How Many Sirtuin Genes Are Out There? Evolution of Sirtuin Genes in Vertebrates With a Description of a New Family Member.

Studying the evolutionary history of gene families is a challenging and exciting task with a wide range of implications. In addition to exploring fundamental questions about the origin and evolution of genes, disentangling their evolution is also critical to those who do functional/structural studies to allow a deeper and more precise interpretation of their results in an evolutionary context. The sirtuin gene family is a group of genes that are involved in a variety of biological functions mostly related to aging. Their duplicative history is an open question, as well as the definition of the repertoire of sirtuin genes among vertebrates. Our results show a well-resolved phylogeny that represents an improvement in our understanding of the duplicative history of the sirtuin gene family. We identified a new sirtuin gene family member (SIRT3.2) that was apparently lost in the last common ancestor of amniotes but retained in all other groups of jawed vertebrates. According to our experimental analyses, elephant shark SIRT3.2 protein is located in mitochondria, the overexpression of which leads to an increase in cellular levels of ATP. Moreover, in vitro analysis demonstrated that it has deacetylase activity being modulated in a similar way to mammalian SIRT3. Our results indicate that there are at least eight sirtuin paralogs among vertebrates and that all of them can be traced back to the last common ancestor of the group that existed between 676 and 615 millions of years ago.

Temperature and immune challenges modulate the transcription of genes of the ubiquitin and apoptosis pathways in two high-latitude Notothenioid fish across the Antarctic Polar Front.

Thermal variations due to global climate change are expected to modify the distributions of marine ectotherms, with potential pathogen translocations. This is of particular concern at high latitudes where cold-adapted stenothermal fish such as the Notothenioids occur. However, little is known about the combined effects of thermal fluctuations and immune challenges on the balance between cell damage and repair processes in these fish. The aim of this study was to determine the effect of thermal variation on specific genes involved in the ubiquitination and apoptosis pathways in two congeneric Notothenioid species, subjected to simulated bacterial and viral infections. Adult fish of Harpagifer bispinis and Harpagifer antarcticus were collected from Punta Arenas (Chile) and King George Island (Antarctica), respectively, and distributed as follows: injected with PBS (control), LPS (2.5 mg/kg) or Poly I:C (2 mg/kg) and then submitted to 2, 5 and 8 °C. After 1 week, samples of gills, liver and spleen were taken to evaluate the expression by real-time PCR of specific genes involved in ubiquitination (E3-ligase enzyme) and apoptosis (BAX and SMAC/DIABLO). Gene expression was tissue-dependent and increased with increasing temperature in the gills and liver while showing an opposite pattern in the spleen. Studying a pair of sister species that occur across the Antarctic Polar Front can help us understand the particular pressures of intertidal lifestyles and the effect of temperature in combination with biological stressors on cell damage and repair capacity in a changing environment.

Revealing the Salmo salar NLRP3 Inflammasome: Insights from Structural Modeling and Transcriptome Analysis.

The NLRP3, one of the most heavily studied inflammasome-related proteins in mammals, remains inadequately characterized in Atlantic salmon (Salmo salar), despite the significant commercial importance of this salmonid. The NLRP3 inflammasome is composed of the NLRP3 protein, which is associated with procaspase-1 via an adapter molecule known as ASC. This work aims to characterize the Salmo salar NLRP3 inflammasome through in silico structural modeling, functional transcript expression determination in the SHK-1 cell line in vitro, and a transcriptome analysis on Atlantic salmon. The molecular docking results suggested a similar arrangement of the ternary complex between NLRP3, ASC, and caspase-1 in both the Atlantic salmon and the mammalian NLRP3 inflammasomes. Moreover, the expression results confirmed the functionality of the SsNLRP3 inflammasome in the SHK-1 cells, as evidenced by the lipopolysaccharide-induced increase in the transcription of genes involved in inflammasome activation, including ASC and NLRP3. Additionally, the transcriptome results revealed that most of the inflammasome-related genes, including ASC, NLRP3, and caspase-1, were down-regulated in the Atlantic salmon following its adaptation to seawater (also known as parr-smolt transformation). This is correlated with a temporary detrimental effected on the immune system. Collectively, these findings offer novel insights into the evolutionarily conserved role of NLRP3.

