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Genome-wide identification of the HSP70 superfamily in tropical sea cucumber Stichopus monotuberculatus and their expression analysis under low-salinity stress.

Heat shock proteins (HSPs) are a group of evolutionarily conserved molecular chaperones that serve as indispensable core regulators in preserving cellular homeostasis and orchestrating organismal stress responses. The tropical sea cucumber Stichopus monotuberculatus, a high-value aquaculture species, is sensitive to fluctuations in environmental salinity-a challenge that has emerged as a critical bottleneck limiting its large-scale commercial cultivation. However, no systematic investigation has been conducted to characterize the HSP70 superfamily in S. monotuberculatus and elucidate its functional roles in salinity adaptation. In the present study, we performed a comprehensive genome-wide scan and identified 19 HSP70 superfamily genes in the S. monotuberculatus genome, with the HSP70IV subfamily showing remarkable gene expansion, containing 8 distinct copies. Phylogenetic analysis, conserved motif identification, and gene structure characterization demonstrated high evolutionary conservation within each HSP subfamily. These genes were unevenly distributed across the chromosomes of S. monotuberculatus, and prediction of cis-acting elements revealed that their upstream regulatory regions were enriched with numerous functional elements associated with stress response and immune regulation. Salinity stress experiments revealed that under severe low-salinity conditions (18‰), the expression levels of SmHSPA14L and multiple HSP70IV subfamily members were significantly elevated, while SmHYOU1D was significantly downregulated; in contrast, only subtle changes were detected in the expression of most HSP70 genes under moderate low-salinity stress (24‰). These findings strongly suggest that HSP70 genes, particularly the expanded HSP70IV subfamily, may act as key modulators in the low-salinity stress response. This work provides valuable insight into the molecular mechanisms underlying salinity adaptation in tropical sea cucumbers.

Animals

Low-salinity stress alters growth, histology, physiology, and transcriptomic profiles of the gills and antennal glands in Macrobrachium rosenbergii.

Salinity is a major abiotic constraint in freshwater aquaculture of the giant freshwater prawn Macrobrachium rosenbergii, yet the coordinated roles of the gills and antennal glands, the two primary osmoregulatory organs in decapod crustaceans, under low-salinity stress remain poorly characterized. Here, we integrated histological, physiological, and transcriptomic analyses to characterize the adaptive responses of M. rosenbergii to acute (96 h) and chronic (8 weeks) exposure to salinity 5. Chronic low-salinity stress significantly impaired growth performance and decreased the survival rate. Acute stress induced thinning of the gill filaments, partial disorganization of pillar cells, and dilation of the intermicrovillar space in the antennal glands, whereas chronic stress caused gill vacuolization, cuticle thinning, and adaptive folding of antennal gland microvilli. In parallel, acute exposure significantly decreased hemolymph sodium and potassium ion concentrations but increased magnesium ion concentration, whereas chronic exposure increased hemolymph sodium and potassium ion concentrations, upregulated gill Na+/K+-ATPase activity, and enhanced hepatopancreatic antioxidant capacity. Transcriptomic analyses revealed distinct tissue-specific responses. Under acute stress, the gills preferentially activated pathways associated with cytoskeletal remodeling, motor proteins, and tight junctions, whereas chronic acclimation shifted the transcriptional response toward the renin-angiotensin system and glutathione metabolism. In the antennal glands, acute stress rapidly activated the renin secretion pathway, whereas chronic exposure promoted membrane remodeling by enriching pathways related to lipid and glycan metabolism. These findings reveal tissue-specific functional differentiation and synergistic coordination between the gills and antennal glands that underpin M. rosenbergii's adaptive response to low-salinity stress.

Animals

Exchanges of sodium and chloride at low salinities by Nereis diversicolor (Annelida, Polychaeta).

