Divergent life histories among organisms are linked to variations in thermal response. Although there have been extensive studies on interpopulation variations in thermal tolerance, the ecological relevance of the tolerance to life history remains largely unexplored. We compared critical thermal maximum (CTmax) among two Pacific salmon species with different freshwater life histories, masu salmon (Oncorhynchus masou) and chum salmon (O. keta), across two local Japanese populations. CTmax was higher for masu salmon fry (26.9 °C) than for chum salmon fry (26.1 °C). Although the mean CTmax differed by 1.1 °C among masu salmon populations, the difference among chum salmon populations was much smaller, at 0.17 °C. This high interpopulation variation was well explained by variation in river temperatures during freshwater rearing. In contrast, the low interpopulation variation of chum salmon fry might reflect their immediate ocean entry after emergence from the riverbed, which leads to them experiencing similar ocean temperatures across populations. Our findings provide crucial evidence that interpopulation variation in thermal tolerance might be most pronounced for Pacific salmon species that are highly dependent on freshwater environments.

Long non-coding RNAs (lncRNAs) are emerging regulators of diverse biological processes, but their mechanistic roles in molluscan biomineralization remain incompletely understood. Here, we identified a novel 635-nt lncRNA, lncRNA7379, from mantle transcriptome data of the pearl oyster Pinctada fucata martensii. lncRNA7379 showed a mantle-enriched expression pattern and dynamic responses after shell notching and under elevated temperature. Because shell injury and thermal exposure elicit broad physiological responses, these expression changes were treated as complementary contextual evidence rather than as biomineralization-specific readouts. RNA interference-mediated depletion of lncRNA7379 altered shell ultrastructure and increased the transcript abundance of MSX and Smad1/5. Subcellular fractionation and RNA FISH showed that lncRNA7379 was predominantly cytoplasmic. In heterologous 293 T cells, lncRNA7379 overexpression reduced Smad1/5 mRNA and protein levels, while RNA immunoprecipitation assays showed enrichment of lncRNA7379 in Smad1/5 immunoprecipitates. Together, these data support an association of lncRNA7379 with shell formation and suggest that it may modulate the PfSmad1/5-related regulatory network.

The testicular functions of neuropeptide Y are largely unexplored in non-mammalian vertebrates as the few reports available remain restricted to fishes. Current research work reveals the testicular presence of npy and npyr in Hemidactylus flaviviridis and its variation in expression depending upon reproductive phases, marking the first documentation of such findings in reptilian species. Expression analysis of npy/npyr across different reproductive phases showed peak levels of both ligand and receptor during the recrudescent period, while the lowest expression occurred in regression. The investigation also examined the influence of NPY on markers of testicular spermatogenesis and steroidogenesis in H. flaviviridis. The in vitro exposure of wall lizard testes to NPY during recrudescent phase stimulated bcl 2, while simultaneously reduced caspase 3. NPY treatment also stimulated testicular expression of scf, c-kit, pcna, bmp-15 and gdf-9, along with gonadotropin receptor (fshr), and sex-steroid receptors (er-α, er-β, and ar). Further, NPY elevated star expression and promoted testosterone synthesis in wall lizard. Regarding the regulation of testicular npy and its receptor, npyr the expression of both ligand and its receptor was upregulated by the neuropeptide, kisspeptin, adipokines like leptin and nesfatin-1, and sex steroids such as DHT, and E2, while they were suppressed by the gonadotropin, FSH and the neuropeptide, substance P. Overall, this investigation provides a detailed documentation of the presence, phase-specific pattern of expression, role, and hormonal regulation of NPY and its receptor in wall lizard testes.