Transcriptional modulation of immune genes in gut of Sub-Antarctic notothenioid fish Eleginops maclovinus challenged with Francisella noatunensis subsp. noatunensis.

The search for functional foods that improve the immune response has traditionally been focused on lymphoid tissue and the intestinal mucosa. However, it is unknown whether there is a different immune response in different portions of the gut following exposure to a bacterial pathogen. We challenged Eleginops maclovinus intraperitoneally (i.p) with Francisella noatunensis subsp. noatunensis and measured mRNA transcripts related to innate and adaptive immune responses in different parts of the gut (foregut, midgut and hindgut). We used control (i.p only with bacterial culture medium), low dose (i.p of F. noatunensis at 1 × 101 bact/µL), medium dose (i.p of F. noatunensis at 1 × 105 bact/µL) and high dose (i.p of F. noatunensis at 1 × 1010 bact/µL) groups in our experiments. We sampled fish at days 1, 3, 7, 14, 21, and 28 post-injection. We observed tissue-specific expression of TLR1, TLR5, TLR8, MHCI, MHCII and IgM, and transcription of these immune markers was lower in foregut and higher in midgut and hindgut. We detected Francisella genetic material (DNA) in fish stimulated with a high dose from day 1-28 in foregut, midgut, and hindgut. However, we could only detect Francisella DNA in fish stimulated the medium and low dose at later timepoints in the foregut (21-28 days post injection "dpi") and hindgut (low dose from day 7-28 dpi). Our results suggest that the immune responses to bacterial pathogens occur throughout the gut, but certain segments may be more susceptible to infection because of their cellular morphology (anterior, middle and posterior).

Effects of temperature on the innate immune response on Antarctic and sub-Antarctic fish Harpagifer antarcticus and Harpagifer bispinis challenged with two immunostimulants, LPS and Poly I:C: In vivo and in vitro approach.

Rising ocean temperatures due to climate change combined with the intensification of anthropogenic activity can drive shifts in the geographic distribution of species, with the risks of introducing new diseases. In a changing environment, new host-pathogen interactions or changes to existing dynamics represent a major challenge for native species at high latitudes. Notothenioid fish constitute a unique study system since members of this group are found inside and outside Antarctica, are highly adapted to cold and particularly sensitive to temperature increments. However, data about their immune response remains scarce. Here, we aimed to evaluate the innate immune response under thermal stress in two species of Notothenioid fish, Harpagifer antarcticus and Harpagifer bispinis. Adult individuals from both species were collected on King George Island (Antarctica), and Punta Arenas (Chile), respectively. Specimens were assigned to a control group or injected with one of two agents (LPS and Poly I:C) to simulate either a bacterial or viral infection, and subjected to three different temperatures 2, 5 and 8 °C for 1 week. In parallel, we established leukocytes primary cell cultures from head kidney, which were also subjected to the immunostimulants at the same three temperatures, and incubated for 0.5, 1, 3, 6, 12, 24, and 48 h. We evaluated the relative gene expression of genes involved in the innate immune response (TLR1, TLR3, NF-kB, MYD88, IFNGR e IL-8) through real time qPCR. We found differences between species mainly in vivo, where H. antarcticus exhibited upregulation at high temperatures and H. bispinis seemed to have reached their physiological minimum at 2 °C. Although temperature had a strong effect during the in vivo assay for both species, it was negligible for primary cell cultures, which responded primarily to condition and time. Moreover, while leukocytes responded with fluctuations across time points, in vivo both species manifested strong and clear patterns of gene expression. These results highlight the importance of evaluating the effect of multiple stressors and set a precedent for future research.

Brain immunity response of fish Eleginops maclovinus to infection with Francisella noatunensis.

The brain's immune system is selective and hermetic in most species, including fish, favoring immune responses mediated by soluble immunomodulatory factors such as serotonin and the availability of nutrients against infectious processes. Francisella noatunensis coexist with fish such as Eleginops maclovinus, which raises questions about the susceptibility and immune response of the brain of E. maclovinus against Francisella. In this study, we inoculated fish with different doses of Francisella and took samples for 28 days. We detected bacteria in the brain of fish injected with a high concentration of Francisella at all time points. qPCR analysis of immune genes indicated a response mainly in the medium-dose and early expression of genes involved in iron metabolism. Finally, brain serotonin levels were higher than in uninfected fish in all conditions, suggesting possible immunomodulatory participation in an infectious process.