1. Experiments to compare the exchange (total influx) of sodium and chloride in the polychaete Nereis diversicolor in steady-state adaptation to very low salinities are reported. 2. The Na-uptake mechanism shows a high affinity for sodium, reaching half the maximal uptake rate at an external Na-concentration of 8-10 mM/liter (ca. 2% SW), and becomes "saturated" or reaches a plateau of uptake at concentrations of 40-50 mM/liter (ca. 10% SW) up to ca. 350 mM/liter (75% SW), above which Na-exchange is proportional to the external concentration. 3. The Cl-uptake curve differs from the Na-uptake curve in showing a relative depression at very low salinities before reaching "saturation" at Cl-concentrations of 50-60 mM/liter (ca. 10% SW). Cl-uptake becomes proportional to external concentration in salinities of 50% SW or greater, suggestive of passive diffusion in the ionic and osmotic conforming range. 4. It is shown that the permeability of the body wall, both to Na and to Cl, is reduced at very low salinities, thus destroying one of the assumptions upon which a previously-presented balance-sheet for chloride exchanges in N. diversicolor was based (Smith, 1970a). 5. Attempts to demonstrate an activation of the Na-uptake mechanism at very low salinities were inconclusive; reduction of body-wall permeability to sodium masks any possible activation. 6. It is suggested that the inside-negative body-wall potential is related to the depression of the Cl-uptake curve in salinities below 10% SW.

Animals

Transcriptomic and RNAi analyses reveal chloride channel 3-associated osmoregulation in Litopenaeus vannamei under low-salinity stress.

Chloride channels and transporters are important for cellular volume regulation and salinity adaptation in euryhaline crustaceans, yet the intestinal transcriptional relationship between plasma-membrane and intracellular chloride pathways remains unclear in Litopenaeus vannamei. In this study, RNA interference of anoctamin 1 (ANO1) was combined with intestinal transcriptome sequencing under the production-relevant low-salinity condition of salinity 3. ANO1 silencing produced a focused transcriptional response, with 16 differentially expressed genes (DEGs) identified (11 upregulated and 5 downregulated). Functional enrichment indicated that these genes were associated with transporter activity, cytoskeletal organization, extracellular matrix-receptor interaction, membrane lipid metabolism, and vesicular processes. Notably, a transcript encoding chloride channel protein 3 (CLC-3) was significantly upregulated following ANO1 knockdown, suggesting a potential transcriptional relationship between ANO1 and CLC-3 in chloride homeostasis. Based on this finding, CLC-3 was selected for full-length cDNA cloning, sequence characterization, salinity-gradient expression analysis, and RNAi-based functional assessment. The cloned CLC-3 cDNA was 2883 bp in length and encoded an 850 amino acid protein containing a conserved voltage-gated chloride channel (Voltage-CLC) domain and two cystathionine β-synthase domains. Phylogenetic analysis placed LvCLC-3 within the intracellular CLC-c clade, and tissue distribution analysis showed the highest CLC-3 expression in the intestine. Intestinal CLC-3 expression responded nonlinearly to salinity variation, peaking at salinity 20. Under salinity 3, CLC-3 knockdown reduced ANO1, Na+/K+-ATPase alpha subunit, and Na+-K+-2Cl- cotransporter transcript levels, whereas glutamate-gated chloride channel expression increased. Mild hepatopancreatic structural alterations were also observed after CLC-3 knockdown. These findings suggest that CLC-3 is a salinity-responsive intracellular chloride-transporter candidate associated with intestinal ion-transport-related transcriptional responses after ANO1 suppression in L. vannamei, although the underlying physiological mechanism requires further validation.

Animals

The ultrastructure of the posterior midgut caecum of Pachygrapsus crassipes (Decapoda, Brachyura) adapted to low salinity.

The effects of salinity adaptation and of composition and tonicity of fixatives upon the ultrastructure of the posterior midgut caecum (PMC) of Pachygrapsus crassipes have been studied. The PMC epithelium consists of a single layer of columnar cells with a microvillous border. The apical cytoplasm contains numerous mitochondria, lysosomes, and much smooth endoplasmic reticulum. Rough endoplasmic reticulum and Golgi apparatus are situated in the perinuclear cytoplasm. This epithelium resembles other transporting epithelia in that the basal cytoplasm has an extensive system of branched tubules formed from invaginations of the lateral and basal plasma membrane. Numerous mitochondria are associated with the basal tubular system. To determine the possible contribution of the PMC to the osmoregulatory ability of Pachygrapsus, the ultrastructure of the PMC from animals adapted to 40, 50, and 100 and 150% sea water was investigated. Enlargement of basal tubules and intercellular spaces at low salinity, suggestive of fluid-transport activity, was found to be an artifact of fixation. The most consistent response when animals were acclimated to dilute salinities was that some basal mitochondria assume a more complex shape, usually appearing as rings in cross sections of the caecum. A hypothesis concerning the functional significance of these mitochondria is proposed.