Whether marine bivalves, as osmoconformers, actively transport inorganic ions to counter osmotic stress remains unclear due to insufficient direct evidence. The estuarine bivalve Crassostrea hongkongensis offers an ideal model to address this issue, as our previous ultrastructural study hinted at the existence of ionocyte-like cells in the gill. Here, single-cell RNA sequencing revealed that the homologs of key ion transporters in oyster gill, Na[+]/K[+]-ATPase α subunit (CHNKAα), Na[+]/K[+]/2Cl[-] cotransporter 1 (CHNKCC1) and Na[+]/H[+] exchanger 3 (CHNHE3), lack the cluster-specific expression typical of teleost homologs. Instead, their transcripts are broadly distributed across multiple gill epithelial cells, confirmed by RNA fluorescence in situ hybridization. At 8 h post-salinity exposure, CHNKAα and CHNHE3 transcripts increased at salinity 30 (positively correlated with gill Na[+], K[+] and Cl[-] contents), whereas CHNKCC1 was upregulated at salinity 6 (negatively correlated), implicating their involvement in active ion transport. Multiplex immunofluorescence revealed an interesting membrane distribution: CHNKAα predominantly localized to the basal membrane, CHNKCC1 showed an unusual apical localization, and CHNHE3 exhibited dual (apical and basal) localization in different cells. RNA interference-mediated knockdown further indicated that CHNKAα drives Cl[-] uptake while CHNKCC1 mediates Cl[-] secretion, directly linking their active transport to salinity-induced ion content changes. Notably, these observations, particularly membrane distribution and functional contribution to ion transport, do not completely conform to any existing ion transport model in aquatic animals. Our findings suggest that a distributed, flexible network of gill epithelial cells contributes to ion transport in a euryhaline bivalve, revealing a potential ion transport system that is fundamentally distinct from the teleost paradigm and opening new avenues for understanding osmoregulation in marine invertebrate.

Hibernation is an extreme form of seasonal heterothermy that enables animals to survive under harsh environmental conditions and provides valuable natural models for investigating the physiological mechanisms underlying stress tolerance. The Daurian ground squirrel (Spermophilus dauricus) is a small burrowing mammal that exhibits pronounced seasonal changes in activity and naturally undergoes hibernation, making it a useful model for elucidating the molecular mechanisms underlying hibernation. Heat shock proteins (HSPs) are highly conserved molecular chaperones involved in protein homeostasis and cellular stress responses. A total of 40 HSP genes were identified in the Daurian ground squirrel and grouped into six subfamilies, consisting of three small HSPs (sHSPs), 26 HSP40s, six HSP70s, three HSP90s, one HSP60, and one HSP110. Comprehensive analyses of molecular characteristics, conserved domains, gene structures, phylogenetic relationships, and chromosomal distributions revealed substantial evolutionary diversity within the HSP40 family. Although members of the DNAJA, DNAJB, and DNAJC subfamilies shared several conserved structural features, differences were also observed in their molecular characteristics and evolutionary relationships. To investigate their expression dynamics during hibernation, the transcript levels of HSP40 genes in the liver tissues of the Daurian ground squirrels at different hibernation stages were quantified using the real-time quantitative polymerase chain reaction (RT-qPCR). Most of the HSP40 genes examined exhibited broadly similar expression trends throughout the hibernation cycle, although individual genes showed different temporal expression profiles. These findings suggest that HSP40 genes undergo differential transcriptional regulation during hibernation and may be involved in the physiological responses associated with hepatic adaptation to the hibernation state. Collectively, this study provides a systematic characterization of the HSP gene family in a natural mammalian hibernator and offers valuable insights into the potential involvement of HSPs in proteostasis regulation during hibernation.

Regulation of water permeability in the collecting duct is important for osmoregulatory acclimation in teleost fish. In hyperosmotic environments such as seawater (SW), the teleost kidney functions as a site of divalent ion excretion. The collecting ducts reabsorb Na[+], Cl[-], and water, thereby reducing urine volume and producing small amounts of isotonic urine with high concentrations of divalent ions. In hypoosmotic environments such as freshwater (FW) or low-salinity water (LS), the kidney produces large volumes of hypotonic urine and serves as a site of water excretion; under these conditions, the collecting ducts reabsorb Na[+] and Cl[-] but not water. To identify aquaporins (Aqps) involved in regulating collecting duct water permeability, we analyzed renal Aqp expression in a euryhaline marine fish, the Japanese pufferfish (Takifugu rubripes), which possesses 16 Aqp genes in its genome, seven of which (Aqp1aa, 1ab, 3a, 4a, 7, 8bb, and 11a) are expressed in the kidney. Quantitative RT-PCR analysis showed that Aqp1aa and Aqp4a were highly expressed in collecting duct tissues, and that Aqp1aa expression was markedly reduced in fish acclimated to LS (1 ppt). Immunohistochemistry revealed apical localization of Aqp1aa and basolateral localization of Aqp4a in collecting duct cells, with apical Aqp1aa downregulated in LS. These results suggest that Aqp1aa and Aqp4a mediate water reabsorption in SW and that downregulation of Aqp1aa contributes to hypotonic urine production in under LS conditions.