Effect of low-dose Piscirickettsia salmonis infection on haematological-biochemical blood parameters in Atlantic salmon (Salmo salar).

Piscirickettsia salmonis is the etiological agent of Piscirickettsiosis, a severe disease that affects Atlantic salmon (Salmo salar) farmed in Chile and many other areas (Norway, Scotland, Ireland, Canada and the USA). This study investigated the effects of low-dose P. salmonis infection (1 × 102 CFU/ml) on Atlantic salmon. In this study, we challenged fish with an isolated representative of the EM-90 genogroup via intraperitoneal injection for 42 days. Infected fish displayed decreased haematocrit and haemoglobin levels at day 13 post-infection, indicating erythropenia, haemolysis and haemodilution. Conversely, their white blood cell counts increased on days 13 and 21 post-infection. Additionally, their iron levels decreased from day 2 post-infection, indicating iron deficiency and an inability to retrieve stored iron before infection. Their magnesium levels also decreased at day 28 post-infection, possibly due to osmoregulatory problems. Also, we observed an increase in lactate dehydrogenase activity on days 5, 21, and 28 post-infection, suggesting early symptoms of hepatotoxicity. Later analyses determined a decrease in plasma glucose levels from day 2 post-infection. This may be attributed to the hypoxic conditions caused by P. salmonis, leading to an excess utilization of stored carbohydrates. Our results suggest that the blood parameters we studied are useful for monitoring the physiological status of Atlantic salmon infected with P. salmonis.

Is the southern crab Halicarcinus planatus (Fabricius, 1775) the next invader of Antarctica?

The potential for biological colonization of Antarctic shores is an increasingly important topic in the context of anthropogenic warming. Successful Antarctic invasions to date have been recorded exclusively from terrestrial habitats. While non-native marine species such as crabs, mussels and tunicates have already been reported from Antarctic coasts, none have as yet established there. Among the potential marine invaders of Antarctic shallow waters is Halicarcinus planatus (Fabricius, 1775), a crab with a circum-Subantarctic distribution and substantial larval dispersal capacity. An ovigerous female of this species was found in shallow waters of Deception Island, South Shetland Islands in 2010. A combination of physiological experiments and ecological modelling was used to assess the potential niche of H. planatus and estimate its future southward boundaries under climate change scenarios. We show that H. planatus has a minimum thermal limit of 1°C, and that its current distribution (assessed by sampling and niche modelling) is physiologically restricted to the Subantarctic region. While this species is presently unable to survive in Antarctica, future warming under both 'strong mitigation' and 'no mitigation' greenhouse gas emission scenarios will favour its niche expansion to the Western Antarctic Peninsula (WAP) by 2100. Future human activity also has potential to increase the probability of anthropogenic translocation of this species into Antarctic ecosystems.

Evolution of chaperome gene expression and regulatory elements in the antarctic notothenioid fishes.

Confined within the cold-stable Southern Ocean, Antarctic notothenioid fishes have undergone an evolutionary loss of the inducible heat shock response (HSR), while facing perpetual low-temperature challenges to cellular proteostasis. This study examines how evolution in chronic cold has affected the shared cellular apparatus that mediates proteostasis under normal and heat stressed states. To deduce Antarctic-specific changes, we compared native expression levels across the full suite of chaperome genes and assessed the structural integrity of two crucial HSR regulators - Heat Shock Factor 1 (HSF1) that activates HSR, and heat shock elements (HSEs), the binding sites for HSF1 - between Antarctic fishes and the basal temperate notothenioid Eleginops maclovinus. Native expression levels of Antarctic fish chaperomes showed very modest changes overall, contrary to the common view of constitutive upregulation in the cold. Only a few cytosolic HSP70 genes showed greater transcription, with only the ancestrally-inducible HSPA6 strongly upregulated across all Antarctic species. Additionally, the constant cold has apparently not relaxed the selective pressures on maintaining HSF1 and HSEs in Antarctic fish. Instead, we found HSF1 experienced intensified selective pressure, with conserved sequence changes in Antarctic species suggesting optimization for non-heat-stress functional roles. HSEs of the HSP70 gene family have largely remained conserved in canonical sequence motifs and copy numbers as in E. maclovinus, showing limited impact of relaxed selective pressure. This study shows that evolution in chronic cold has led to both subtle and distinctive changes in the cellular apparatus for proteostasis and HSR, with functional consequences amenable to experimental evaluation.

