Physiological preparedness and performance of Atlantic salmon Salmo salar smolts in relation to behavioural salinity preferences and thresholds

This study investigated the relationships between behavioural responses of Atlantic salmon Salmo salar smolts to saltwater (SW) exposure and physiological characteristics of smolts in laboratory experiments. It concurrently described the behaviour of acoustically tagged smolts with respect to SW and tidal cycles during estuary migration. Salmo salar smolts increased their use of SW relative to fresh water (FW) from April to June in laboratory experiments. Mean preference for SW never exceeded 50% of time in any group. Preference for SW increased throughout the course of smolt development. Maximum continuous time spent in SW was positively related to gill Na⁺, K⁺‐ATPase (NKA) activity and osmoregulatory performance in full‐strength SW (measured as change in gill NKA activity and plasma osmolality). Smolts decreased depth upon reaching areas of the Penobscot Estuary where SW was present, and all fish became more surface oriented during passage from head of tide to the ocean. Acoustically tagged, migrating smolts with low gill NKA activity moved faster in FW reaches of the estuary than those with higher gill NKA activity. There was no difference in movement rate through SW reaches of the estuary based on gill NKA activity. Migrating fish moved with tidal flow during the passage of the lower estuary based on the observed patterns in both vertical and horizontal movements. The results indicate that smolts select low‐salinity water during estuary migration and use tidal currents to minimize energetic investment in seaward migration. Seasonal changes in osmoregulatory ability highlight the importance of the timing of stocking and estuary arrival.     

Two NHX‐type transporters from Helianthus tuberosus improve the tolerance of rice to salinity and nutrient deficiency stress

The NHX‐type cation/H⁺ transporters in plants have been shown to mediate Na⁺(K⁺)/H⁺ exchange for salinity tolerance and K⁺ homoeostasis. In this study, we identified and characterized two NHX homologues, HtNHX1 and HtNHX2 from an infertile and salinity tolerant species Helianthus tuberosus (cv. Nanyu No. 1). HtNHX1 and HtNHX2 share identical 5′‐ and 3′‐UTR and coding regions, except for a 342‐bp segment encoding 114 amino acids (L₂₇₂ to Q₃₈₅) which is absent in HtNHX2. Both hydroponics and soil culture experiments showed that the expression of HtNHX1 or HtNHX2 improved the rice tolerance to salinity. Expression of HtNHX2, but not HtNHX1, increased rice grain yield, harvest index, total nutrient uptake under K⁺‐limited salt‐stress or general nutrient deficiency conditions. The results provide a novel insight into NHX function in plant mineral nutrition.     

Microtubule‐dependent changes in morphology and localization of chloride transport proteins in gill mitochondria‐rich cells of the tilapia,Oreochromis mossambicus

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na⁺/Cl^? cotransporter (NCC) and Na⁺/K⁺/2Cl^? cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria‐rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time‐course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule‐dependent cellular regulating processes was used. SW‐acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine‐FW) for 96 h. Compared with the FW‐treatment group, in the MR cells of colchicine‐FW‐treatment group, (1) the average size was significantly larger, (2) only wavy‐convex‐subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule‐dependent. J. Morphol. 277:1113–1122, 2016. © 2016 Wiley Periodicals, Inc.     

Identification of the interaction between bta‐miR‐370 and OLR1 gene in bovine adipocyte

It has been shown that the oxidized low density lipoprotein receptor 1 (OLR1) gene plays an important role in the degradation of oxidized low density lipoprotein. Previous studies found a SNP in the 3′‐untranslated region (3′‐UTR) of the OLR1 gene associated with milk production traits in different dairy cattle populations and with loin eye area and marbling depth in beef cattle. MicroRNAs can regulate gene expression by binding the 3′‐UTR of target genes to degrade or to repress the translation of target genes. Bioinformatics have shown that there is a binding site of bta‐miR‐370 in the 3′‐UTR of the OLR1 gene, and a previous luciferase reporter assay system showed that the A/C mutation occurring in the 3′‐UTR of this gene caused the binding sites of bta‐miR‐370 to disappear in HEK293 cells. To further validate whether OLR1 was the target gene of bta‐miR‐370, the over‐expression and interference expression of bta‐miR‐370 were determined by transfecting bta‐miR‐370 mimics and inhibitor supplementations into bovine adipocyte. The qRT‐PCR result showed that the relative expression of OLR1 gene significantly decreased in the mimics group compared to the control, whereas the expression level in inhibitor group was higher than its control group. The above results were further verified by a Western blot at the protein level. In addition, lipid formation analysis of bovine adipocytes was performed via oil red O staining, and we found that cytoplasm lipid droplets in the inhibitor group showed a tendency to increase compared to the control group, whereas in the mimics group, we observed an obvious decrease of cytoplasm lipid droplets compared to the control and inhibitor groups. Taken together, our data here suggest that bta‐miR‐370 has a negative regulation role for OLR1 both at the gene expression and protein levels and bovine adipocytes cytoplasm lipid droplets formation, which provides a reference for illustrating how the OLR1 gene affects milk production and beef quality traits in cattle.     

