Intermittent administration of peracetic acid is a mild environmental stressor that elicits mucosal and systemic adaptive responses from Atlantic salmon post-smolts.

BACKGROUND: Fish encounter oxidative stress several times during their lifetime, and it has a pervasive influence on their health and welfare. One of the triggers of oxidative stress in fish farming is the use of oxidative disinfectants to improve rearing conditions, especially in production systems employing recirculation technology. Here we report the physiological and morphological adaptive responses of Atlantic salmon (Salmo salar L.) post-smolts to intermittent exposure to a potent oxidative agent peracetic acid (PAA). Fish reared in semi-commercial scale brackish water recirculating aquaculture system (RAS) were exposed to 1 ppm PAA every 3 days over 6 weeks. Mucosal and systemic responses were profiled before exposure, 22 and 45 days during the intermittent PAA administration. RESULTS: Oxidative stress was likely triggered as plasma antioxidant capacity increased significantly during the exposure period. Adaptive stress response to the periodic oxidant challenge was likewise demonstrated in the changes in plasma glucose and lactate levels. PAA-induced alterations in the transcription of antioxidants, cytokines, heat shock proteins and mucin genes showed a tissue-specific pattern: downregulation was observed in the gills and olfactory rosette, upregulation occurred in the skin, and no substantial changes in the liver. Further, PAA exposure resulted in histological changes in key mucosal organs (i.e. olfactory rosette, skin and gills); pathological alterations were predominant in the gills where cases of epithelial lifting, hypertrophy and clubbing were prevalent. In addition, intermittent PAA administration resulted in an apparent overproduction of mucus in the nasal mucosa. Lastly, PAA did not dramatically alter the ability of salmon to mount a physiological stress response in the presence of a secondary stressor, though some subtle interference was documented in the kinetics and magnitude of plasma cortisol and glucose response post-stress. CONCLUSIONS: The present study collectively demonstrated that intermittent oxidant exposure was a mild environmental stressor that salmon could mount strong adaptive responses at systemic and mucosal levels. The results will be valuable in optimising the rearing conditions of post-smolts in RAS, especially in adopting water treatment strategies that do not considerably interfere with fish health and welfare.

Consequences of continuous ozonation on the health and welfare of Atlantic salmon post-smolts in a brackish water recirculating aquaculture system.

This study investigated the biological consequences of 45-day continuous ozonation on Atlantic salmon (Salmo salar) post-smolts in a brackish water recirculating aquaculture system (RAS). There was no significant difference in survival, operational welfare indicators, and average weight at termination between the ozone-treated and control groups. Plasma biochemical analyses revealed that the creatinine level was significantly higher in the ozone-treated group than in the control at termination. Histological evaluation of skin health showed no significant difference between the two groups. On the other hand, quantitative histopathology disclosed that the ozone group exhibited a better gill health status than did the control group, particularly at the end of the trial. Mucosal transcriptomics revealed a distinct response profile between the gills and skin. At day 45, there were no differentially expressed genes (DEG) identified in the skin, in contrast to 242 ozone-induced DEGs in the gills. Assessment of the transcriptomic profiles over time revealed that temporal effects were of greater impact in skin compared to gills, regardless of the treatment. The treatment did not result in metabolomic dysregulation and the overall profile lent support to the transcriptomics data that temporal effects had a greater influence on the changes observed. Exposure to handling-confinement stress revealed that ozone treatment did not alter the ability of post-smolts to respond to a secondary stressor. In summary, the suite of health and welfare indicators collectively indicated that continuous ozonation resulted in minimal physiological perturbations in salmon post-smolts. The results are expected to contribute to optimising the rearing conditions for post-smolts in RAS.

Oxidative Chemical Stressors Alter the Physiological State of the Nasal Olfactory Mucosa of Atlantic Salmon.