Vitellogenesis in the Patagonian toothfish (Dissostichus eleginoides) conditioned to a recirculating aquaculture system.

The Patagonian toothfish (Dissostichus eleginoides) is a new promising fish species for diversifying the aquaculture industry in Chile because of its high economic value and high international demand. However, when attempting to start aquaculture of a new species, one of the major challenges is successfully achieving conditions to reproduce them. This is particularly difficult when the information on the biology and physiology of the reproduction process of the species in question is scarce, as is the case with D. eleginoides. Additionally, female reproductive dysfunction is more prevalent under culture conditions and it is very important to have tools to evaluate the progress of oocyte maturation. Therefore, evaluation of the vitellogenesis process in addition to measuring gonadosomatic index (GSI) and oocyte diameter is an important parameter for allowing the monitoring of females from a broodstock that will spawn in the reproductive season. This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) specific for the Patagonian toothfish (D. eleginoides) vitellogenine (Vtg) and quantify the plasma level in the fishes, maintained in a recirculation aquaculture system (RAS), throughout their reproductive cycle. A polyclonal antibody was prepared using the isolated major egg protein as antigen. This antibody was specific to the major plasma phosphoprotein identified as Vtg and was used to develop and standardize an indirect ELISA assay. The assay standard curve was linear from 0.1 to 1 µg/ml purified egg yolk protein and the average r2 was 0.995. We corroborated our ELISA assay by demonstrating a strong correlation between high levels of plasma Vtg obtained by the assay and the intensity of the corresponding bands in both SDS-PAGE coomassie stained gels and Western Blot. During the two reproductive seasons analyzed, the highest Vtg plasma level was obtained in the majority of the females in the last three months before spawning (December-January). This differs from the wild population in which the spawning occurs during the austral winter (June-September). Therefore, the RAS condition established to maintain in captivity the D. eleginoides allows females to develop mature oocytes normally, as was evidenced by picks of Vtg plasma levels.

Effects of warming rates on physiological and molecular components of response to CTMax heat stress in the Antarctic fish Harpagifer antarcticus.

Maximum and minimum Critical thermal limits (CTMax and CTMin) have been studied extensively to assess thermal tolerance in ectotherms by means of ramping assays. Notothenioid fish have been proposed as particularly sensitive to temperature increases related to global climate change. However, there are large gaps in our understanding of the thermal responses of these extreme cold-adapted fish in assays with heating rates. We evaluated the effects of two commonly used heating rates (0.3 and 1 °C/min) on the cellular stress responses in the intertidal Antarctic fish Harpagifer antarcticus immediately after CTMax was reached, and at 2 and 4 h of recovery time in ambient water. We compared CTMax values, the relative transcript expression of genes relvant to heat shock response (Hsc70, Hsp70, Grp78), hypoxia (Hif1-α, LDHa, GR), ubiquitination (Ube2), and apoptosis (SMAC/DIABLO), and five plasma parameters - glucose, lactate, total protein, osmolality and cortisol. CTMax values between the two heating rates are not significantly different, and both rates elicited a similar stress response at molecular and physiological levels. We found a lack of up-regulated response of heat shock proteins, consistent with other Antarctic notothenioids. The general transcriptional pattern trended to downregulation, which was more evident in the slower 0.3 °C/min rate, and instances of upregulation were mainly related to ubiquitination. The faster 1 °C/min rate, rarely used for Antarctic fish, can be suitable for studying cold-adapted stenothermic fish without overestimating thermal tolerance or inducing damage from longer heat exposure.

