Article

Ectoparasite Caligus rogercresseyi modifies the lactate response in Atlantic salmon ( Salmo salar ) and Coho salmon ( Oncorhynchus kisutch )

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Abstract

Although Caligus rogercresseyi negatively impacts Chilean salmon farming, the metabolic effects of infection by this sea louse have never been completely characterized. Therefore, this study analyzed lactate responses in the plasma, as well as the liver/muscle lactate dehydrogenase (LDH) activity and gene expression, in Salmo salar and Oncorhynchus kisutch infested by C. rogercresseyi. The lactate responses of Atlantic and Coho salmon were modified by the ectoparasite. Both salmon species showed increasing in plasma levels, whereas enzymatic activity increased in the muscle but decreased in the liver. Gene expression was overexpressed in both Coho salmon tissues but only in the liver for Atlantic salmon. These results suggest that salmonids need more energy to adapt to infection, resulting in increased gene expression, plasma levels, and enzyme activity in the muscles. The responses differed between both salmon species and over the course of infection, suggesting potential species-specific responses to sea-lice infection.

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... This is of great importance, mainly due to the characteristics of the tissues used (muscle and liver), since they are not precisely immunological, they presented gene expressions related to the fish immune system. This can be due to the high-energy rate, that is to say lactate, which is used by these organs before infestation with C. rogercresseyi, which could activate the immune system in both species [37], or also by an iron regulation that could affect infestation, conferring a type of nutritional immunity [23]. ...
... In addition, the expression of MHCII, which is directly regulated by NLRC5 in mammals, has been evaluated by binding sequences that activate the transcription of this gene, through the enhancer of binding sites [39]. No significant differences in MHCII mRNA expression were observed in Atlantic salmon liver, this is probably due to a differential regulation in this organ or because the energy is used for the energetic metabolism during infestation with C. rogercresseyi [37,38]. It was even possible to determine that mRNA expression decreased in Coho salmon liver during the infestation process, which could be possible due to different signaling pathways, and MHCII mRNA was not detected in the muscle of both salmonid species. ...
... It has been described that teleost fishes are exposed to stress by modified parasite energy metabolites that modulate the immune responses against pathogens [45][46][47][48]. In addition, the muscle of salmonids needs more energy to be able to adapt to an infestation [37]. Therefore, the muscles of these two salmonids have a decreased immune reaction against an infestation with C. rogercresseyi. ...
Article
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Caligus rogercresseyi, a marine ectoparasite, causes notable economic losses for the Chilean salmonid industry. Nevertheless, the immunological responses of infected fish remain poorly understood, including proinflammatory cytokine generation and the respective modulatory effects of various cytokine receptors. This study evaluated mRNA expression of the NLRC5, major histocompatibility complex (MHC) class II, I-kappa-B-alpha, a regulatory that inhibits NF-kappa-B, and proinflammatory cytokines (IL-1β and IL-18) in the liver and muscle of Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch) during a time-course C. rogercresseyi infestation trial. All assessed mRNA were strongly regulated during infestation, but S. salar showed up-regulated expression, possibly accounting for the high infestation vulnerability of this salmonid. In conclusion, this work helps to understand the modulation of the expression of different transcripts involved over short periods of C. rogercresseyi infestation in two salmonid species (S. salar and O. kisutch).
... During fish farming there are also biotic factors that affect fish growth, such as stocking density (Mancera et al., 2008;Vargas-Chacoff et al., 2014) or disease produced by bacteria, viruses, or parasites (González et al., 2015;Martínez et al., 2017bMartínez et al., , 2017a. Parasites, such as endoparasites (with minimal effect on fish farming) and ectoparasites, can substantially affect hosts by impacting behavioral, physiological, and morphological traits as well as by damaging the integument (Vargas-Chacoff et al., 2017Wendelaar Bonga, 1997). These ectoparasites activate the stress systems and can induce changes in fish physiology responses (Barton, 2002;Vargas-Chacoff et al., 2014;Wendelaar Bonga, 1997). ...
... These ectoparasites activate the stress systems and can induce changes in fish physiology responses (Barton, 2002;Vargas-Chacoff et al., 2014;Wendelaar Bonga, 1997). Several authors indicated that some teleost fish exposed to parasitism stress can mobilize energy metabolites to modulate immunological responses to pathogens (Barton, 2002;Vargas-Chacoff et al., 2017;Wendelaar Bonga, 1997), with glucose and lactate being the most important energetic metabolites, but it is not clear which are the endocrine effects. In Atlantic salmon (Salmo salar) the effects of sea lice (Lepeophtheirus salmonis) on serotoninergic brain activity have been described; finding significant effects on the brain stem (Øverli et al., 2014). ...
... The same specimens and experimental procedures used in Vargas-Chacoff et al. (Vargas-Chacoff et al., 2017) were applied in the present study. Briefly, groups of juvenile post-smolt Atlantic salmon (166.4 ± 17.5 g body weight, n = 250) and Coho salmon (161.2 ± 15.8 g body weight, n = 250) were purchased from the Puerto Phillipi and Chaparano fish farms, respectively. ...
Article
The aquaculture industry has many problems associated with bacteria, viruses, and parasites, and the stress caused by these infections can modulate the physiological response in fish. In Chile, the sea lice Caligus rogercresseyi constitutes a major problem affecting the Chilean salmonid industry, having a strong negative effect on salmon production. For this reason, the aim of this study was to investigate the neuroendocrine and stress response of the most commonly cultured salmonid species in Chile, the Atlantic salmon (Salmo salar)and the Coho salmon (Oncorynchus kisutch), which were experimentally infested with the sea lice Caligus rogercresseyi. We analyzed the monoamine response in the telencephalon, optic tectum, and hypothalamus, as well as cortisol levels in plasma. In the liver and muscle we analyzed the cellular response by measuring heat shock proteins (60, 70, 90)and glucocorticoid mRNA transcription. After 14 days post infection C. rogercresseyi infestation modified the neuroendocrine and stress response in Atlantic and Coho salmon, with Coho salmon presenting a faster and higher response than the Atlantic salmon.
... During fish farming there are also biotic factors that affect fish growth, such as stocking density (Mancera et al., 2008;Vargas-Chacoff et al., 2014) or disease produced by bacteria, viruses, or parasites (González et al., 2015;Martínez et al., 2017bMartínez et al., , 2017a. Parasites, such as endoparasites (with minimal effect on fish farming) and ectoparasites, can substantially affect hosts by impacting behavioral, physiological, and morphological traits as well as by damaging the integument (Vargas-Chacoff et al., 2017Wendelaar Bonga, 1997). These ectoparasites activate the stress systems and can induce changes in fish physiology responses (Barton, 2002;Vargas-Chacoff et al., 2014;Wendelaar Bonga, 1997). ...
... These ectoparasites activate the stress systems and can induce changes in fish physiology responses (Barton, 2002;Vargas-Chacoff et al., 2014;Wendelaar Bonga, 1997). Several authors indicated that some teleost fish exposed to parasitism stress can mobilize energy metabolites to modulate immunological responses to pathogens (Barton, 2002;Vargas-Chacoff et al., 2017;Wendelaar Bonga, 1997), with glucose and lactate being the most important energetic metabolites, but it is not clear which are the endocrine effects. In Atlantic salmon (Salmo salar) the effects of sea lice (Lepeophtheirus salmonis) on serotoninergic brain activity have been described; finding significant effects on the brain stem (Øverli et al., 2014). ...
... The same specimens and experimental procedures used in Vargas-Chacoff et al. (Vargas-Chacoff et al., 2017) were applied in the present study. Briefly, groups of juvenile post-smolt Atlantic salmon (166.4 ± 17.5 g body weight, n = 250) and Coho salmon (161.2 ± 15.8 g body weight, n = 250) were purchased from the Puerto Phillipi and Chaparano fish farms, respectively. ...