The olfactory organs of fish have vital functions for chemosensory and defence. Though there have been some ground-breaking discoveries of their involvement in immunity against pathogens in recent years, little is known about how they respond to non-infectious agents, such as exogenous oxidants, which fish encounter regularly. To this end, we employed Atlantic salmon (Salmo salar) as a model to study the molecular responses at the nasal olfactory mucosa of a teleost fish when challenged with oxidants. Microarray analysis was employed to unravel the transcriptional changes at the nasal olfactory mucosa following two types of in vivo exposure to peracetic acid (PAA), a highly potent oxidative agent commonly used in aquaculture: Trial 1: periodic and low dose (1 ppm, every 3 days over 45 days) to simulate a routine disinfection; and Trial 2: less frequent and high dose (10 ppm for 30 min, every 15 days, 3 times) to mimic a bath treatment. Furthermore, leukocytes from the olfactory organ were isolated and exposed to PAA, as well as to hydrogen peroxide (H2O2) and acetic acid (AA)-the two other components of PAA trade products-to perform targeted cellular and molecular response profiling. In the first trial, microarrays identified 32 differentially expressed genes (DEG) after a 45-day oxidant exposure. Erythrocyte-specific genes were overly represented and substantially upregulated following exogenous oxidant exposure. In Trial 2, in which a higher dose was administered, 62 DEGs were identified, over 80% of which were significantly upregulated after exposure. Genes involved in immune response, redox balance and stress, maintenance of cellular integrity and extracellular matrix were markedly affected by the oxidant. All chemical stimuli (i.e., PAA, H2O2, AA) significantly affected the proliferation of nasal leukocytes, with indications of recovery observed in PAA- and H2O2-exposed cells. The migration of nasal leukocytes was promoted by H2O2, but not much by PAA and AA. The three chemical oxidative stressors triggered oxidative stress in nasal leukocytes as indicated by an increase in the intracellular reactive oxygen species level. This resulted in the mobilisation of antioxidant defences in the nasal leukocytes as shown by the upregulation of crucial genes for this response network. Though qPCR revealed changes in the expression of selected cytokines and heat shock protein genes following in vitro challenge, the responses were stochastic. The results from the study advance our understanding of the role that the nasal olfactory mucosa plays in host defence, particularly towards oxidative chemical stressors.

A broad mutational target explains a fast rate of phenotypic evolution.

The rapid evolution of a trait in a clade of organisms can be explained by the sustained action of natural selection or by a high mutational variance, that is the propensity to change under spontaneous mutation. The causes for a high mutational variance are still elusive. In some cases, fast evolution depends on the high mutation rate of one or few loci with short tandem repeats. Here, we report on the fastest evolving cell fate among vulva precursor cells in Caenorhabditis nematodes, that of P3.p. We identify and validate causal mutations underlying P3.p's high mutational variance. We find that these positions do not present any characteristics of a high mutation rate, are scattered across the genome and the corresponding genes belong to distinct biological pathways. Our data indicate that a broad mutational target size is the cause of the high mutational variance and of the corresponding fast phenotypic evolutionary rate.

Heritable characteristics or traits of a group of organisms, for example the large brain size of primates or the hooves of a horse, are determined by genes, the environment, and by the interactions between them. Traits can change over time and generations when enough mutations in these genes have spread in a species to result in visible differences. However, some traits, such as the large brain of primates, evolve faster than others, but why this is the case has been unclear. It could be that a few specific genes important for that trait in question mutate at a high rate, or, that many genes affect the trait, creating a lot of variation for natural selection to choose from. Here, Besnard, Picao-Osorio et al. studied the roundworm Caenorhabditis elegans to better understand the causes underlying the different rates of trait evolution. These worms have a short life cycle and evolve quickly over many generations, making them an ideal candidate for studying mutation rates in different traits. Previous studies have shown that one of C. elegans' six cells of the reproductive system evolves faster than the others. To investigate this further, Besnard, Picao-Osorio et al. analysed the genetic mutations driving change in this cell in 250 worm generations. The results showed that five mutations in five different genes ­ all responsible for different processes in the cells ­ were behind the supercharged evolution of this particular cell. This suggests that fast evolution results from natural selection acting upon a collection of genes, rather than one gene, and that many genes and pathways shape this trait. In conclusion, these results demonstrate that how traits are coded at the molecular level, in one gene or many, can influence the rate at which they evolve.

A Single MicroRNA-Hox Gene Module Controls Equivalent Movements in Biomechanically Distinct Forms of Drosophila.

Movement is the main output of the nervous system. It emerges during development to become a highly coordinated physiological process essential to survival and adaptation of the organism to the environment. Similar movements can be observed in morphologically distinct developmental stages of an organism, but it is currently unclear whether or not these movements have a common molecular cellular basis. Here we explore this problem in Drosophila, focusing on the roles played by the microRNA (miRNA) locus miR-iab4/8, which we previously showed to be essential for the normal corrective response displayed by the fruit fly larva when turned upside down (self-righting). Our study shows that miR-iab4 is required for normal self-righting across all three Drosophila larval stages. Unexpectedly, we also discover that this miRNA is essential for normal self-righting behavior in the adult fly, an organism with different morphology, neural constitution, and biomechanics. Through the combination of gene expression, optical imaging, and quantitative behavioral approaches, we provide evidence that miR-iab4 exerts its effects on adult self-righting behavior in part through repression of the Hox gene Ultrabithorax (Ubx) in a specific set of adult motor neurons, the NB2-3/lin15 neurons. Our results show that miRNA controls the function, rather than the morphology, of these neurons and demonstrate that post-developmental changes in Hox gene expression can modulate behavior in the adult. Our work reveals that a common miRNA-Hox genetic module can be re-deployed in different neurons to control functionally equivalent movements in biomechanically distinct organisms and describes a novel post-developmental role of the Hox genes in adult neural function.

Pervasive Behavioral Effects of MicroRNA Regulation in Drosophila.