Freshening effect on the osmotic response of the Antarctic spiny plunderfish Harpagifer antarcticus.

Global warming is having a significant impact around the world, modifying environmental conditions in many areas, including in zones that have been thermally stable for thousands of years, such as Antarctica. Stenothermal sedentary intertidal fish species may suffer due to warming, notably if this causes water freshening from increased freshwater inputs. Acute decreases in salinity, from 33 down to 5, were used to assess osmotic responses to environmental salinity fluctuations in Antarctic spiny plunderfish Harpagifer antarcticus, in particular to evaluate if H. antarcticus is able to cope with freshening and to describe osmoregulatory responses at different levels (haematological variables, muscle water content, gene expression, NKA activity). H. antarcticus were acclimated to a range of salinities (33 as control, 20, 15, 10 and 5) for 1 week. At 5, plasma osmolality and calcium concentration were both at their lowest, while plasma cortisol and percentage muscle water content were at their highest. At the same salinity, gill and intestine Na+ -K+ -ATPase (NKA) activities were at their lowest and highest, respectively. In kidney, NKA activity was highest at intermediate salinities (15 and 10). The salinity-dependent NKA mRNA expression patterns differed depending on the tissue. Marked changes were also observed in the expression of genes coding membrane proteins associated with ion and water transport, such as NKCC2, CFTR and AQP8, and in the expression of mRNA for the regulatory hormone prolactin (PRL) and its receptor (PRLr). Our results demonstrate that freshening causes osmotic imbalances in H. antarcticus, apparently due to reduced capacity of both transport and regulatory mechanisms of key organs to maintain homeostasis. This has implications for fish species that have evolved in stable environmental conditions in the Antarctic, now threatened by climate change.

Warming and freshening activate the transcription of genes involved in the cellular stress response in Harpagifer antarcticus.

Thermal and saline variations of the Southern Ocean are important signs of climate change which can alter the physiological responses of stenotic species residing at high latitudes. Our study aimed to evaluate the cellular stress response (CSR) of Harpagifer antarcticus subjected to increased ambient temperature and decreased salinity. The fish were distributed in different thermal (2, 5, 8, 11, and 14 °C) and saline (23, 28, and 33 psu) combinations for 10 days. We used qPCR analysis to evaluate the transcription of genes involved in the thermal shock response (HSP70, HSC70, HSP90, and GRP78), ubiquitination (E2, E3, ubiquitin, and CHIP), 26S proteasome complex (PSMA2, PSMB7, and PSMC1), and apoptosis (SMAC/Diablo and BAX) in the liver and gill. The expression profiles were tissue-specific and mainly dependent on temperature rather than salinity in the gill; meanwhile, in the liver, both conditions modulated the expression of these genes. Transcription of markers involved in the heat shock response was much higher in the liver than in the gill and was higher when salinity decreased and the temperature increased. Similarly, the genes involved in the ubiquitination pathway, 26S complex of the proteasome, and the apoptotic pathway showed the same pattern, being mainly induced in the liver rather than in the gill. This is the first study to show that this Antarctic fish can induce the cellular stress response in their tissues when subjected to these thermal/saline combinations.

Salmo salar glucocorticoid receptors analyses of alternative splicing variants under stress conditions.

Cortisol is the main corticosteroid in teleosts, exerting multiple functions by activating glucocorticoid receptors (GR). Most teleost species have two GR genes, gr-1 and gr-2. Some teleost also presents two splice variants for gr-1; gr-1a and gr-1b. In this study, we report for first time the presence of 2 homeologous genes for gr-1 and gr-2, located on chromosomes 4q-13q (gr-1) and 5p-9q (gr-2) of the Salmo salar genome. Furthermore, our results describe gr-1 splice variants derived from chromosome 4 and 13, sharing typical teleost GR elements such as the 9 amino acid insertion in the DNA binding domain (DBD) and variations in the length of the ligand binding domain (LBD). Three splice variants were predicted for the gr-2 homeologous gene in chromosome 5, with differences of a 5 amino acid insertion in the DBD. We also identified an uncommon truncated gr-2 gene in chromosome 9 in salmon, which lacked the DBD and LBD domains. Finally, by designing specific primers for each predicted splice variant, we validated and evaluated the expression of their transcripts in S. salar subjected to stress caused by stocking density. Differences were observed in the expression of all identified mRNAs, revealing that gr-1 and gr-2 splice variants were upregulated in head kidney and gills of post-stressed fish. In conclusion, our findings suggest that from specific salmonid genomic duplication (125 MYA), two gene copies of each GR receptor were generated in S. salar. The identified splice variants could contribute to the variability of GR receptor complex modulation expression during stressful events, leading to variations in physiological responses in fish.