Poster
The sea lice Caligus rogercresseyi constitute a major problem affecting the Chilean salmonids industry, having a strong negative effect over the production of Atlantic salmon (Salmo salar) and Rainbow trout (Oncorynchus mykiss). Caligidosis has been described affecting Atlantic salmon and rainbow trout more than Coho salmon (Onchorynchus kisutch).The aim of this study was to analyze the effects of C. rogercresseyi infestation over the stress response in these two related species, Atlantic salmon and Coho salmon. Through a time course experiment individuals from both species were infested with C. rogercresseyi and sampled at 0, 1, 3, 7 and 14 days after infestation. Plasma cortisol levels were measured as a physiological indicator of stress and gene expression of the Heat­shock Proteins (Hsp60 and Hsp70) was measured through RT­qPCR in muscle and liver tissues as a molecular marker of cellular stress response. Plasma cortisol levels presented highest values at day 1 in Coho salmon, whereas the peak was at day 3 in specimens of Atlantic salmon. Moreover, the peak of cortisol was up to two­fold higher in Coho salmon than the maximum peak observed for Atlantic salmon. Differences were found in liver and muscle Hsps gene expression in both species, where coho salmon showed an earlier and higher response compared to Atlantic salmon. Our results, suggest that Coho salmon could be experiencing a significantly faster stress response associated to the presence of the parasite, compared to the observed in Atlantic salmon, which would be then allowing a greater infestation by the sea lice in Atlantic salmon. The present study could help in understanding, in a comparative manner, the effects of this parasite on salmonids species and explain the differences observed in their response to its presence. This study was financed by INNOVA CORFO 13IDL2 23565, Fondecyt 11121498, 1160877, and PAI 7912010009, DID­UACh.
... In addition, the abundance of tlr13 in infested fish, the increased expression of this receptor in the skin, and the phylogenetic similarity of tlr22 and tlr13, suggest the possible role of tlr13 in the immune response to ectoparasites (Valenzuela-Muñoz et al. 2016). In S. salar and Oncorhynchus kisutch Walbaum, 1792, infestation by C. rogercresseyi caused an increase in levels of lactate dehydrogenase in muscles, and a decrease in the liver (Vargas-Chacoff et al. 2017). ...
Article
Full-text available
This review surveyed information on Caligus Müller, 1785 to identify global infestation patterns and geographic distribution in teleost fishes, as well as physiological and histopathological data and description of treatment strategies. A total 990 samples of Caligus spp. (N = 212 species) obtained of 233 scientific papers on farmed and wild teleost species from 99 families and 30 orders were used, and the highest number of occurrences was on Carangidae. Caligus spp. was predominantly found in marine environments, and only Caligus lacustris and Caligus epidemicus were found in teleost fish of freshwater environments. There was a high prevalence of Caligus spp. on hosts and infestation occurred predominantly in both the tegument and the gills. Caligus species are distributed across different countries and some particularities were identified and discussed. Caligus elongatus and Caligus bonito bonito had the broadest geographic distribution. Histomorphological and hematological disorders caused by infestation by Caligus spp. were reported and discussed, as well as chemotherapeutic products used for controlling and treating the infestations. Variation in the distribution and geographic patterns of Caligus spp. were little evident in many ecosystems and due to the limited data on the infestation of these sea lice on teleost populations in different regions.
... Host behavior, physiology, fitness, health, and metabolism are influenced by parasite infections [69][70][71][72]. To avoid the harmful effects of parasite infection, the aquaculture industry employs different strategies, such as the use of formulated diet, selective breeding, and vaccines, among others [73][74][75][76][77]. ...
Article
Full-text available
The sea louse Caligus rogercresseyi genome has opened the opportunity to apply the reverse vaccinology strategy for identifying antigens with potential effects on lice development and its application in sea lice control. This study aimed to explore the efficacy of three sea lice vaccines against the early stage of infestation, assessing the transcriptome modulation of immunized Atlantic salmon. Therein, three experimental groups of Salmo salar (Atlantic salmon) were vaccinated with the recombinant proteins: Peritrophin (prototype A), Cathepsin (prototype B), and the mix of them (prototype C), respectively. Sea lice infestation was evaluated during chalimus I-II, the early-infective stages attached at 7-days post infestation. In parallel, head kidney and skin tissue samples were taken for mRNA Illumina sequencing. Relative expression analyses of genes were conducted to identify immune responses, iron transport, and stress responses associated with the tested vaccines during the early stages of sea lice infection. The vaccine prototypes A, B, and C reduced the parasite burden by 24, 44, and 52% compared with the control group. In addition, the RNA-Seq analysis exhibited a prototype-dependent transcriptome modulation. The high expression differences were observed in genes associated with metal ion binding, molecular processes, and energy production. The findings suggest a balance between the host’s inflammatory response and metabolic process in vaccinated fish, increasing their transcriptional activity, which can alter the early host–parasite interactions. This study uncovers molecular responses produced by three vaccine prototypes at the early stages of infestation, providing new knowledge for sea lice control in the salmon aquaculture.
... Myosins and troponins have previously been identified as genes that respond to sea lice attachment in salmon skin (Holm et al., 2015). Further, Caligus infection is known to induce increased enzyme activity in muscle tissue (Vargas-Chacoff et al., 2017), and behavioral changes in the fish such as flashing and jumping are associated with ectoparasite removal (Furevik et al., 1993;Magnhagen et al., 2008). It has been recently reported that inactivity or reduced swimming activity contribute to resistance to sea lice (Bui, 2017), so it is possible that the high lice counts of susceptible fish in this study are due to higher activity levels with associated expression of muscle contraction related genes. ...
Preprint
Full-text available
Background Sea lice are parasitic copepods that cause large economic losses to salmon aquaculture worldwide. Frequent chemotherapeutic treatments are typically required to control this parasite, and alternative measures such as breeding for improved host resistance are desirable. Insight into the host-parasite interaction and mechanisms of host resistance can lead to improvements in selective breeding, and potentially novel treatment targets. In this study, RNA sequencing was used to study the skin transcriptome of Atlantic salmon parasitized with sea lice ( C. rogercresseyi ). The overall aims were to compare the transcriptomic profile of skin at louse attachment sites and ‘healthy’ skin, and to assess differences between animals with varying levels of resistance to the parasite. Results Atlantic salmon were challenged with C. rogercresseyi , growth and lice count measurements were taken for each fish. 21 animals were selected and RNA-Seq was performed on skin from a louse attachment site, and skin distal to attachment sites for each animal. These animals were classified into family-balanced groups according to the traits of resistance (high vs low lice count), and growth during infestation (an indication of tolerance). Overall comparison of skin from louse attachment sites versus healthy skin showed that 4,355 genes were differentially expressed, indicating local up-regulation of several immune pathways and activation of tissue repair mechanisms. Comparison between resistant and susceptible animals highlighted expression differences in several immune response and pattern recognition genes, and also myogenic and iron availability factors. Genomic regions showing signs of differentiation between resistant and susceptible fish were identified using an Fst analysis. Conclusions Comparison of the skin transcriptome between louse attachment sites and healthy skin has yielded a detailed profile of genes and pathways with putative roles in the local host immune response to C. rogercresseyi . The difference in skin gene expression profile between resistant and susceptible animals led to the identification of several immune and myogenic pathways potentially involved in host resistance. Components of these pathways may be targets for studies aimed at improved or novel treatment strategies, or to prioritise candidate functional polymorphisms to enhance genomic selection for host resistance in commercial salmon farming.
... Myosins and troponins have previously been identified as genes that respond to sea lice attachment in salmon skin (Holm et al., 2015). Further, Caligus infection is known to induce increased enzyme activity in muscle tissue (Vargas-Chacoff et al., 2017), and behavioral changes in the fish such as flashing and jumping are associated with ectoparasite removal (Furevik et al., 1993;Magnhagen et al., 2008). It has been recently reported that inactivity or reduced swimming activity contribute to resistance to sea lice (Bui, 2017), so it is possible that the high lice counts of susceptible fish in this study are due to higher activity levels with associated expression of muscle contraction related genes. ...