Origins and functional diversification of salinity‐responsive Na+, K+ ATPase α1 paralogs in salmonids

The Salmoniform whole‐genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity‐responsive paralogs of the Na⁺, K⁺ ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue‐specific expression patterns. We found that these paralogs arose during a small‐scale duplication event prior to the Salmoniform, but after the teleost, whole‐genome duplication. The ‘freshwater paralog’ (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the ‘saltwater paralog’ (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na⁺, K⁺ ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole‐genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in fishes.     

Immunolocalization of Na+/K+‐ATPase,Na+/K+/2Cl− co‐transporter (NKCC) and mRNA expression of Na+/K+‐ATPase α‐subunit during short‐term salinity transfer in the gills of Persian sturgeon (Acipenser persicus,Borodin, 1897) juveniles

The study tests the physiological responses of Persian sturgeon, Acipenser persicus, during the abrupt release of juveniles from freshwater (FW) into brackish waters (BW = 11‰) of the Caspian Sea. Fish weight at release was 2‐3 g (2.55 ± 0.41 g; 8.8 ± 0.58 cm TL). Totals of 160 individuals were randomly distributed into four fiber‐glass aerated tanks (volume 60‐L). Two tanks served as controls (FW groups), and two as exposure tanks for BW (Caspian Sea water = CSW). Fish were sampled at 0, 3, 6, 12, 24, 48 and 96 hr after abrupt transfer to CSW. Plasma osmolality, immunolocalization of Na⁺, K⁺ ‐ATPase (NKA) and Na⁺/K⁺/2Cl^– (NKCC) Co‐transporter, NKA activity and the NKA α‐subunit mRNA expression were analyzed. Blood osmolality of fish transferred from FW to CSW increased significantly within hours post‐transfer (p < .05) and remained at a high level for up to 96 hr. Immunolocalization of NKCC indicated co‐localization with NKA in the chloride cells in the gill epithelium. A partial sequence of the NKA α‐subunit (632 bp) is described. Its expression levels were up‐regulated at 12 and 48 hr following salinity transfer (p < .05). However, NKA activity sharply increased in CSW specimens by almost 2.8‐fold (p < .05) between 48 and 96 hr after transfer. Gill NKCC co‐transporter abundance increased, coinciding with increased gill NKA activity. The increased activity of NKCC during salt excretion in CSW may lead to an influx of Na⁺ into the chloride cells. Consequently, NKA activity increases to maintain intracellular Na⁺ homeostasis.     

Early life stage salinity tolerance of wild and hatchery-reared juvenile pink salmon Oncorhynchus gorbuscha

Salinity tolerance in wild (Glendale) and hatchery (Quinsam) pink salmon Oncorhynchus gorbuscha (average mass 0·2 g) was assessed by measuring whole body [Na⁺] and [Cl^?] after 24 or 72 h exposures to fresh water (FW) and 33, 66 or 100% sea water (SW). Gill Na⁺, K⁺‐ATPase activity was measured following exposure to FW and 100% SW and increased significantly in both populations after a 24 h exposure to 100% SW. Whole body [Na⁺] and whole body [Cl^?] increased significantly in both populations after 24 h in 33, 66 and 100% SW, where whole body [Cl^?] differed significantly between Quinsam and Glendale populations. Extending the seawater exposure to 72 h resulted in no further increases in whole body [Na⁺] and whole body [Cl^?] at any salinity, but there was more variability among the responses of the two populations. Per cent whole body water (c. 81%) was maintained in all groups of fish regardless of salinity exposure or population, indicating that the increase in whole body ion levels may have been related to maintaining water balance as no mortality was observed in this study. Thus, both wild and hatchery juvenile O. gorbuscha tolerated abrupt salinity changes, which triggered an increase in gill Na⁺, K⁺‐ATPase within 24 h. These results are discussed in terms of the preparedness of emerging O. gorbuscha for the marine phase of their life cycle.     