Adaptation, Physiological

Effects of low-sodium salines upon the atrio-ventricular propagation of the turtle heart.

The perfused heart in situ and strip preparations were used. In the in situ preparation, low-Na hypoosmotic saline had a positive inotropic effect, shortened the A-V interval and abolished arrhythmia or A-V blocking. The return to normal saline caused a transient off-response: further acceletation of the heartbeat and further shortening of the A-V interval. Low Na isoosmotic saline (sodium replaced by sucrose) increased pacemaker activity but the effect on the length of the A-V interval was variable. In the strip preparation, Low-Na hypoosmotic saline increased the rate of rise of the action potentials of atrioventricular ring (R-potentials) and shortened its latency of activation. The effects of the isoosmotic saline varied with the substitute used, either sucrose or lithium. Sucrose saline gave unpredictable results, though the rate of rise of R-potentials increased in 14 out of 24 experiments. Lithium saline decreased the rate of rise of R-potentials and blocked atrioventricular propagation in all experiments. Tetrodotoxin (10- minus 5 g/ml) did not abolish R-potentials. An increase of calcium concentration to 3 and 5 times the normal decreased the rate of rise of R-potentials and blocked atrio-ventricular propagation. While the Low-Na saline blocked pacemaker activity at the sinus venosus, it stimulated the generation of R-potentials, so that throughtout low-Na treatment, the site of pacemaker activity shifted to the A-V ring. The situation was reversed when normal saline was restored. The data are discussed according to the current ideas of action potential generation.

Action Potentials

Hypoosmolarity inhibits ammonia oxidation by terrestrial and freshwater Nitrosopumilaceae members.

Salinity strongly influences the physiology and distribution of nitrifying microorganisms, yet the effects of low salinity remain understudied. This study investigates the impact of hypoosmolarity on different groups of ammonia oxidizers in soil and freshwater reservoirs, as well as in pure culture isolates. In soil microcosms amended with ammonium, at low salinity levels (~120 μS/cm), comparable to values commonly found in pristine terrestrial and freshwater environments, the abundance of ammonia-oxidizing bacteria (AOB), dominated by Nitrosomonas oligotropha, significantly increased. In contrast, the growth of ammonia-oxidizing archaea (AOA), dominated by "Candidatus Nitrosotenuis" of the Nitrosopumilaceae family, was stimulated by high salinity (~760 μS/cm). In ammonium-fed freshwater microcosms, the abundance of AOB, dominated by N. oligotropha, significantly increased under both low (~170 μS/cm) and high salinity (~850 μS/cm) conditions. In the presence of allylthiourea (50 μM), used to inhibit bacterial ammonia oxidation, AOA were sensitive to low salinity in both soil and freshwater microcosms. Consistently, culture-dependent studies revealed marked growth inhibition of terrestrial AOA, especially members of Nitrosopumilaceae, under hypoosmolarity, unlike AOB and complete ammonia oxidizer (comammox) strains. Genomic analyses, along with transcriptomic studies, suggested that the sensitivity of AOA to hypoosmolarity stress was possibly due to a lack of osmoregulatory transport systems and their S-layer cell wall structure. Overall, this study indicates hypoosmolarity as an important factor shaping the ecological niches and distribution of ammonia oxidizers, as well as nitrification activities, in terrestrial and freshwater environments that are increasingly affected by intensified water cycles due to global change.

Ammonia

Adaptations with respect to salinity.