Article
Full-text available
Sea lice are parasitic copepods that cause large economic losses to salmon aquaculture worldwide. Frequent chemotherapeutic treatments are typically required to control this parasite, and alternative measures such as breeding for improved host resistance are desirable. Insight into the host–parasite interaction and mechanisms of host resistance can lead to improvements in selective breeding, and potentially novel treatment targets. In this study, RNA sequencing was used to study the skin transcriptome of Atlantic salmon (Salmo salar) parasitized with sea lice (Caligus rogercresseyi). The overall aims were to compare the transcriptomic profile of skin at louse attachment sites and “healthy” skin, and to assess differences in gene expression response between animals with varying levels of resistance to the parasite. Atlantic salmon pre-smolts were challenged with C. rogercresseyi, growth and lice count measurements were taken for each fish. 21 animals were selected and RNA-Seq was performed on skin from a louse attachment site, and skin distal to attachment sites for each animal. These animals were classified into family-balanced groups according to the traits of resistance (high vs. low lice count), and growth during infestation. Overall comparison of skin from louse attachment sites vs. healthy skin showed that 4,355 genes were differentially expressed, indicating local up-regulation of several immune pathways and activation of tissue repair mechanisms. Comparison between resistant and susceptible animals highlighted expression differences in several immune response and pattern recognition genes, and also myogenic and iron availability factors. Components of the pathways involved in differential response to sea lice may be targets for studies aimed at improved or novel treatment strategies, or to prioritize candidate functional polymorphisms to enhance genomic selection for host resistance in commercial salmon breeding programs.
Chapter
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This review surveys the available literature on the efficacy and ecotoxicology of chemotherapeutic agents for the control of sea lice (Copepoda: Caligidae) infestations in marine aquaculture. To date, treatment for the disease has relied almost exclusively on the use of the organophosphorus pesticides, trichlorfon and dichlorvos. While use of dichlorvos and trichlorfon appears to have little, if any, environmental impact, protracted use in some areas has led to reduced efficacy. Compounds proposed for sea lice control currently under field evaluation include azamethiphos (organophosphate), ivermectin (avermectin), pyrethrum (pyrethrin) and hydrogen peroxide. Although all of these compounds are effi cacious, at present published information on marine environmental toxicology is scarce and is limited to in vitro toxicity testing. Other compounds that have been considered for sea lice control include formalin, malathion (organophosphate), carbaryl (carbamate), diflubenzuron (insect growth regulator) and natural remedies (i.e. onions and garlic). In general, formaldehyde (aldehyde), malathion and natural compounds show either poor efficacy or unsuitable therapeutic margins. Although carbaryl and diflubenzuron are both efficacious and exhibit suitable therapeutic margins, available data suggests that the compounds, due to undesirable environmental toxicological characteristics, would be unsuitable for sea lice control.
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Hydrogen peroxide (H2O2) was the only effective alternative treatment to emamectin benzoate (EMB, SLICE, Schering Plough) for the sea louse Caligus rogercresseyi in Chile during the period from February to September 2007. This introduction was due to the loss of sensitivity of lice to EMB after 7 years of exclusive use. A detailed field appraisal of treatments was carried out on salmon farms at two sites near Puerto Montt (Lat 42ºS) in August 2007 to evaluate the efficacy of hydrogen peroxide in the control of C. rogercresseyi. A skirt was used on one farm for treatments and an enclosed tarpaulin in the other. Hydrogen peroxide was applied by bath at a concentration of 1.5 g l−1 for 20 min treatment but this did not kill C. rogercresseyi. Most of the parasites recovered from the treatment and were available to infest new hosts. Treatments were carried out in tanks under laboratory conditions to test the findings obtained in the field and these gave similar results. Caligus recovered completely after 30 min post treatment.
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Atlantic salmon Salmo salar were infected with two levels of sea lice Lepeophtheirus salmonis (0·13 ± 0·02 and 0·02 ± 0·00 sea lice g−1). Once sea lice became adults, the ventral aorta of each fish was fitted with a Doppler cuff to measure cardiac output (Q̇), heart rate (fH) and stroke volume (VS) during swimming. Critical swimming speeds (Ucrit) of fish with higher sea lice numbers [2·1 ± 0·1 BL (body lengths) s−1] were significantly lower (P < 0·05) than fish with lower numbers (2·4 ± 0·1 BL s−1) and controls (sham infected, 2·6 ± 0·1 BL s−1). After swimming, chloride levels in fish with higher sea lice numbers (184·4 ± 11·3 mmol l−1) increased significantly (54%) from levels at rest and were significantly higher than fish with fewer lice (142·0 ± 3·7 mmol l−1) or control fish (159·5 ± 3·5 mmol l−1). The fH of fish with more lice was 9% slower than the other two groups at Ucrit. This decrease resulted in Q̇ not increasing from resting levels. Sublethal infection by sea lice compromised the overall fitness of Atlantic salmon. The level of sea lice infection used in the present study was lower than has previously been reported to be detrimental to wild Atlantic salmon.
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Cortisol is the principal corticosteriod in teleost fishes and its plasma concentrations rise dramatically during stress. The relationship between this cortisol increase and its metabolic consequences are subject to extensive debate. Much of this debate arises from the different responses of the many species used, the diversity of approaches to manipulate cortisol levels, and the sampling techniques and duration. Given the extreme differences in experimental approach, it is not surprising that inconsistencies exist within the literature. This review attempts to delineate common themes on the physiological and metabolic roles of cortisol in teleost fishes and to suggest new approaches that might overcome some of the inconsistencies on the role of this multifaceted hormone. We detail the dynamics of cortisol, especially the exogenous and endogenous factors modulating production, clearance and tissue availability of the hormone. We focus on the mechanisms of action, the biochemical and physiological impact, and the interaction with other hormones so as to provide a conceptual framework for cortisol under resting and/or stressed states. Interpretation of interactions between cortisol and other glucoregulatory hormones is hampered by the absence of adequate hormone quantification, resulting in correlative rather than causal relationships.The use of mammalian paradigms to explain the teleost situation is generally inappropriate. The absence of a unique mineralocorticoid and likely minor importance of glucose in fishes means that cortisol serves both glucocorticoid and mineralocorticoid roles; the unusual structure of the fish glucocorticoid receptor may be a direct consequence of this duality. Cortisol affects the metabolism of carbohydrates, protein and lipid. Generally cortisol is hyperglycaemic, primarily as a result of increases in hepatic gluconeogenesis initiated as a result of peripheral proteolysis. The increased plasma fatty acid levels during hypercortisolaemia may assist to fuel the enhanced metabolic rates noted for a number of fish species. Cortisol is an essential component of the stress response in fish, but also plays a significant role in osmoregulation, growth and reproduction. Interactions between cortisol and toxicants may be the key to the physiology of this hormone, although cortisol's many important housekeeping functions must not be ignored. Combining molecular approaches with isolated cell systems and the whole fish will lead to an improved understanding of the many faces of this complex hormone in an evolutionary and environmental framework.
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A survey of parasitic copepods carried out in three introduced salmonid species in Southern Chile and 5 native fish species commonly found in the vicinity of the fish farm cages yielded 5 caligid species. Caligus flexispina, formerly found in the oceanic islands of the Pacific ocean, is currently the dominant species found in continental waters on native fish species (Eleginops maclovinus and Odonthestes regia) as well as on cultured trout Oncorhynchus mykiss from the south of Chile. Coho salmon O. kisutch and Atlantic salmon Salmo salar are more resistant to this parasite. Among the native fish species associated with netpen aquaculture, E. maclovinus harbours 4 caligid copepods, namely Lepeophtheirus mugiloidis, C. flexispina, C. teres and C. cheilodactylus (in the same order of ranked abundance). Odonthestes regia only harbours C. flexispina, whereas Paralichthys microps is a host of Lepeophtheirus edwardsi and C. flexispina. It is highly likely that the transfer of C. flexispina to salmonids is mediated by the presence of these native fish species.
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Atlantic salmon Salmo salar L. artificially infected with salmon lice Lepeophtheirus salmonis (Krøyer 1837) recovered from detrimental physiological changes and skin damage induced by preadult lice as the parasites matured. Growth rates of Atlantic salmon remained unaffected by lice infection, but food consumption decreased with increasing feeding and movement of the lice prior to and post-mating, correlating with the appearance of head erosions and detrimental changes in physiological integrity. Food consumption of the fish increased as the lice moulted to the adult stage and gravid female lice settled in a posterior location on the fish, subsequently reducing the impact of infection and allowing recovery of the skin damage. However, the impact of preadults was limited, as the decrease in food consumption of fish at 21 d post-infection had no effect on either the specific growth rate or condition factor of the fish. Furthermore, the intensity of lice infections at each of the sample days was not correlated with food consumption, specific growth rate or any of the haematological or physiological parameters measured, either before or after infection, indicating that lice intensity was independent of social dominance/subordinance. This work has provided the first evidence that infected fish can recover from the detrimental changes caused by lice infection, even when they are still infected with lice. If fish can survive the preadult stage of lice, then the mortal impact of lice infections is greatly reduced.