Chinese tongue sole (Cynoglossus semilaevis) is a unique model for studying the mechanisms of sex determination and sexual size dimorphism (SSD). Preliminary transcriptome and Weighted Gene Co-expression Network Analysis (WGCNA) analyses have identified the neurotrophic tropomyosin receptor kinase 2 gene (ntrk2), mutations in which lead to disorders such as obesity and developmental delays. To better understand the role of neurotrophic tropomyosin receptor kinase (ntrk) family genes in C. semilaevis SSD, we characterized the ntrk gene family and the ligand of ntrk2, brain-derived neurotrophic factor (bdnf). As a result, six ntrk homologous genes (ntrk1, ntrk2a-w, ntrk2a-z, ntrk2b, ntrk3a, ntrk3b) were identified in C. semilaevis at the genome-wide level. Expression profiling revealed predominant brain and pituitary expression of ntrk2 and ntrk3, with ntrk2a-w exhibiting female-specific patterns in the telencephalon and hypothalamus, and ntrk2a-z/ntrk2b widely expressed in all five brain regions. Six different transcripts for bdnf, the primary ligand for ntrk2, were also characterized. Quantitative RT-PCR further revealed sex-specific expression patterns of bdnf1-6 in the five brain regions. Interaction network prediction showed that ntrk could interact with key molecules related to growth and development including BDNF, NGFa, NGFb, and FGFR. Knockdown of ntrk2 and bdnf in C. semilaevis brain cells significantly reduced the expression of pro-opiomelanocortin (pomc), AMP-activated protein kinase α1 subunit (prkaa1), and leptin receptor (lepr)-genes encoding key components of the leptin-melanocortin energy homeostasis pathway. This suggests that the BDNF/NTRK2 signaling axis may centrally regulate feeding and metabolism in C. semilaevis. Our findings provide molecular and functional clues for exploring the neuroendocrine mechanisms underlying sexual size dimorphism in this flatfish.

Temperature is a crucial environmental factor determining the survival and growth of Apostichopus japonicus. Thermotaxis represents an innate behavioral strategy that enables poikilotherms to migrate toward thermally favorable microhabitats. However, the molecular mechanisms underlying temperature sensation and thermal behavioral responses in A. japonicus remain unclear. Here, we investigated the functional roles of transient receptor potential vanilloid (TRPV) channels in regulating the thermotactic behavior of A. japonicus larvae. Both AjTRPVA and AjTRPVB were highly expressed during the auricularia larval stage, with expression levels 4.05-fold and 1.46-fold higher than those at the gastrula stage, respectively. Whole mount in situ hybridization showed that both AjTRPVA and AjTRPVB were predominantly localized in the ciliary bands of auricularia larvae. Sequence alignment identified highly conserved residues potentially responsible for capsaicin and heat activation, which are clustered in intracellular S4-S5 linker and S6 transmembrane domain. Behavioral assays demonstrated that A. japonicus auricularia larvae exhibit positive thermotaxis toward warm temperatures (18-27 °C). Capsaicin (a canonical TRPV activator) significantly reduced larval aggregation in preferred high-temperature zones (H2: 21-24 °C, H3: 24-27 °C), decreasing the total occupancy rate by 22.08%. Application of RR (a TRPV inhibitor) fully rescued the impaired thermotactic performance and restored normal larval thermal preference when released from high-temperature site. Collectively, these pharmacological results indicate that TRPV channels may contribute to positive thermotaxis in A. japonicus larvae.

