Scaling features in high-concentrations PM2.5 evolution: the Ignored factor affecting scarlet fever incidence.

As an acute respiratory disease, scarlet fever has great harm to public health. Some evidence indicates that the time distribution pattern of heavy PM2.5 pollution occurrence may have an impact on health risks. This study aims to reveal the relation between scaling features in high-concentrations PM2.5 (HC-PM2.5) evolution and scarlet fever incidence (SFI). Based on the data of Hong Kong from 2012 to 2019, fractal box-counting dimension (D) is introduced to capture the scaling features of HC-PM2.5. It has been found that index D can quantify the time distribution of HC-PM2.5, and lower D values indicate more cluster distribution of HC-PM2.5. Moreover, scale-invariance in HC-PM2.5 at different time scales has been discovered, which indicates that HC-PM2.5 occurrence is not random but follows a typical power-law distribution. Next, the exposure-response relationship between SFI and scale-invariance in HC-PM2.5 is explored by Distributed lag non-linear model, in conjunction with meteorological factors. It has been discovered that scale-invariance in HC-PM2.5 has a nonlinear effect on SFI. Low and moderate D values of HC-PM2.5 are identified as risk factors for SFI at small time-scale. Moreover, relative risk shows a decreasing trend with the increase of exposure time. These results suggest that exposure to short-term clustered HC-PM2.5 makes individual more prone to SFI than exposure to long-term uniform HC-PM2.5. This means that individuals in slightly-polluted regions may face a greater risk of SFI, once the PM2.5 concentration keeps rising. In the future, it is expected that the relative risk of scarlet fever for a specific region can be estimated based on the quantitative analysis of scaling features in high-concentrations PM2.5 evolution.

Integrated mRNA and miRNA analysis reveals the regulatory network of oxidative stress and inflammation in Coilia nasus brains during air exposure and salinity mitigation.

BACKGROUND: Air exposure is an inevitable source of stress that leads to significant mortality in Coilia nasus. Our previous research demonstrated that adding 10‰ NaCl to aquatic water could enhance survival rates, albeit the molecular mechanisms involved in air exposure and salinity mitigation remained unclear. Conversely, salinity mitigation resulted in decreased plasma glucose levels and improved antioxidative activity. To shed light on this phenomenon, we characterized the transcriptomic changes in the C. nasus brain upon air exposure and salinity mitigation by integrated miRNA-mRNA analysis. RESULTS: The plasma glucose level was elevated during air exposure, whereas it decreased during salinity mitigation. Antioxidant activity was suppressed during air exposure, but was enhanced during salinity mitigation. A total of 629 differentially expressed miRNAs (DEMs) and 791 differentially expressed genes (DEGs) were detected during air exposure, while 429 DEMs and 1016 DEGs were identified during salinity mitigation. GO analysis revealed that the target genes of DEMs and DEGs were enriched in biological process and cellular component during air exposure and salinity mitigation. KEGG analysis revealed that the target genes of DEMs and DEGs were enriched in metabolism. Integrated analysis showed that 24 and 36 predicted miRNA-mRNA regulatory pairs participating in regulating glucose metabolism, Ca2+ transport, inflammation, and oxidative stress. Interestingly, most of these miRNAs were novel miRNAs. CONCLUSION: In this study, substantial miRNA-mRNA regulation pairs were predicted via integrated analysis of small RNA sequencing and RNA-Seq. Based on predicted miRNA-mRNA regulation and potential function of DEGs, miRNA-mRNA regulatory network involved in glucose metabolism and Ca2+ transport, inflammation, and oxidative stress in C. nasus brain during air exposure and salinity mitigation. They regulated the increased/decreased plasma glucose and inhibited/promoted antioxidant activity during air exposure and salinity mitigation. Our findings would propose novel insights to the mechanisms underlying fish responses to air exposure and salinity mitigation.

Integration of metagenome and metabolome analysis reveals the correlation of gut microbiota, oxidative stress, and inflammation in Coilia nasus under air exposure stress and salinity mitigation.