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Rainbow trout (Oncorhynchus mykiss) retain the majority of lactate produced during exhaustive exercise within white muscle. Previous studies have suggested that this retention is partially via a re-uptake of released lactate. The purpose of this work was to study lactate uptake using trout white muscle sarcolemmal vesicles. Lactate uptake by trout white muscle is partially through a low-affinity, high-capacity carrier (apparent K(m)=55.6 mmol l(-)(1) and V(max)=44.5 nmol mg(-)(1 )protein min(-)(1)). At high concentrations (20 and 50 mmol l(-)(1)), pyruvate partially (up to 39 %) inhibited lactate uptake, suggesting the involvement of a monocarboxylate carrier. The anion transport inhibitor 4-acetoamido-4'-isothiocyanstilbene-2,2'-disulphonic acid (SITS) and the monocarboxylate transport inhibitor (&agr;)-cyano-4-hydroxycinnamate (CHC) stimulated apparent lactate uptake. The model developed suggests that lactate is taken up by the vesicles, at least in part by a pyruvate-sensitive monocarboxylate carrier, and that its subsequent efflux is inhibited by SITS and CHC, suggesting that lactate export from trout white muscle is also carrier-mediated.
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To evaluate the effects of endurance training on the expression of monocarboxylate transporters (MCT) in human vastus lateralis muscle, we compared the amounts of MCT1 and MCT4 in total muscle preparations (MU) and sarcolemma-enriched (SL) and mitochondria-enriched (MI) fractions before and after training. To determine if changes in muscle lactate release and oxidation were associated with training-induced changes in MCT expression, we correlated band densities in Western blots to lactate kinetics determined in vivo. Nine weeks of leg cycle endurance training [75% peak oxygen consumption (VO(2 peak))] increased muscle citrate synthase activity (+75%, P < 0.05) and percentage of type I myosin heavy chain (+50%, P < 0.05); percentage of MU lactate dehydrogenase-5 (M4) isozyme decreased (-12%, P < 0.05). MCT1 was detected in SL and MI fractions, and MCT4 was localized to the SL. Muscle MCT1 contents were consistent among subjects both before and after training; in contrast, MCT4 contents showed large interindividual variations. MCT1 amounts significantly increased in MU, SL, and MI after training (+90%, +60%, and +78%, respectively), whereas SL but not MU MCT4 content increased after training (+47%, P < 0.05). Mitochondrial MCT1 content was negatively correlated to net leg lactate release at rest (r = -0.85, P < 0.02). Sarcolemmal MCT1 and MCT4 contents correlated positively to net leg lactate release at 5 min of exercise at 65% VO(2 peak) (r = 0.76, P < 0.03 and r = 0. 86, P < 0.01, respectively). Results support the conclusions that 1) endurance training increases expression of MCT1 in muscle because of insertion of MCT1 into both sarcolemmal and mitochondrial membranes, 2) training has variable effects on sarcolemmal MCT4, and 3) both MCT1 and MCT4 participate in the cell-cell lactate shuttle, whereas MCT1 facilitates operation of the intracellular lactate shuttle.
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The "lactate shuttle hypothesis" holds that lactate plays a key role in the distribution of carbohydrate potential energy that occurs among various tissue and cellular compartments such as between: cytosol and mitochondria, muscle and blood, blood and muscle, active and inactive muscles, white and red muscles, blood and heart, arterial blood and liver, liver and other tissues such as exercising muscle, intestine and portal blood, portal blood and liver, zones of the liver, and skin and blood. Studies on resting and exercising humans indicate that most lactate (75-80%) is disposed of through oxidation, with much of the remainder converted to glucose and glycogen. Lactate transport across cellular membranes occurs by means of facilitated exchange along pH and concentration gradients involving a family of lactate transport proteins, now called monocarboxylate transporters (MCTs). Current evidence is that muscle and other cell membrane lactate transporters are abundant with characteristics of high Km and Vmax. There appears to be long-term plasticity in the number of cell membrane transporters, but short-term regulation by allosteric modulation or phosphorylation is not known. In addition to cell membranes, mitochondria also contain monocarboxylate transporters (mMCT) and lactic dehydrogenase (mLDH). Therefore, mitochondrial monocarboxylate uptake and oxidation, rather than translocation of transporters to the cell surfaces, probably regulate lactate flux in vivo. Accordingly, the "lactate shuttle" hypothesis has been modified to include a new, intracellular component involving cytosolic to mitochondrial exchange. The intracellular lactate shuttle emphasizes the role of mitochondrial redox in the oxidation and disposal of lactate during exercise and other conditions.
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This study was undertaken to quantitatively account for the metabolic disposal of lactate in skeletal muscle exposed to an elevated lactate concentration during rest and mild-intensity contractions. The gastrocnemius plantaris muscle group (GP) was isolated in situ in seven anesthetized dogs. In two experiments, the muscles were perfused with an artificial perfusate with a blood lactate concentration of ~9 mM while normal blood gas/pH status was maintained with [U-(14)C]lactate included to follow lactate metabolism. Lactate uptake and metabolic disposal were measured during two consecutive 40-min periods, during which the muscles rested or contracted at 1.25 Hz. Oxygen consumption averaged 10.1 +/- 2.0 micromol. 100 g(-1). min(-1) (2.26 +/- 0.45 ml. kg(-1). min(-1)) at rest and 143.3 +/- 16.2 micromol. 100 g(-1). min(-1) (32.1 +/- 3.63 ml. kg(-1). min(-1)) during contractions. Lactate uptake was positive during both conditions, increasing from 10.5 micromol. 100 g(-1). min(-1) at rest to 25.0 micromol. 100 g(-1). min(-1) during contractions. Oxidation and glycogen synthesis represented minor pathways for lactate disposal during rest at only 6 and 15%, respectively, of the [(14)C]lactate removed by the muscle. The majority of the [(14)C]lactate removed by the muscle at rest was recovered in the muscle extracts, suggesting that quiescent muscle serves as a site of passive storage for lactate carbon during high-lactate conditions. During contractions, oxidation was the dominant means for lactate disposal at >80% of the [(14)C]lactate removed by the muscle. These results suggest that oxidation is a limited means for lactate disposal in resting canine GP exposed to elevated lactate concentrations due to the muscle's low resting metabolic rate.
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Lactic acid is produced as an end product of glycolysis in rainbow trout white muscle following exhaustive exercise. The metabolically produced lactic acid causes an intramuscular acidosis that must be cleared, either via net transport out of the muscle or by conversion to glycogen, thereby replenishing the muscle energy store. Trout muscle has been shown to retain lactate and utilise it as a substrate for in situ glycogen resynthesis. The giant sarcolemmal vesicle preparation was used to characterise the potential for lactate loss from white muscle of rainbow trout. Minimal lactate loss was expected due to the requirement within the intramuscular compartment of lactate for glycogen resynthesis. The sarcolemma was found to be highly resistant to lactate loss, with efflux rates approximately 500-fold lower than influx rates [0.049+/-0.006 nmol mg(-1) min(-1) (N=21) versus 26.4+/-6.3 nmol mg(-1) min(-1) (N=5), respectively, at 25 mmol l(-1) lactate concentration]. Lactate efflux was linear over the range 10-250 mmol l(-1) lactate, and greatest under conditions when intravesicular pH was lower than extravesicular pH, but was unaffected by alpha-cyano-4-hydroxycinnamate, a known inhibitor of lactate transport. These results suggest that lactate is relatively impermeant to the trout white muscle membrane and any lactate loss occurs via passive diffusion. This resistance to lactate diffusion can explain why trout muscle retains lactate post-exercise, despite transmembrane gradients that should favour net efflux.