Trade‐offs in osmoregulation and parallel shifts in molecular function follow ecological transitions to freshwater in the Alewife

Adaptation to freshwater may be expected to reduce performance in seawater because these environments represent opposing selective regimes. We tested for such a trade‐off in populations of the Alewife (Alosa pseudoharengus). Alewives are ancestrally anadromous, and multiple populations have been independently restricted to freshwater (landlocked). We conducted salinity challenge experiments, whereby juvenile Alewives from one anadromous and multiple landlocked populations were exposed to freshwater and seawater on acute and acclimation timescales. In response to acute salinity challenge trials, independently derived landlocked populations varied in the degree to which seawater tolerance has been lost. In laboratory‐acclimation experiments, landlocked Alewives exhibited improved freshwater tolerance, which was correlated with reductions in seawater tolerance and hypo‐osmotic balance, suggesting that trade‐offs in osmoregulation may be associated with local adaptation to freshwater. We detected differentiation between life‐history forms in the expression of an ion‐uptake gene (NHE3), and in gill Na⁺/K⁺‐ATPase activity. Trade‐offs in osmoregulation, therefore, may be mediated by differentiation in ion‐uptake and salt‐secreting pathways.     

Molecular cloning and expression analysis of a myosin light chain 1 (MLC‐1) gene from Indian meal moth Plodia interpunctella (Lepidoptera: Pyralidae)

Myosin light chain 1 (MLC‐1) protein acts in the organization, dynamics and transport processes associated with the cytoskeleton. In this work, an MLC‐1 gene was cloned and characterized from the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae). The isolated PiMLC‐1 cDNA is 913 bp, including a 5′‐untranslated region (UTR) of 79 bp, 3′‐UTR of 381 bp and an open reading frame (ORF) of 453 bp encoding a polypeptide of 150 amino acids, which contains two calcium binding domains (EF‐hands). The deduced PiMLC‐1 protein sequence has 39–94% comparison with other individuals. The qPCR analysis revealed that PiMLC‐1 was expressed in the four developmental stages (egg, larva, pupa and adult) and in all tissues tested, suggesting that it plays an important role in development of P. interpunctella. Based on the MLC‐1 amino acids, phylogenetic analysis showed a similar topology with the traditional classification, suggesting the potential value of the MLC‐1 protein in phylogenetic inference.     

Fine‐tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system

A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA‐2, termed CPMV‐HT, in which the sequence to be expressed is positioned between a modified 5′ UTR and the 3′ UTR has been successfully used for the plant‐based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5′ UTR can dramatically influence expression levels, the role of the 3′ UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3′UTR of CPMV RNA‐2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress‐HT‐GFP. The results showed that the presence of a 3′ UTR in the CPMV‐HT system is important for achieving maximal expression levels. Removal of the entire 3′ UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y‐shaped secondary structure formed by nucleotides 125–165 of the 3′ UTR plays a key role in its function; mutations that disrupt this Y‐shaped structure have an effect equivalent to the deletion of the entire 3′ UTR. Our results suggest that the Y‐shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5′ and 3′ UTRs in CPMV‐HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.     

MOLECULAR CHARACTERIZATION OF AN APOLIPOPHORIN‐III GENE FROM THE CHINESE OAK SILKWORM,Antheraea pernyi (LEPIDOPTERA: SATURNIIDAE)

Apolipophorin‐III (ApoLp‐III) acts in lipid transport, lipoprotein metabolism, and innate immunity in insects. In this study, an ApoLp‐III gene of Antheraea pernyi pupae (Ap‐ApoLp‐III) was isolated and characterized. The full‐length cDNA of Ap‐ApoLp‐III is 687 bp, including a 5′‐untranslated region (UTR) of 40 bp, 3′‐UTR of 86 bp and an open reading frame of 561 bp encoding a polypeptide of 186 amino acids that contains an Apolipophorin‐III precursor domain (PF07464). The deduced Ap‐apoLp‐III protein sequence has 68, 59, and 23% identity with its orthologs of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap‐apoLp‐III was close to that of Bombycoidea. qPCR analysis revealed that Ap‐ApoLp‐III expressed during the four developmental stages and in integument, fat body, and ovaries. After six types of microorganism infections, expression levels of the Ap‐ApoLp‐III gene were upregulated significantly at different time points compared with control. RNA interference (RNAi) of Ap‐ApoLp‐III showed that the expression of Ap‐ApoLp‐III was significantly downregulated using qPCR after injection of E. coli. We infer that the Ap‐ApoLp‐III gene acts in the innate immunity of A. pernyi.