Intermediary metabolic response and gene transcription modulation on the Sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1930) injected with two strains of Piscirickettsia salmonis.

Pathogen interactions with cultured fish populations are well studied, but their effects on native fishes have not been characterized. In Chile, the disease caused by bacterial species Piscirickettsia salmonis represents one of the main issues and is considered to be one of the important pathogens in the field of aquaculture. They have been found to infect native fish. Therefore, it is necessary to understand the impact of P. salmonis on native species of local commercial value, as well as the potential impact associated with the emergence of antibiotic-resistant strains of P. salmonis. Due to this purpose, the native fish Eleginops maclovinus was used in our study. Fish were randomly distributed in tanks and intraperitoneally inoculated with two strains of P. salmonis. No mortality was recorded during the experiment. Cortisol, glucose and total α-amino acid levels increased in fish injected with AUSTRAL-005 strain compared to sham-injected and LF-89-inoculated fish. Moreover, results showed an increase in the activity of carbohydrates and lipids metabolism in liver; and an increase in the carbohydrates, lipids and total α-amino acid metabolism in muscle after injection with AUSTRAL-005. Our results suggest that P. salmonis modulates the physiology of E. maclovinus and the physiological impact increase in the presence of the antibiotic-resistant strain AUSTRAL-005.

Francisella noatunensis subsp. noatunensis triggers calcium metabolism gene modulation in Eleginops maclovinus.

Francisella noatunensis subsp. noatunensis is the responsible agent of Francisellosis, a bacterial disease that affects an important amount of aquatic farmed species. Eleginops maclovinus is a fish that cohabits with salmonids cages in Chile and can also act as a vector of this bacterial disease. In the present study, we evaluated calcium metabolism in the liver of E. maclovinus injected intraperitoneally with different doses of F. noatunensis subsp. noatunensis (low 1.5 × 101, medium 1.5 × 105 and high doses 1.5 × 1010 cells/µL). Fish were sampled at 1, 3, 7, 14, 21 and 28 days post injection (dpi). No mortalities nor clinical signs were observed. Plasma calcium levels were higher in the high doses group of F. noatunensis subsp. noatunensis at day 7 and 14 compared to the control group (fish injected with bacterial medium alone). Hypercalcemic factors increased at day 14 and 21 for the medium and low dose (parathyroid hormone-related protein precursor), while vitamin D3 receptor increased its expression at times 1, 3 and 7 for the low dose. On the other hand, hypocalcemic factors such as calcitonin receptor and stanniocalcin increased its expression at time 7 and 14, respectively. Calmodulin involved in calcium storage decreased its expression during all experimental days in fish subjected to high bacterial dose. Proteins involved in calcium transport, such as L-type voltage-gated calcium channel and trpv5 increased their transcription at day 1 and 14, compared to calcium sensing-receptor and plasma membrane Ca2 +- ATPase that showed peak expression at times 14 and 28. The results suggest a clear alteration of calcium metabolism, mainly in high bacterial doses. This study provides new knowledge about the calcium metabolism in fish infected with bacteria.

Water temperature affects osmoregulatory responses in gilthead sea bream (Sparus aurata L.).

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psµ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na+,K+-ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na+,K+-ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (Vmax) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na+,K+-ATPase capacity appears fixed and activity appears to be determined by temperature.

Novel microsatellite markers discovery in Patagonian toothfish (Dissostichus eleginoides) using high-throughput sequencing.