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Physiological, immunological and biochemical parameters of blood and mucus, as well as skin histology, were compared in 3 salmonid species (rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon O. kisutch) following experimental infection with sea lice Lepeophtheirus salmonis. The 3 salmonid species were cohabited in order to standardize initial infection conditions. Lice density was significantly reduced on coho salmon within 7 to 14 d, while lice persisted in higher numbers on rainbow trout and Atlantic salmon. Lice matured more slowly on coho salmon than on the other 2 species, and maturation was slightly slower on rainbow trout than on Atlantic salmon. Head kidney macrophages from infected Atlantic salmon had diminished respiratory burst and phagocytic capacity at 14 and 21 d post-infection (dpi), while infected rainbow trout macrophages had reduced respiratory burst and phagocytic capacities at 21 dpi, compared to controls. The slower development of lice, coupled with delayed suppression of immune parameters, suggests that rainbow trout are slightly more resistant to lice than Atlantic salmon. Infected rainbow trout and Atlantic salmon showed increases in mucus lysozyme activities at 1 dpi, which decreased over the rest of the study. Mucus lysozyme activities of infected rainbow trout, however, remained higher than controls over the entire period. Coho salmon lysozyme activities did not increase in infected fish until 21 dpi. Mucus alkaline phosphatase levels were also higher in infected Atlantic salmon compared to controls at 3 and 21 dpi. Low molecular weight (LMW) proteases increased in infected rainbow trout and Atlantic salmon between 14 and 21 dpi. Histological analysis of the outer epithelium revealed mucus cell hypertrophy in rainbow trout and Atlantic salmon following infection. Plasma cortisol, glucose, electrolyte and protein concentrations and hematocrit all remained within physiological limits for each species, with no differences occurring between infected and control fish. Our results demonstrate that significant differences in mucus biochemistry and numbers of L. salmonis occur between these species.
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Since its first documented observation in exhausted animal muscle in the early 19th century, the role of lactate (lactic acid) has fascinated muscle physiologists and biochemists. Initial interpretation was that lactate appeared as a waste product and was responsible in some way for exhaustion during exercise. Recent evidence, and new lines of investigation, now place lactate as an active metabolite, capable of moving between cells, tissues and organs, where it may be oxidised as a fuel or reconverted to form pyruvate or glucose. The questions now to be asked concern the effects of lactate at the systemic and cellular level on metabolic processes. Does lactate act as a metabolic signal to specific tissues, becoming a metabolite pseudo-hormone?Does lactate have a role in whole-body coordination of sympathetic/parasympathetic nerve system control? And, finally, does lactate play a role in maintaining muscle excitability during intense muscle contraction? The concept of lactate acting as a signalling compound is a relatively new hypothesis stemming from a combination of comparative, cell and whole-organism investigations. It has been clearly demonstrated that lactate is capable of entering cells via the monocarboxylate transporter (MCT) protein shuttle system and that conversion of lactate to and from pyruvate is governed by specific lactate dehydrogenase isoforms, thereby forming a highly adaptable metabolic intermediate system. This review is structured in three sections,the first covering pertinent topics in lactate's history that led to the model of lactate as a waste product. The second section will discuss the potential of lactate as a signalling compound, and the third section will identify ways in which such a hypothesis might be investigated. In examining the history of lactate research, it appears that periods have occurred when advances in scientific techniques allowed investigation of this metabolite to expand. Similar to developments made first in the 1920s and then in the 1980s, contemporary advances in stable isotope, gene microarray and RNA interference technologies may allow the next stage of understanding of the role of this compound, so that, finally, the fundamental questions of lactate's role in whole-body and localised muscle function may be answered.
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Between 2006 and 2009, the Chilean salmon industry suffered a major sanitary crisis caused by outbreaks of infectious salmon anemia virus and Caligus rogercresseyi. Additionally, Piscirickettsia salmonis, the causative agent of piscirickettsiosis, and the parasite C. rogercresseyi continue to impact the salmon industry. This study evaluates the effects of both C. rogercresseyi infestation and P. salmonis infection concurrently on the blood parameters of Salmo salar (Atlantic salmon) under field conditions, providing an estimated parasite threshold at which the least impact occurs on host physiology. The presence of P. salmonis and C. rogercresseyi increased S. salar hematocrit, plasma glucose, and pCO2 levels but decreased hemoglobin and pO2 levels. Significant threshold values of parasite abundances were estimated for glucose, hematocrit, hemoglobin, and lymphocytes. This is the first study to evaluate the effects of C. rogercresseyi on S. salar physiology and to provide estimated abundance threshold values under farm conditions. Although the threshold estimations will likely be of great value for surveillance and control programs, they are an initial approximation that requires further confirmation. The prevalence of C. rogercresseyi and P. salmonis in Chilean salmon farms necessitates further research on the development and establishment of on-farm pathogen indicators.
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In this study we assessed the influence of three different environmental salinities (5, 15 and 31 psu during 90 days) on growth, osmoregulation, energy metabolism and digestive capacity in juveniles of the Notothenioid fish Eleginops maclovinus. At the end of experimental time samples of plasma, liver, gill, intestine, kidney, skeletal muscle, stomach and pyloric caeca were obtained. Growth, weight gain, hepatosomatic index and specific growth rate increased at 15 and 31 psu and were lower at 5 psu salinity. Gill Na ? , K ?-ATPase (NKA) activity presented a ''U-shaped'' relationship respect to salinity, with its minimum rates at 15 psu, while this activity correlated negatively with salinity at both anterior and posterior intestinal portions. No significant changes in NKA activity were observed in kidney or mid intestine. Large changes in plasma, metabolite levels and enzymatic activities related to energy metabolism in liver, gill, intestine, kidney and muscle were generally found in the groups exposed to 5 and 31 psu compared to the 15 psu group. Only the pepsin activity (digestive enzymes) assessed enhanced with environmental salinity, while pyloric caeca trypsin/ chymotrypsin ratio decreased. This study suggests that juvenile of E. maclovinus presents greater growth near its iso-osmotic point (15 psu) and hyperosmotic environment (31 psu). Acclimation to low salinity increased the osmoregulatory expenditure as seen by the gill and anterior intestine results, while at high salinity, branchial osmoregulatory activity was also enhanced. This requires the mobilization of lipid stores and amino acids, thereby holding the growth of fish back. The subsequent reallocation of energy sources was not sufficient to maintain the growth rate L. Vargas-Chacoff (&) Á E. Saavedra Á R. Oyarzún Á J. P. Pontigo Á E. Ortiz Á of fish exposed to 5 psu. Thus, E. maclovinus juveniles present better growth efficiencies in salinities above the iso-osmotic point and hyperosmotic environment of this species, showing their best performance at 15 psu as seen by the main osmoregulatory and energy metabolism enzymatic activities.
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In this study we assessed changes in the osmoregulatory system of juvenile sub-Antarctic Eleginops maclovinus submitted to different environmental salinities (5, 15, 32 and 45 psu) using two different acclimation trials: (1) an end-point experiment (exposure for 14 days) and (2) a time course experiment (specimens were sampled on days 1, 3, 7 and 14 post-transfer). Plasma osmolality, cortisol and metabolites (glucose, lactate and protein) values as well as Na+, K+-ATPase (NKA) activity were assessed in several osmoregulatory tissues (gills, kidney and intestine). In both trials, acclimation to different environmental salinities for 14 days induced changes in plasma metabolites (glucose, lactate and proteins) as well as cortisol values related to salinity challenges. Plasma osmolality and gill NKA activity presented a direct and positive relationship with respect to environmental salinity, while kidney NKA activity showed a “U-shaped” relationship. Anterior intestinal NKA activity increased in response to environmental salinity and apparently did not change in the middle portion of this organ, while it was enhanced in the posterior portion in environmental salinities different than seawater. Plasma metabolite values increased under hypo- and hypersaline conditions, indicating the importance of these energy substrates in extreme environments. The time course study revealed that specimens of E. maclovinus are able to accommodate their osmotic and metabolic system to respond to osmoregulatory challenges by allostatic changes.