The effects of microRNA (miRNA) regulation on the genetic programs underlying behavior remain largely unexplored. Despite this, recent work in Drosophila shows that mutation of a single miRNA locus (miR-iab4/iab8) affects the capacity of the larva to correct its orientation if turned upside down (self-righting, SR), suggesting that other miRNAs might also be involved in behavioral control. Here we explore this possibility, studying early larval SR behavior in a collection of 81 Drosophila miRNA mutants covering almost the entire miRNA complement of the late embryo. Unexpectedly, we observe that >40% of all miRNAs tested significantly affect SR time, revealing pervasive behavioral effects of miRNA regulation in the early larva. Detailed analyses of those miRNAs affecting SR behavior (SR-miRNAs) show that individual miRNAs can affect movement in different ways, suggesting that specific molecular and cellular elements are affected by individual miRNA mutations. Furthermore, gene expression analysis shows that the Hox gene Abdominal-B (Abd-B) represents one of the targets deregulated by several SR-miRNAs. Our work thus reveals pervasive effects of miRNA regulation on a complex innate behavior in Drosophila and suggests that miRNAs may be core components of the genetic programs underlying behavioral control in other animals too.

Molecular Regulation of Alternative Polyadenylation (APA) within the Drosophila Nervous System.

Alternative polyadenylation (APA) is a widespread gene regulatory mechanism that generates mRNAs with different 3'-ends, allowing them to interact with different sets of RNA regulators such as microRNAs and RNA-binding proteins. Recent studies have shown that during development, neural tissues produce mRNAs with particularly long 3'UTRs, suggesting that such extensions might be important for neural development and function. Despite this, the mechanisms underlying neural APA are not well understood. Here, we investigate this problem within the Drosophila nervous system, focusing on the roles played by general cleavage and polyadenylation factors (CPA factors). In particular, we examine the model that modulations in CPA factor concentration may affect APA during development. For this, we first analyse the expression of the Drosophila orthologues of all mammalian CPA factors and note that their expression decreases during embryogenesis. In contrast to this global developmental decrease in CPA factor expression, we see that cleavage factor I (CFI) expression is actually elevated in the late embryonic central nervous system, suggesting that CFI might play a special role in neural tissues. To test this, we use the UAS/Gal4 system to deplete CFI proteins from neural tissue and observe that in this condition, multiple genes switch their APA patterns, demonstrating a role of CFI in APA control during Drosophila neural development. Furthermore, analysis of genes with 3'UTR extensions of different length leads us to suggest a novel relation between 3'UTR length and sensitivity to CPA factor expression. Our work thus contributes to the understanding of the mechanisms of APA control within the developing central nervous system.

MicroRNA-encoded behavior in Drosophila.

The relationship between microRNA (miRNA) regulation and the specification of behavior is only beginning to be explored. We found that mutation of a single miRNA locus (miR-iab4/iab8) in Drosophila larvae affects the animal's capacity to correct its orientation if turned upside down (self-righting). One of the miRNA targets involved in this behavior is the Hox gene Ultrabithorax, whose derepression in two metameric neurons leads to self-righting defects. In vivo neural activity analysis reveals that these neurons, the self-righting node (SRN), have different activity patterns in wild type and miRNA mutants, whereas thermogenetic manipulation of SRN activity results in changes in self-righting behavior. Our work thus reveals a miRNA-encoded behavior and suggests that other miRNAs might also be involved in behavioral control in Drosophila and other species.

The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system.

The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

The genetic content of chromosomal inversions across a wide latitudinal gradient.

There is increasing evidence regarding the role of chromosomal inversions in relevant biological processes such as local adaptation and speciation. A classic example of the adaptive role of chromosomal polymorphisms is given by the clines of inversion frequencies in Drosophila subobscura, repeatable across continents. Nevertheless, not much is known about the molecular variation associated with these polymorphisms. We characterized the genetic content of ca. 600 individuals from nine European populations following a latitudinal gradient by analysing 19 microsatellite loci from two autosomes (J and U) and the sex chromosome (A), taking into account their chromosomal inversions. Our results clearly demonstrate the molecular genetic uniformity within a given chromosomal inversion across a large latitudinal gradient, particularly from Groningen (Netherlands) in the north to Málaga (Spain) in the south, experiencing highly diverse environmental conditions. This low genetic differentiation within the same gene arrangement across the nine European populations is consistent with the local adaptation hypothesis for th evolutionof chromosomal polymorphisms. We also show the effective role of chromosomal inversions in maintaining different genetic pools within these inverted genomic regions even in the presence of high gene flow. Inversions represent thus an important barrier to gene flux and can help maintain specific allelic combinations with positive effects on fitness. Consistent patterns of microsatellite allele-inversion linkage disequilibrium particularly in loci within inversions were also observed. Finally, we identified areas within inversions presenting clinal variation that might be under selection.