Amino acids contribute up to about 50% of the intracellular osmotic pressure of aquatic invertebrates. Since their concentration varies according to the salinity of the medium (high in sea water, low in fresh water) euryhaline invertebrates are good models for studying the mechanisms involved in the control of amino acid metabolism. During hyperosmotic stress CO2 production and O2 consumption decrease whereas the reverse is true when the animal is submitted to a hypo-osmotic stress. Nitrogen excretion (as NH3) increases in media of low salinity and the concentration of cyclic AMP increases during hyperosmotic stress. Moreover, blood proteins and haemocyanin are more concentrated in individuals adapted to media of low salinity. To explain the situation, three main mechanisms can be considered: (a) hydrolysis and synthesis of blood proteins; (b) transport of amino acids across the cell membrane; (c) control of the turnover rate of some amino acids. Results obtained on whole animals as well as on isolated tissues indicate that some amino acids are released from the cells and carried via the haemolymph to the posterior pairs of gills where they are oxidized (mechanisms b) or to an organ (hepatopancreas ?) Where they are used for blood protein synthesis (mechanism a). The use of labelled substrates demonstrates that the turnover rate of amino acids is controlled by the salinity of the environment (mechanism c). It is suggested that inorganic ions trigger the metabolic response by directing reducing equivalents toward oxygen or 2-oxo acids through control of the catalytic activity of dehydrogenases.

Adaptation, Physiological

Ecological and evolutionary implications of a mobile genetic element-rich haloarchaeon with unique osmotic resilience.

We isolated a novel halophilic archaeon, strain DSL9, representing the proposed new species Haloliberatus hailidukes gen. nov., sp. nov., from Dishui Lake, China. Unlike most obligate halophiles, DSL9 survives in low salinity, even distilled water, without lysis. Genomic analysis revealed dual salinity adaptation strategies: salt-in and compatible solutes, including a complete trehalose biosynthesis pathway. The strain harbors multiple plasmids, notably a 111,311 bp large plasmid (pHdsl9-3) encoding replication (Orc1/Cdc6, SSB), transcription (TFIIB), transmission (T4SS cluster, ArdC-like protein), and recombination (XerA) modules. pHdsl9-3 provides auxiliary functions such as defense, genome diversification, ion detoxification, and suggests active horizontal gene transfer. Similar elements are widespread in Halobacteriales, highlighting their role in haloarchaeal genetic diversity and plasticity. The encoded XerA hinted at a function beyond DNA dimer resolution, suggesting it may have been adapted by other archaeal mobile genetic elements. These findings underscore the need to investigate plasmid-driven evolution and environmental adaptation mechanisms in haloarchaea.IMPORTANCEThis study reports the isolation and characterization of DSL9, a novel halophilic archaeon from a freshwater lake. Remarkably, DSL9 defies the typical obligate halophilic lifestyle by surviving in low-salinity environments, including distilled water, without cell lysis. A key discovery is the identification of a 111,311 bp large plasmid harboring essential modules for replication, transcription, transmission, and integration. Widespread distribution of similar elements across Halobacteriales suggests their crucial role in haloarchaeal genetic diversity and plasticity, warranting further study of plasmid-mediated evolution and adaptation strategies.

Lakes

[Relationship between the ionic composition of blood and urine and the salinity of the external environment of the crab Hemigrapsus sanguineus].

Studies have been made on the dependence of sodium, potassium, magnesium and calcium concentrations of the blood and urine on the salinity of the external milieu in the crab H. sanguineus. Effective regulation of sodium and potasssium balance at low salinities was found. Within the salinity range investigated, magnesium level in the blood is maintained at lower level as compared to that in the environment. At low salinities, regulation of potassium and sodium concentrations in the blood is monitored by extrarenal mechanisms. Uber high salinity conditions, regulation of magnesium and potassium concentrations in the blood is accomplished at the expense of the activity of antennal glands. Calcium concentration in the blood is regulated by extra-renal mechanisms. The antennal glands affect regulation of calcium balance.

Animals

[Adenosine triphosphatase activity in the organs of the crab Hemigrapsus sanguineus, acclimated to sea water of different salinity].

In crabs acclimated to low salinity, the activity of Na, K-ATPase from the gills increases; the activity also increases in the antennal glands after acclimation of the animals to high salinity. The activity of Na, K-ATPase in the abdominal ganglion and in the heart does not depend on the salinity to which crabs had been acclimated. Changes in the activity of Mg-ATPase in the gills and antennal glands associated with acclimation of crabs to sea water with different salinity correspond to those in the activity of Na, K-ATPase.