Due to the strong response to air exposure, high mortality was occurred in Coilia nasus. Previous studies reported that 10 ‰ NaCl could significantly reduce mortality in C. nasus under air exposure. To investigate the mechanisms that 10 ‰ NaCl can alleviate stress, community structure and metabolism of the intestinal flora of C. nasus were detected via metagenome and metabolome. In this study, C. nasus were divided into control group (C), air exposure group without 10 ‰ NaCl (AE), and air exposure group with 10 ‰ NaCl (AES). After air exposure stress and salinity mitigation, the mortality, intestinal microorganisms, metabolites, and physiological biomarkers were analyzed. The results showed that the mortality rate of C. nasus was reduced after salinity reduction; the antioxidant capacity was elevated compared to the AE group; and anti-inflammatory capacity was increased in the AES group compared to the AE group. Metagenomic sequencing results showed that the levels of harmful bacteria (E. coli, Aeromonas) in the Candida nasus gut increased after air exposure; beneficial bacteria (Actinobacteria, Corynebacteria) in the C. nasus gut increased after salinity reduction. Metabolomics analyses showed that AE decreased the expression of beneficial metabolites and increased the expression of harmful metabolites; AES increased beneficial metabolites and decreased harmful metabolites. Correlation analysis showed that in the AE group, beneficial metabolites were negatively correlated with oxidative stress and inflammatory response, while harmful metabolites were positively correlated with oxidative stress and inflammatory response, and were associated with bacterial communities such as Gillisia, Alkalitalia, Avipoxvirus, etc.; the correlation of metabolites with oxidative stress and inflammatory response was opposite to that of AE in the case of AES, and was associated with Lentilactobacillus, Cyanobacterium, and other bacterial communities. Air exposure caused damage to Candida rhinoceros and 10 ‰ salinity was beneficial in alleviating C. nasus stress. These results will provide new insights into methods and mechanisms to mitigate stress in fish.

Cortisol enhances aerobic metabolism and locomotor performance during the transition to land in an amphibious fish.

Amphibious fishes on land encounter higher oxygen (O2) availability and novel energetic demands, which impacts metabolism. Previous work on the amphibious mangrove killifish (Kryptolebias marmoratus) has shown that cortisol becomes elevated in response to air exposure, suggesting a possible role in regulating metabolism as fish move into terrestrial environments. We tested the hypothesis that cortisol is the mechanism by which oxidative processes are upregulated during the transition to land in amphibious fishes. We used two groups of fish, treated fish (+metyrapone, a cortisol synthesis inhibitor) and control (-metyrapone), to determine the impact of cortisol during air exposure (0 and 1 h, 7 days) on O2 consumption, terrestrial locomotion, the phenotype of red skeletal muscle, and muscle lipid concentration. Metyrapone-treated fish had an attenuated elevation in O2 consumption rate during the water to air transition and an immediate reduction in terrestrial exercise performance relative to control fish. In contrast, we found no short- (0 h) or long-term (7 days) differences between treatments in the oxidative phenotype of red muscles, nor in muscle lipid concentrations. Our results suggest that cortisol stimulates the necessary increase in aerobic metabolism needed to fuel the physiological changes that amphibious fishes undergo during the acclimation to air, although further studies are required to determine specific mechanisms of cortisol regulation.

Chromosome-Level Genome Assembly of Protosalanx chinensis and Response to Air Exposure Stress.

Protosalanx chinensis is a suitable particular species for genetic studies on nearly scaleless skin, transparency and high sensitivity to hypoxia stress. Here, we generated a high-quality chromosome-level de novo assembly of P. chinensis. The final de novo assembly yielded 379.47 Mb with 28 pseudo-chromosomes and a scaffold N50 length of 14.52 Mb. In total, 21,074 protein-coding genes were predicted. P. chinensis, Esox lucius and Hypomesus transpacificus had formed a clade, which diverged about 115.5 million years ago. In the air exposure stress experiment, we found that some genes play an essential role during P. chinensis hypoxia, such as bhlh, Cry1, Clock, Arntl and Rorb in the circadian rhythm pathway. These genomic data offer a crucial foundation for P. chinensis ecology and adaptation studies, as well as a deeper understanding of the response to air exposure stress.

ROS-induced moderate autophagy of haemocytes confers resistance of Mercenaria mercenaria to air exposure stress.