Patagonian toohfish (Dissostichus eleginoides), is a sub Antartic notothenioid fish key in the marine ecosystem that sustains fishery of higher commercial value in the world. However, there are a scarce knowledge or information about its population genetic background, product of the almost null information of molecular markers available for this species. Here, we use high-throughput sequencing technology (Illumina platform) to develop 1071 microsatellite loci, of which 22 loci were selected to evaluation. Polymorphism and genetic diversity of each locus was assessed in two locations distant by 2370 km. Considering both locations, a mean PIC value of 0.748 was estimated. Selected microsatellite loci showed among two to seventeen alleles by locus in the first location and two to twelve in the second. The observed heterozygosity varied from 0.18 to 0.91 and from 0.12 to 0.87 for the first and second location, respectively. While, the expected heterozygosity ranged from 0.15 to 0.92 and from 0.11 to 0.90. Three loci were monomorphic in only one location. Microsatellite markers developed here will be useful in future studies on conservation, fishery and population genetics of this species.

Mechanosensory system of the lateral line in the subantarctic Patagonian blenny Eleginops maclovinus.

This study describes the cephalic and trunk lateral line systems in Patagonian blenny Eleginops maclovinus juveniles, providing morphological details for pores, canals and neuromasts. Eleginops maclovinus juveniles possess a complete laterodorsal lateral line that extends from the upper apex of the gill opening along the trunk as far as the caudal fin. The lateral line was ramified through pores and canals. The following pores were recorded: four supraorbital pores, with two along the eye border and two on the snout; seven infraorbital pores, with three on the lacrimal bone and four being infraorbital; five postorbital pores, with three along the preopercular border (upper preoperculum branch) and two on the bone curvature (inferior preoperculum branch); and four mandibular pores aligned along the jaw. Furthermore, five narrow-simple and interconnected canals were found (i.e. preopercular, mandibular, supraorbital and infraorbital canals). Histologically, the dorsal lateral line presented thin neuromasts (350 µm) with short hair cells. By contrast, the cranial region presented long, thick neuromasts. Infraorbital and mandibular neuromasts had a major axis length of 260 µm and respective average diameters of 200 and 185 µm. Sensory system variations would be due to a greater concentration of neuromasts in the cranial region, allowing for a greater perception of changes in water pressure. Scarce morphological information is available for the lateral sensory system in Eleginopsidae, particularly compared to Channichthyidae, Bovichthydae, Artedidraconidae and Bathydraconidae. Therefore, the presented results form a fundamental foundation of knowledge for the lateral-line system in juvenile E. maclovinus and provide a basis for future related research in this taxon as well as within the Notothenioidei suborder.

Evolution in chronic cold: varied loss of cellular response to heat in Antarctic notothenioid fish.

BACKGROUND: Confined within the freezing Southern Ocean, the Antarctic notothenioids have evolved to become both cold adapted and cold specialized. A marked signature of cold specialization is an apparent loss of the cellular heat shock response (HSR). As the HSR has been examined in very few notothenioid species to-date, it remains unknown whether HSR loss pervades the Antarctic radiation, or whether the broader cellular responses to heat stress has sustained similar loss. Understanding the evolutionary status of these responses in this stenothermal taxon is crucial for evaluating its adaptive potential to ocean warming under climate change. RESULTS: In this study, we used an acute heat stress protocol followed by RNA-Seq analyses to study the evolution of cellular-wide transcriptional responses to heat stress across three select notothenioid lineages - the basal temperate and nearest non-Antarctic sister species Eleginops maclovinus serving as ancestral proxy, the cryopelagic Pagothenia borchgrevinki and the icefish Chionodraco rastrospinosus representing cold-adapted red-blooded and hemoglobinless Antarctic notothenioids respectively. E. maclovinus displayed robust cellular stress responses including the ER Unfolded Protein Response and the cytosolic HSR, cementing the HSR as a plesiomorphy that preceded Antarctic notothenioid radiation. While the transcriptional response to heat stress was minimal in P. borchgrevinki, C. rastrospinosus exhibited robust responses in the broader cellular networks especially in inflammatory responses despite lacking the classic HSR and UPR. CONCLUSION: The disparate patterns observed in these two archetypal Antarctic species indicate the evolutionary status in cellular ability to mitigate acute heat stress varies even among Antarctic lineages, which may affect their adaptive potential in coping with a warming world.