallest zooplankton. Importantly, feeding efficiency is significantly positively correlated with the number of gill rakers when using small zooplankton, also when species identity is statistically controlled for. Our results support the hypothesis that a larger number of gill rakers are of adaptive significance for feeding on zooplankton and pro- vide one of the first experimental demonstrations of trait utility of gill raker number when fish feed on zooplankton. These results are consistent with the suggested importance of divergent selection driven feeding adaptation during adaptive radiation of fish in post-glacial lakes.

Phosphorylation of serine 4642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.

Migratory Urge and Gill Na(+),K(+)-ATPase Activity of Hatchery-Reared Atlantic Salmon Smolts from the Dennys and Penobscot River Stocks, Maine

Hatchery-reared Atlantic salmon Salmo salar smolts produced from captive-reared Dennys River and sea-run Penobscot River broodstock are released into their source rivers in Maine. The adult return rate of Dennys smolts is comparatively low, and disparity in smolt quality between stocks resulting from genetic or broodstock rearing effects is plausible. Smolt behavior and physiology were assessed during sequential 14-d trials conducted in seminatural annular tanks with circular flow. "Migratory urge'' (downstream movement) was monitored remotely using passive integrated transponder tags, and gill Na(+),K(+)-ATPase activity was measured at the beginning and end of the trials to provide an index of smolt development. The migratory urge of both stocks was low in early April, increased 20-fold through late May, and declined by the end of June. The frequency and seasonal distribution of downstream movement were independent of stock. In March and April, initial gill Na(+),K(+)-ATPase activities of Penobscot River smolts were lower than those of Dennys River smolts. For these trials, however, Penobscot River smolts increased enzyme activity after exposure to the tank, whereas Dennys River smolts did not, resulting in similar activities between stocks at the end of all trials. There was no clear relationship between migratory urge and gill Na(+),K(+)-ATPase activity. Gill Na(+),K(+)-ATPase activity of both stocks increased in advance of migratory urge and then declined while migratory urge was increasing. Maximum movement was observed from 2 h after sunset through 1 h after sunrise but varied seasonally. Dennys River smolts were slightly more nocturnal than Penobscot River smolts. These data suggest that Dennys and Penobscot River stocks are not markedly different in either physiological or behavioral expression of smolting.

Spatial distribution and impact of the gill-parasitic Mazocraes alosae (Monogenea Polyopisthocotylea) on Alosa alosa and A. fallax (Actinopterygii, Clupeidae)

Are the distribution of Mazocraes alosae and its impact on the host similar between Alosa alosa and A. fallax according to their resemblances? Parasites were numbered on each gill of shads sampled in North-East Atlantic coastal waters and connected rivers. Their impact on host condition was measured using girth, gonado-somatic ratio, C/N ratio, and Fulton’s K. Prevalence and mean intensity of M. alosae were significantly higher for A. alosa than for A. fallax, including in sympatric conditions. The mean intensity varied among sites whatever fish species; it was higher in coastal–estuarine versus fresh waters only for A. fallax. The distribution of M. alosae was aggregated in the host population whatever species. At the host individual level, some gills (second and third for A. alosa, second for A. fallax) were significantly more inhabited than others, probably in relation with larger water volumes flowing on these gills and mazocraeid sedentary lifestyle. Despite high prevalence and intensity, no negative impact of M. alosae was demonstrated on the host condition whatever the index considered. Our study underlines the major occurrence of M. alosae on shads and the potential use of such benign parasite as biological tag to discriminate closely related host species. © 2015, Springer International Publishing Switzerland.

Chemical Composition of Micrometer-Sized Filaments in an Aragonite Host by a Miniature Laser Ablation/Ionization Mass Spectrometer

Detection of extraterrestrial life is an ongoing goal in space exploration, and there is a need for advanced instruments and methods for the detection of signatures of life based on chemical and isotopic composition. Here, we present the first investigation of chemical composition of putative microfossils in natural samples using a miniature laser ablation/ionization time-of-flight mass spectrometer (LMS). The studies were conducted with high lateral (similar to 15 mu m) and vertical (similar to 20-200 nm) resolution. The primary aim of the study was to investigate the instrument performance on micrometer-sized samples both in terms of isotope abundance and element composition. The following objectives had to be achieved: (1) Consider the detection and calculation of single stable isotope ratios in natural rock samples with techniques compatible with their employment of space instrumentation for biomarker detection in future planetary missions. (2) Achieve a highly accurate chemical compositional map of rock samples with embedded structures at the micrometer scale in which the rock matrix is easily distinguished from the micrometer structures. Our results indicate that chemical mapping of strongly heterogeneous rock samples can be obtained with a high accuracy, whereas the requirements for isotope ratios need to be improved to reach sufficiently large signal-to-noise ratio (SNR). Key Words: Biogenicity-Biomarkers-Biosignatures-Filaments-Fossilization. Astrobiology 15, 669-682.

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

Gill tissue masses and oxygen consumption of Gobionotothen gibberifrons, Notothenia coriiceps and Zoarces viviparus

Mechanisms responsive to hypercapnia (elevated CO2 concentrations) and shaping branchial energy turnover were investigated in isolated perfused gills of two Antarctic Notothenioids (Gobionotothen gibberifrons, Notothenia coriiceps). Branchial oxygen consumption was measured under normo- versus hypercapnic conditions (10,000 ppm CO2) at high extracellular pH values. The fractional costs of ion regulation, protein and RNA synthesis in the energy budgets were determined using specific inhibitors. Overall gill energy turnover was maintained under pH compensated hypercapnia in both Antarctic species as well as in a temperate zoarcid (Zoarces viviparus). However, fractional energy consumption by the examined processes rose drastically in G. gibberifrons (100-180%), and to a lesser extent in N. coriiceps gills (7-56%). In conclusion, high CO2 concentrations under conditions of compensated acidosis induce cost increments in epithelial processes, however, at maintained overall rates of branchial energy turnover.