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
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Studies of the biology of sea lice have been conducted from various perspectives for two decades. For Lepeophtheirus spp., most of the published literature has centred on the economically important Lepeophtheirus salmonis, while for Caligus spp., research has focused on a wider range of species. The most numerous species of Caligus in North Atlantic waters, however, is Caligus elongatus, which is also economically important to salmon fanning. Since the last review by Pike, A. W., and Wadsworth, S. L. (1999. Sea lice on salmonids: their biology and control. Advances in Parasitology, 44: 234-337.), research on sea lice has developed considerably, including the application of genetic methods. This new research has focused on life history biology, studying developmental stages under different environmental conditions (e.g. temperature and salinity), behaviour, distribution and the dispersal of free-living stages, monitoring practices, population structure, and modelling. The results of this research have informed risk analyses and allowed the refinement of management strategies to reduce sea lice infestations in wild and fanned populations of anadromous salmonids. Molecular techniques have been used to describe population structure and identify differences in genetic characterization of geographically separate populations and population markers. Research has been initiated to understand the parasite-host relationship at a molecular level and to develop a vaccine against sea lice. (c) 2006 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.
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For wild, sea-run brown trout (Salmo trutta) smolts, the physiological consequences of abrupt transfer to seawater and simultaneous challenge with copepodid larvae of the sea louse, Lepeophtheirus salmonis (Krøyer, 1837), were investigated in the laboratory. Analysis of osmoregulatory, metabolic, and stress markers allowed the derivation of a sublethal threshold burden of L. salmonis, above which the host suffers major physiological stress. Noticeable lice effects, consistent across all measured markers, were not observed until L. salmonis developed to the mobile preadult and adult stages. Preadult L. salmonis caused significant increases in plasma chloride, osmolality, glucose, lactate, and cortisol and a significant reduction in haematocrit. Piecewise linear statistical approaches allowed the determination of abrupt changes in these physiological markers, attributable to the intensity of L. salmonis infestation on individual fish, and identification of overall threshold lice burdens. Thirteen mobile lice·fish–1 (weight range 19–70 g) was a consistent breakpoint across several physiological measures. This information will provide a valuable, objectively derived tool to aid in the formulation of effective wild fisheries management policy concerning S. trutta conservation.
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A monitoring program for the prevalence and intensity of sea lice infestations of wild and escaped farmed salmon has been underway on the Magaguadavic River since 1992. Fish are screened in a fish ladder trap located in freshwater a short distance above the head of tide. No trends with time were evident in observed sea lice burdens, and in all years the majority of salmon, both wild and escapees, had no or low levels of infestation with sea lice. In the spring of 2002, 23 landlocked salmon moving to sea from the Magaguadavic River were acoustically tagged. Two fish returned to the river after a brief period of residence in Passamaquoddy Bay, with significant dermal damage from sea lice. These fish were tracked to areas close to commercial salmon farms.
Article
Seasonal variations in osmoregulatory and metabolic parameters were assessed in juvenile gilthead sea bream (Sparus auratus) cultured in earthen ponds under a natural photoperiod and temperature. Specimens were sampled, and the plasma, gill, kidney and liver were collected during winter 2005 and 2006 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Plasma osmoregulatory parameters showed higher values in summer, while metabolic parameters presented different patterns of variations. Gill Na+,K+-ATPase activity decreased significantly in winter, while gill metabolite levels showed different patterns of variations among seasons. The enzymatic activities tested did not present a clear pattern of variation [(glutamate dehydrogenase (EC 1.4.1.2) (GDH) and hexokinase (EC 2.7.1.11) (HK)] or significant differences along seasons [glucose 6-phosphate dehydrogenase (EC 1.1.1.49)]. Kidney Na+,K+-ATPase activity decreased during summer and autumn. Different patterns of variation were observed in kidney metabolite levels while all the enzymatic activities assessed [lactate dehydrogenase-oxidase (EC 1.1.1.27) (LDH-O), HK and GDH] presented the highest values during summer. In the liver, metabolite levels and enzymatic activities did not show significant variations or present clear patterns of variation along different seasons. These results indicated seasonal variations in the osmoregulatory and metabolic parameters of different organs (blood, gill, kidney and liver) in earthen pond-cultured gilthead sea bream (S. auratus), which could be mainly attributed to seasonal changes in temperature.
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Salmon sea lice represent one of the most important threats to salmon farming throughout the world. Results of private monitoring efforts have shown an increase in the number of positive cages and cage-level abundance of sea lice in southern Chile since 2004. As a consequence, the Chilean Fisheries Service implemented an Official Surveillance Program in the main salmon production area of southern Chile to assess the situation of sea lice in fish farms. Results showed that the prevalence of sea lice in the fish farms was 53.4%, ranging from 3.5% in Puerto Aysén to 100% in the Seno de Reloncaví zone. The average sea lice abundance was 11.8 per fish (Geometrical mean (GM)=8.61, 95% CI (2.1-6.9)). The highest levels were found in Seno de Reloncaví (GM=24.99, 95% CI (15.9-39.2)), Hornopirén (GM=14.7, 95% CI (10.4-20.8)) and Chiloé norte (GM=9.75, 95% CI (1-1.9)), and the lowest loads were observed in Puerto Aysén (GM=1.35, 95%CI (1-1.9)) and Puerto Cisnes (GM=1.67, 95%CI (1.1-2.6)). Salmo salar and Oncorhynchus mykiss had the highest abundance levels (GM=6.93, 95% CI (5.7-8.5), and (GM=5.55, 95% CI (3.6-8.5), respectively). O. kisutch showed lower levels (GM=1.34, 95% CI (1-1.7)), apparently being more resistant to infestation. Sea lice in farmed salmon are widely distributed in different zones of southern Chile, and are becoming a serious threat to this industry. Prevalence and abundance levels were found to be generally high, decreasing in southern zones.
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Monocarboxylate transporter (MCT) isoforms 1-4 catalyze the proton-linked transport of monocarboxylates such as L-lactate across the plasma membrane, whereas MCT8 and MCT10 are thyroid hormone and aromatic amino acid transporters, respectively. The importance of MCTs is becoming increasingly evident as their extensive physiological and pathological roles are revealed. MCTs 1-4 play essential metabolic roles in most tissues with their distinct properties, expression profile, and subcellular localization matching the particular metabolic needs of a tissue. Important metabolic roles include energy metabolism in the brain, skeletal muscle, heart, tumor cells, and T-lymphocyte activation, gluconeogenesis in the liver and kidney, spermatogenesis, bowel metabolism of short-chain fatty acids, and drug transport. MCT8 is essential for thyroid hormone transport across the blood-brain barrier. Genetic perturbation of MCT function may be involved in disease states such as pancreatic β-cell malfunction (inappropriate MCT1 expression), chronic fatigue syndromes (impairment of muscle MCT function), and psychomotor retardation (MCT8 mutation). MCT expression can be regulated at both the transcriptional and post-transcriptional levels. Of particular importance is the upregulation of muscle MCT1 expression in response to training and MCT4 expression in response to hypoxia. The latter is mediated by hypoxia inducible factor 1α and often observed in tumor cells that rely almost entirely on glycolysis for their energy provision. The recent discovery of potent and specific MCT1 inhibitors that prevent proliferation of T-lymphocytes confirms that MCTs may be promising pharmacological targets including for cancer chemotherapy.
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Physical, chemical and perceived stressors can all evoke non-specific responses in fish, which are considered adaptive to enable the fish to cope with the disturbance and maintain its homeostatic state. If the stressor is overly severe or long-lasting to the point that the fish is not capable of regaining homeostasis, then the responses themselves may become maladaptive and threaten the fish's health and well-being. Physiological responses to stress are grouped as primary, which include endocrine changes such as in measurable levels of circulating catecholamines and corticosteroids, and secondary, which include changes in features related to metabolism, hydromineral balance, and cardiovascular, respiratory and immune functions. In some instances, the endocrine responses are directly responsible for these secondary responses resulting in changes in concentration of blood constituents, including metabolites and major ions, and, at the cellular level, the expression of heat-shock or stress proteins. Tertiary or whole-animal changes in performance, such as in growth, disease resistance and behavior, can result from the primary and secondary responses and possibly affect survivorship. Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol in actinopterygian fishes, that occur following a stressful event. The characteristic elevation in circulating cortisol during the first hour after an acute disturbance can vary by more than two orders of magnitude among species and genetic history appears to account for much of this interspecific variation. An appreciation of the factors that affect the magnitude, duration and recovery of cortisol and other physiological changes caused by stress in fishes is important for proper interpretation of experimental data and design of effective biological monitoring programs.