Small amphibians active on dry, sunlit substrates face a physiological conflict: exposure to solar radiation and heat transfer from sun-warmed substrates can increase body temperature and activity, but permeable skin makes such exposure costly through rapid evaporative water loss. Most amphibians reduce this cost by restricting water loss or by remaining in moist microhabitats. The endangered tepui summit toad Oreophrynella quelchii, however, is frequently observed basking for extended periods on dry, exposed sandstone, raising the question of whether this species copes with coupled heat-desiccation stress by suppressing evaporative water loss or by sustaining high evaporative flux and recovering from acute body mass loss. We investigated upper thermal tolerance, behavioural thermal avoidance, and water-loss dynamics in O. quelchii during a standardised dynamic heat-desiccation challenge. To provide ecological context, we also report limited observations from the sympatric frog Pristimantis aureoventris, which is associated with more buffered vegetation microhabitats. Oreophrynella quelchii exhibited CTmax values averaging 32.6 °C. Although this value is not exceptional among amphibians as a whole, it combines with previously reported extremely low CTmin values to produce a broad thermal tolerance breadth for a tropical amphibian. Behavioural avoidance occurred below CTmax, indicating a margin between voluntary retreat and critical thermal failure. During dynamic heat-desiccation exposure, evaporative flux increased with body temperature and vapour pressure deficit and approached passive evaporation estimates from a plaster-model reference, suggesting limited restriction of water loss under fixed-posture exposure. Despite this high evaporative flux, individuals reached the predefined acute body-mass-loss endpoint of approximately 10% initial mass and recovered after rehydration. Limited observations from P. aureoventris indicated lower thermal limits and lower evaporative flux under the same experimental framework, but trials were stopped at a lower safety endpoint and these data are interpreted as contextual rather than comparative because of the small sample size. Overall, our results suggest that O. quelchii does not cope with exposed tepui summit conditions primarily by strongly suppressing evaporative water loss. Instead, its physiological profile appears to combine broad thermal tolerance, behavioural avoidance of critical overheating, high evaporative flux, and the capacity to sustain and recover from acute body mass loss. This tolerance-based strategy may help explain how a small amphibian remains active on exposed tepui sandstone, while highlighting the need to integrate thermal and hydric traits when interpreting amphibian activity in extreme microhabitats.

The incubation period (I) for bird eggs varies among species and is used in establishing allometric relationships. Research on variations in I shed light on the evolutionary mechanisms that gave rise to the differentiation of embryonic development in distinct taxa of birds. Here, using a sampling of 444 images from 444 avian species, 89 families and 30 orders, we calculated their major geometric dimensions: volume (V) and surface area (S). An assessment of the relationship between I and the measured and calculated egg parameters demonstrated the closest and most significant correlation (R = -0.760) between I and the S/V ratio that was adopted as a conditional indicator and reflects the embryo's metabolic rate. Approximation of the values of these parameters made it possible to derive a power-law dependence for the prediction of I depending on the S/V value of a particular egg (R[2] = 0.757). The prediction accuracy was higher (R[2] = 0.783) if the eggs of the family Procellariiformes (petrels), whose I value is characterized by a longer time, were removed from the general sampling computation. We conclude that the value of the S/V ratio can characterize both the metabolism of an embryo and the conditional thermal conductivity of an egg, which aids in ensuring the temperature regime of egg incubation.

Alanyl-tRNA synthetase 1 (AARS1) has been identified as an enzyme that can recognise L-lactate directly and function as a lactyltransferase. However, its potential regulatory role in hypoxia tolerance remains unclear. In this study, we generated aars1 mutants in zebrafish using the CRISPR/Cas9 technique. We found that homozygous loss of aars1 was lethal, whereas heterozygous loss significantly enhanced hypoxia tolerance. Further investigation revealed that AARS1 negatively regulates HIF1α-mediated hypoxia signaling by promoting HIF1α degradation. Loss of AARS1 enhanced HIF1α stability and hypoxia-responsive gene expression; however, AARS1 restoration reversed this effect. This regulation mainly depended on the autophagy-lysosome pathway, revealing a non-canonical role for AARS1 in modulating HIF1α stability and adaptation to hypoxia.

Sex differences in the neural mechanisms underlying energy balance are a fundamental aspect of avian integrative physiology, yet remain poorly understood. The 26RFamide/pyroglutamylated RFamide peptide (26RFa/QRFP) is a hypothalamic orexigenic neuropeptide whose precise role and regulation in songbirds are unknown due to the lack of species-specific analytical tools. To investigate the 26RFa/QRFP system in the zebra finch (Taeniopygia guttata), we developed and validated a novel, high-affinity rabbit antiserum directed against a specific fragment of zebra finch 26RFa/QRFP. The specificity of this reagent was rigorously confirmed through a competitive enzyme-linked immunosorbent assay and cross-validated by the neuroanatomical colocalization of 26RFa/QRFP-like immunoreactive cell bodies and mRNA transcripts in the ventromedial and lateral hypothalamic areas. Using this validated platform, we identified striking female-biased dimorphism in the zebra finch diencephalon, with approximately 3.9-fold higher mature peptide content and 1.5-fold higher mRNA expression in females than in males (P < 0.05). These findings suggest that zebra finches employ sex-specific neuroendocrine strategies for metabolic regulation.