Adaptation, Physiological

Micropuncture studies of the osmoregulation in the nauplius of Artemia salina.

The osmoregulation of the nauplius of the brine shrimp, Artemia salina, was investigated using micropuncture and microanalytical techniques. The naupliar body fluid, hemolymph was hyposmotic to and had lower Na concentrations than the suspending medium for the range of medium salinities from 80 to 4,900 mM NaCl. In medium containing 20 mM NaCl, the hemolymph was hyperosmotic to the medium, with osmolarity of 101 +/- 8 mosmol/1 and with [Na] of 49 +/- 11 meq/1. Whereas the maximal observed NaCl concentration gradient between hemolymph and medium was 4,785 mM, during the incubation of nauplii in artificial seawater (osmolarity: 932 mosmol/1; and [Na]: 502 meq/1) the osmolarity and [Na] of the naupliar hemolymph were 161 +/- SD 16 mosmol/1 and 86 +/- 14 meq/1, respectively. The influx and efflux of Na between medium and hemolymph were measured using 22Na. The fluxes of this ion were temperature dependent. The main site of efflux of 22Na was the neck organ as was shown by experiments of differential recovery of 22Na introduced in the hemolymph. These studies demonstrate that the nauplius of A. salina has the ability to osmoregulate not only against high environmental salinities but also against low salinities approaching those of freshwater.

Animals

Incidence of Salmonella spp., Clostridium botulinum, and Vibrio parahaemolyticus in an estuary.

A study of the incidence of Salmonella spp., Vibrio parahaemolyticus-like organisms, and clostridium botulinum in samples collected at five stations located in the Upper Chesapeake Bay, a major estuary on the Atlantic Coast of the United States, was conducted in December 1973 through December 1974. C. botulinum types B and E were detected in 12.3% of the total sediment samples examined. V. parahaemolyticus was recovered from 10.4% of a total of 86 water, sediment, and suspended sediment samples. Of 131 samples examined for the presence of Salmonella spp., approximately 3% were found to be positive for serologically confirmed Salmonella isolates. Shellfish examined during the investigation were also found to be free of enteric pathogens. The low frequency of occurrence of V. parahaemolyticus was attributed to the low salinities encountered at the sites included in the study. A low incidence of Salmonella spp. in the Upper Chesapeake Bay samples was found, whereas the distribution of C. botulinum appeared to be both random and autochthonous. A strong relationship between presence of potential pathogens and other generally accepted microbiological indicators of pollution was not observed.

Animals

Decoding the trajectory of antibiotic resistance genes in saline and alkaline soils: Insights from different fertilization regimes.

The soil salinity and alkalinity play an important role in the occurrence and proliferation of antibiotic resistance genes (ARGs). Yet, little is known the underlying mechanism by which soil salinity and alkalinity affect antibiotic resistance evolution. Here we investigated the ARGs variation in soil salinity and alkalinity environments created by different fertilization, and explored the biological mechanisms that salinity and alkalinity alter the evolutionary paradigm of antibiotic resistance. The results showed the soil treated by organic fertilizer exhibited a low salinity, neutral level (TSD 239.20 μS/cm, pH 7.17). The ARG abundance in the OF treatment was the highest, keeping an average of 67.83 TPM. Beside the effect of direct input of organic fertilizer at the beginning, it was important to note that, ARGs abundance during planting showed significant correlations with pH and electric conductivity. We observed that changes in microbial survival strategies under different salinity and alkalinity conditions further affected ARG hosts abundance. Indoor experiments demonstrated that there was a survival trade-off between the growth of resistant bacteria and the evolution of antibiotic resistance in salinity and alkalinity environments. Meta-genomic and Meta-transcriptomic analysis consistently demonstrated bacterial antibiotic resistance was primarily associated with pyruvate, energy and lipid metabolic pathways. The functional gene related to salinity and alkalinity, like cysH, cysK, plsB and plsC showed negative correlations with MDR. Prokaryotic transcription assays validated these relations. This study well explains the prevalence of soil ARGs after different fertilization regimes and will give a deeper understanding for the effect of soil salinity and alkalinity on antibiotic resistance evolution.

Soil