Article
The gilthead sea bream Sparus aurata is a euryhaline and euritherm species with the capacity of living under different environmental conditions of salinity and temperature. The influence of acclimation to different environmental salinities (5, 38 and 55 per thousand) and temperatures (12 degrees , 19 degrees and 26 degrees C) for seven weeks was analyzed in plasma and tissues (liver, gills and kidney) of gilthead sea bream assessing levels of metabolites and enzyme activities related to energy metabolism. Changes observed in specimens acclimated to different environmental salinities agree with previous results reported for this species. The temperature alone did also affect metabolic parameters in a way similar to that previously described. A significant interaction of salinity with temperature was found in most parameters assessed in tissues suggesting that the metabolic effects of salinity are different depending on the temperature of acclimation. The interactions were different among tissues and parameters displaying different patterns of changes. In general, the acclimation to extreme temperatures (especially low) alters the metabolic responses to different salinities thus suggesting that the energy demand of increased osmoregulatory work is not so important under temperature conditions different from those commonly found in nature and in those used in culture.
Article
Temperature and salinity are important factors that affect several physiological processes in aquatic organisms, which could be produced by variation of certain hormones. In this study, the expression of pituitary hormones involved in the acclimation to different temperatures and salinities was examined in Sparus aurata, a euryhaline and eurytherm species, by Q-Real Time RT-PCR and Western blot analyses for mRNA and protein expression, respectively. Three different experimental conditions were designed with specimens (10 per treatment) acclimated to: a) low salinity water; b) sea water; and c) high salinity water. Additionally, fish under different salinities were acclimated to three different temperatures: 12, 19 and 26 degrees C. Animals were maintained seven weeks before sampling pituitary glands. Our results provided enough evidence for a differential expression of PRL, GH and SL in the pituitary of gilthead sea bream, under different temperature and salinity regimes.
Article
The seasonal variation of PRL, GH and SL gene and protein expression has been analyzed in gilthead sea bream (Sparus aurata) pituitaries using Real-Time Q-PCR and Western Blots, respectively. Animals were cultured in earthen ponds under natural photoperiod, temperature and salinity conditions. Samples were taken during winter 2005 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Beta-actin, used as the housekeeping gene both for Q-RT-PCR and Western analysis, did not present significant differences among seasons. Higher expression was observed during spring and autumn for PRL, summer and winter for GH, and spring for SL. Expression of PRI, GH and SL, presented seasonal variation, suggesting that these hormones could play a role in the molecular signal transduction of environmental factors (especially of photoperiod and temperature) in eurythermal fish.
Article
In the development of integrated pest management (IPM) plans for the control of sea lice there are some components that are common to many areas. However, effective plans must be tailored to regionally varying environmental and biological factors affecting the severity of sea lice infections. This paper describes factors that would be involved in the development of an IPM plan for sea lice in the Broughton Archipelago, British Columbia. Temperature, salinity and currents affect the production, dispersion and competence of larvae of sea lice, Lepeophtheirus salmonis (Krøyer), as they develop to the infective copepodid stage. This information can be coupled with oceanographic conditions in the Broughton Archipelago and emerging computer models to define zones of infection where infections of new hosts are most likely. Salinity and temperature depend, in part, on river discharge in estuarine systems. River discharge depends on precipitation, snow pack and ambient temperatures, which can be monitored to help forecast the intensity of sea lice infections associated with both farmed and wild hosts. One of the goals of IPM planning is to reduce reliance on pesticides to avoid development of resistance in targeted parasites and to minimize environmental residues. Recommendations for developing an IPM plan specific to the Broughton Archipelago are provided along with a discussion of the additional information needed to refine IPM plans in this and other areas.
Article
This study used an isolated-perfused tail-trunk preparation of rainbow trout to examine the uptake and release of lactate (Lac) and metabolic protons (delta H+M) in resting and exercised fish white muscle. In exercised muscle, L(+)-Lac efflux was inhibited (approximately 40%) by 5 mM alpha-cyano-4-hydroxycinnamate (CIN), but not by 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) or 0.1 mM amiloride. These results suggest that Lac release occurs through a Lac(-)-H- symport and the free diffusion of lactic acid (HLac) or Lac-, but not via the Lac-/HCO3(-)-Cl- antiporter. Lac efflux was accompanied by delta H+m influx in all treatments, and increased delta H+m influx occurred after SITS treatment. In resting muscle, Lac uptake rates were greater than Lac efflux rates in the postexercise preparation. L-Lac influx exhibited partial saturation kinetics, whereas D(-)-Lac influx was linearly related to its extracellular concentration (0-32 mM). At 16 mM extracellular L-Lac, with a negligible transmembrane L-HLac gradient and an outwardly directed not driving force on L-Lac-, CIN, and SITS reduced net L-Lac uptake by 75 and 45%, respectively. At 16 mM extracellular concentration, D-Lac influx was 64% of the net L-Lac influx. These results suggest that in trout muscle at 16 mM extracellular L-Lac, the Lac -H+ symport accounts for 30-36%, the Lac-/HCO3(-)-Cl- antiport for 39-45%, and diffusion for 19-25% of uptake, although the latter is probably overestimated and the former underestimated for methodological reasons. Net L-Lac efflux was not affected by extracellular D-Lac concentration and/or D-Lac influx, implying the existence of a concurrent L-Lac efflux during L-Lac influx. The D-Lac influx kinetics data indicated that the Lac-/HCO3 antiport was not saturable in the extracellular D-Lac concentration range of 0-32 mM. This study clearly demonstrates the involvement of carrier-mediated transport in transmembrane Lac movement in fish muscle and supports the "active lactate retention" mechanism proposed by Turner and Wood (J. Exp. Biol. 105: 895-401, 1983).
Article
The stress response in teleost fish shows many similarities to that of the terrestrial vertebrates. These concern the principal messengers of the brain-sympathetic-chromaffin cell axis (equivalent of the brain-sympathetic-adrenal medulla axis) and the brain-pituitary-interrenal axis (equivalent of the brain-pituitary-adrenal axis), as well as their functions, involving stimulation of oxygen uptake and transfer, mobilization of energy substrates, reallocation of energy away from growth and reproduction, and mainly suppressive effects on immune functions. There is also growing evidence for intensive interaction between the neuroendocrine system and the immune system in fish. Conspicuous differences, however, are present, and these are primarily related to the aquatic environment of fishes. For example, stressors increase the permeability of the surface epithelia, including the gills, to water and ions, and thus induce systemic hydromineral disturbances. High circulating catecholamine levels as well as structural damage to the gills and perhaps the skin are prime causal factors. This is associated with increased cellular turnover in these organs. In fish, cortisol combines glucocorticoid and mineralocorticoid actions, with the latter being essential for the restoration of hydromineral homeostasis, in concert with hormones such as prolactin (in freshwater) and growth hormone (in seawater). Toxic stressors are part of the stress literature in fish more so than in mammals. This is mainly related to the fact that fish are exposed to aquatic pollutants via the extensive and delicate respiratory surface of the gills and, in seawater, also via drinking. The high bioavailability of many chemicals in water is an additional factor. Together with the variety of highly sensitive perceptive mechanisms in the integument, this may explain why so many pollutants evoke an integrated stress response in fish in addition to their toxic effects at the cell and tissue levels. Exposure to chemicals may also directly compromise the stress response by interfering with specific neuroendocrine control mechanisms. Because hydromineral disturbance is inherent to stress in fish, external factors such as water pH, mineral composition, and ionic calcium levels have a significant impact on stressor intensity. Although the species studied comprise a small and nonrepresentative sample of the almost 20,000 known teleost species, there are many indications that the stress response is variable and flexible in fish, in line with the great diversity of adaptations that enable these animals to live in a large variety of aquatic habitats.