Investigating effects of co-occurring biotic and abiotic stressors on freshwater fishes is crucial to understand how they are coping with environmental change. In this study, we used disturbance cue produced from conspecifics subjected to a simulated predator chase to examine impacts of predation risk on the thermal- and hypoxia tolerance of female Pseudocrenilabrus multicolor, a mouth-brooding African cichlid. Using a series of thermal and hypoxia tolerance trials, we tested 1) how acute exposure to disturbance cue impacts thermal tolerance, 2) how acute exposure to disturbance cue impacts hypoxia tolerance, and 3) how concurrent exposure to warming, hypoxia, and predation risk alters their response. Acute exposure to disturbance cue during normoxia thermal tolerance trials increased critical thermal maximum (CTMax) and decreased agitation temperature (TAg) compared to the water control. When fish were exposed to disturbance cue during hypoxia tolerance trials, the dissolved oxygen level at which they initiated aquatic surface respiration (ASR) was lower than when exposed to water. Finally, acute exposure to disturbance cue during thermal tolerance trials run under hypoxia did not impact CTMax and TAg but elicited a lower temperature at first ASR and increased use of ASR compared to when given the water control. Overall, this suggests that exposure to disturbance cue may increase both thermal and hypoxia tolerance of P. multicolor. However, when these stressors co-occur, exposure to disturbance cue no longer conferred them a thermal tolerance advantage, and the fish appeared more stressed as they relied on ASR more often and at lower temperatures. Therefore, predation risk may be perceived and reacted to differently by prey when multiple abiotic stressors are also present.

An animal's metabolism is a vital trait for understanding their lifestyle and behavior in their environment. Metabolism scales with mass with an exponent of approximately ¾ for whole organisms across a variety of taxa, but this value alone cannot contextualize whether the animal is being constrained by morphological features. For water-breathing ectotherms, the balance between metabolic demand and supply is vital due to the low concentration of oxygen in water compared to air. Here we calculated the size dependence of the ratio of metabolic rate to gill surface area (the S metric) for both standard (SMR) and maximum (MMR) metabolic rate for two crustaceans: the Caribbean king crab (Maguimithrax spinosissimus) and Caribbean spiny lobster (Panulirus argus). The S metric scaling exponents for SMR and MMR were 0.11 and 0.17, and - 0.003 and - 0.175 for P. argus and M. spinosissimus, respectively. All S metric scaling exponents were not significantly different from 0, indicating that the gills may not be limiting metabolic demand for either species, although these results alone cannot definitely determine oxygen limitation. Crabs had a more negative S metric exponent during MMR suggesting that as size increases, the balance of metabolic supply and demand could become more constrained with a larger mass. It is important to understand if metabolic demand is being constrained by respiratory surface area as it can better contextualize the behavioral mechanisms these animals use in their environment.

Ayu Plecoglossus altivelis, one of the most important inland fishery species in Japan, exhibits a semelparous annual life cycle. Although native populations are primarily amphidromous, landlocked ayu from Lake Biwa have been widely released into rivers nationwide. This landlocked population diverged from amphidromous ancestor approximately 100,000 years ago and exhibits distinctive morphological and physiological traits. In order to assess how translocation of landlocked ayu may affect native populations, we examined the mechanisms underlying the differences in seawater acclimation capacity between the amphidromous and landlocked forms, with particular focus on the effects of water temperature. Juvenile and larval ayu from both populations were reared in freshwater at 18 °C or 24 °C and subsequently transferred to seawater. In the juvenile stage, the landlocked form exhibited a markedly lower capacity for seawater acclimation (lower survival rate and higher plasma osmolality after seawater transfer) than the amphidromous form, primarily due to the delayed appearance of seawater-type ionocytes in the gills. While temperature had little effect on the amphidromous population, landlocked fish acclimated to seawater at 24 °C exhibited a tendency toward increased plasma osmolality and mortality. In embryos and larvae, both populations possessed seawater-type ionocytes prior to hatching, indicating inherent preparatory adaptation for seawater migration. However, following seawater transfer, landlocked larvae showed significantly reduced survival at high temperature, whereas amphidromous larvae were unaffected by temperature. These findings demonstrate that landlocked ayu retain fundamental mechanisms of seawater acclimation, but their salinity tolerance considerably decreases under elevated temperatures during the larval stage.