Article
The 'crossover' and 'lactate shuttle' concepts of substrate utilization in humans during exercise are extended to describe metabolic responses on other mammalian species. The 'crossover concept' is that lipid plays a predominant role in sustaining efforts requiring half or less aerobic capacity (VO2max); however, greater relative efforts depend increasingly on blood glucose and muscle glycogen as substrates. Thus, as exercise intensity increases from mild to moderate and hard, fuel selection switches (crosses over) from lipid to carbohydrate dependence. Glycogen and glucose catabolic rates are best described as exponential functions of exercise intensity, but with a greater gain in slope of the glycogen than glucose response. In contrast, plasma free fatty acid flux is described as an inverted hyperbola with vertex at approximately 50% VO2max. Both endocrine and intra-cellular factors play critical roles in determining substrate balance during sustained exercise. Moreover, genotypic adaptation for aerobic capacity as well as phenotypic adaptations to short- and long-term chronic activity affect the balance of substrate utilization during exercise. The concept of a 'lactate shuttle' is that during hard exercise, as well as other conditions of accelerated glycolysis, glycolytic flux in muscle involves lactate formation regardless of the state of oxygenation. Further, according to the lactate shuttle concept, lactate represents a major means of distributing carbohydrate potential energy for oxidation and gluconeogenesis. In humans and other mammals, the formation, distribution and disposal of lactate (not pyruvate) represent key steps in the regulation of intermediary metabolism during sustained exercise.
Article
Experiments were conducted to determine the effects of sea lice, Lepeophtheirus salmonis, on non-specific defence mechanisms in Atlantic salmon, Salmo salar, by experimentally infesting hatchery-reared 1 and 2 year old post-smolts, S1 and S2, with laboratory grown infective copepodids at moderate to high infection intensities ranging from 15-285 lice per fish. The effects of sea lice-induced stress were investigated by measuring the blood levels of cortisol and glucose as indicators of primary and secondary stress responses, and by changes in macrophage respiratory burst activity and phagocytosis as indicators of tertiary stress responses as well as non-specific defence mechanisms. Fish were sampled prior to sea lice infestation at day 0 and at days 3, 7, 14 and 21 post-infestation. Sea lice were at copepodid stage at day 3, at chalimus stages at days 7 and 14, and at pre-adult stage at day 21. Blood levels of cortisol and glucose were found to be significantly increased at day 21 in fish-infested with the highest levels. Macrophage respiratory burst and phagocytic activities were found to be significantly decreased only at day 21. These results indicate that sea lice do not suppress host defence mechanisms during the earlier stages of infestation. They do have effects on the development of chronic stress and on the host non-specific defence mechanisms soon after the lice reach the pre-adult stage.
Article
The study of lactic acid (HLa) and muscular contraction has a long history, beginning perhaps as early as 1807 when Berzelius found HLa in muscular fluid and thought that “the amount of free lactic acid in a muscle [was] proportional to the extent to which the muscle had previously been exercised” (cited in ref. 1). Several subsequent studies in the 19th century established the view that HLa was a byproduct of metabolism under conditions of O2 limitation. For example, in 1891, Araki (cited in ref. 2) reported elevated HLa levels in the blood and urine of a variety of animals subjected to hypoxia. In the early part of the 20th century, Fletcher and Hopkins (3) found an accumulation of HLa in anoxia as well as after prolonged stimulation to fatigue in amphibian muscle in vitro. Subsequently, based on the work of Fletcher and Hopkins (3) as well as his own studies, Hill (and colleagues; ref. 4) postulated that HLa increased during muscular exercise because of a lack of O2 for the energy requirements of the contracting muscles. These studies laid the groundwork for the anaerobic threshold concept, which was introduced and detailed by Wasserman and colleagues in the 1960s and early 1970s (5–7). The basic anaerobic threshold paradigm is that elevated HLa production and concentration during muscular contractions or exercise are the result of cellular hypoxia. Table 1 summarizes the essential components of the anaerobic threshold concept.
Article
The purpose of this study was to determine the effect of epinephrine on net lactate (La(-)) uptake at constant elevated blood La(-) concentration and steady level metabolic rate (O(2) uptake) in the canine gastrocnemius-plantaris muscle in situ. Infusion of La(-)/lactic acid (pH 3.5) established a mean arterial blood La(-) concentration of ~10 mM while normal blood-gas and pH status were maintained as the gastrocnemius-plantaris was stimulated with tetanic trains at a rate of one contraction every 4 s. After steady-state control measures, epinephrine was infused for 35 min at rates that produced a high physiological concentration with (Pro; n = 6) and without (Epi; n = 6) beta-adrenergic-receptor blockade via propranolol. Net La(-) uptake values during the control conditions were not significantly different between trials (Epi: 0.756 +/- 0.043; Pro: 0.703 +/- 0.061 mmol. kg(-1). min(-1)). Steady level O(2) uptake averaged approximately 69.5 ml. kg(-1). min(-1) for both control conditions and did not significantly change over the course of the experiments in either set of trials. Epi experiments resulted in a significantly reduced net La(-) uptake (0.346 +/- 0.088 mmol. kg(-1). min(-1) after 5 min of infusion) compared with control value at all sample times measured. However, net La(-) uptake was not significantly different from control at any time during Pro (0.609 +/- 0.052 mmol. kg(-1). min(-1) after 5 min of infusion). When the change from the respective control values for net La(-) uptake was compared across time for both series of experiments, Epi resulted in a significantly greater change from control than did Pro. This study suggests that epinephrine can have a profound effect on net La(-) uptake by contracting muscle and that these effects are elicited through beta-adrenergic-receptor stimulation.
Article
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Article
For much of the 20th century, lactate was largely considered a dead-end waste product of glycolysis due to hypoxia, the primary cause of the O2 debt following exercise, a major cause of muscle fatigue, and a key factor in acidosis-induced tissue damage. Since the 1970s, a 'lactate revolution' has occurred. At present, we are in the midst of a lactate shuttle era; the lactate paradigm has shifted. It now appears that increased lactate production and concentration as a result of anoxia or dysoxia are often the exception rather than the rule. Lactic acidosis is being re-evaluated as a factor in muscle fatigue. Lactate is an important intermediate in the process of wound repair and regeneration. The origin of elevated [lactate] in injury and sepsis is being re-investigated. There is essentially unanimous experimental support for a cell-to-cell lactate shuttle, along with mounting evidence for astrocyte-neuron, lactate-alanine, peroxisomal and spermatogenic lactate shuttles. The bulk of the evidence suggests that lactate is an important intermediary in numerous metabolic processes, a particularly mobile fuel for aerobic metabolism, and perhaps a mediator of redox state among various compartments both within and between cells. Lactate can no longer be considered the usual suspect for metabolic 'crimes', but is instead a central player in cellular, regional and whole body metabolism. Overall, the cell-to-cell lactate shuttle has expanded far beyond its initial conception as an explanation for lactate metabolism during muscle contractions and exercise to now subsume all of the other shuttles as a grand description of the role(s) of lactate in numerous metabolic processes and pathways.
Article
Despite being restricted to the host's first line of defence (the integument, away from vital organs), ectoparasite damage has a pronounced impact on host fitness. This generalization can be explained by the reduced dependence of most ectoparasites on their individual host, which minimizes the fitness loss linked to host death. This explanation implies that permanent ectoparasites evolve less 'aggressively' than do either nest or field ectoparasites. This, and other determinants of ectoparasitic virulence are discussed here by Tovi Lehmann.
Article
The osmoregulatory response of Senegalese sole (Solea senegalensis, Kaup 1858) to 14-day exposure and throughout 17-day exposure to different environmental salinities was investigated. A linear relationship was observed between environmental salinity and gill Na(+),K(+)-ATPase activity whereas kidney Na(+),K(+)-ATPase activity was unaffected. Two osmoregulatory periods could be distinguished according to variations in plasma osmolality: an adjustment period and a chronic regulatory period. No major changes in plasma osmolality and ions levels were registered at the end of the 14- to 17-day exposure period, indicating an efficient adaptation of the osmoregulatory system. Plasma levels of glucose and lactate were elevated in hypersaline water, indicating the importance of these energy substrates in these environments. Glucose was increased during hyper-osmoregulation but only in the adjustment period. Cortisol proved to be a good indicator of chronic stress and stress induced by transfer to the different osmotic conditions. This work shows that S. senegalensis is able to acclimate to different osmotic conditions during short-term exposure.
  • L Vargas-Chacoff
L. Vargas-Chacoff et al. Veterinary Parasitology 243 (2017) 6-11