Air exposure (AE) is a significant environmental stressor that can lead to desiccation, hypoxia, starvation, and disruption of cellular homeostasis in marine bivalves. Autophagy is a highly conserved catabolic pathway that facilitates the degradation of damaged macromolecules and organelles, thereby supporting cellular stress responses. To date, autophagy-mediated resistance mechanisms to AE stress remain largely elusive in bivalves. In this study, we performed a multi-tool approach to investigate the autophagy-related physiological regulation in hard clams (Mercenaria mercenaria) under different duration of AE (T = 0, 1, 5, 10, 20, 30 days). We observed that autophagy of haemocytes was significantly activated on day 5. However, autophagy activity began to significantly decline from day 10 to day 30. Autophagy was significantly inhibited after antioxidant treatment, indicating that reactive oxygen species (ROS) was an endogenous inducer of autophagy. A significant decline in the survival rate of hard clams was observed after injection of ammonium chloride or carbamazepine during AE stress, suggesting that moderate autophagy was conducive for clam survival under AE stress. We also observed DNA breaks and high levels of apoptosis in haemocytes on day 10. Activation of apoptosis lagged behind autophagy, and the relationship between autophagy and apoptosis might shift from antagonism to synergy with the duration of stress. This study provides novel insights into the stress resistance mechanisms in marine bivalves.

Comparison of the Efficiency of Different Eradication Treatments to Minimize the Impacts Caused by the Invasive Tunicate Styela plicata in Mussel Aquaculture.

In 2017, aquaculture producers of the Albufeira lagoon, Portugal, reported an invasion of tunicates that was disrupting mussel production, particularly the tunicate Styela plicata (Lesueur, 1823). A totally effective eradication method still does not exist, particularly for S. plicata, and the effects of the eradication treatments on bivalves' performance are also poorly understood. Our study examined the effectiveness of eradication treatments using three laboratory trials and five treatments (air exposure, freshwater immersion, sodium hypochlorite, hypersaline solution and acetic acid) for S. plicata, as well as their effects on survival and growth of blue mussel Mytilus edulis Linnaeus, 1758. While air exposure and freshwater immersion caused a 27% mortality rate in S. plicata, the acetic acid treatment was the most effective in eliminating this species (>90% mortality). However, a 33-40% mortality rate was registered in mussels. Both species were not affected by the hypersaline treatment in the last trial, but the sodium hypochlorite treatment led to a 57% mortality rate in mussels. Differences in mussels' growth rates were not detected. These trials represent a step forward in responding to the needs of aquaculture producers. However, further studies are needed to investigate the susceptibility of tunicates to treatments according to sexual maturation, as well as to ensure minimum mussel mortality in the most effective treatments, and to better understand the effects on mussel physiological performance in the long-term.

Transferability and reproducibility of exposed air-liquid interface co-culture lung models.

BACKGROUND: The establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels. RESULTS: Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production. CONCLUSION: The transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.

Emersion and recovery alter oxygen consumption, ammonia and urea excretion, and oxidative stress parameters, but not diffusive water exchange or transepithelial potential in the green crab (Carcinus maenas).

The green crab (Carcinus maenas) is an inshore species affected by intertidal zonation patterns, facing periods of emersion during low tide and submersion during high tide. During these periods of air and subsequent water exposure, these species can face physiological challenges. We examined changes in O2 consumption rate (MO2), and ammonia and urea excretion rates over sequential 14 h periods in seawater (32 ppt, control), in air and during recovery in seawater after air exposure (13°C throughout). At the end of each exposure, the anterior (5th) and posterior (8th) gills and the hepatopancreas were removed for measurements of oxidative stress parameters (TBARs and catalase in the gills and hepatopancreas, and protein carbonyls in the gills). MO2 remained unchanged during air exposure, but increased greatly (3.4-fold above control levels) during the recovery period. Ammonia and urea net fluxes were reduced by 98% during air exposure, but rebounded during recovery to >2-fold the control rates. Exchangeable water pools, rate constants of diffusive water exchange, unidirectional diffusive water flux rates (using tritiated water) and transepithelial potential were also measured during control and recovery treatments, but exhibited no significant changes. Damage to proteins was not observed in either gill. However, lipid damage occurred in the anterior (respiratory) gill after the air exposure but not in the posterior (ionoregulatory) gill or hepatopancreas. Catalase activity also decreased significantly in recovery relative to levels during air exposure in both the anterior gill and hepatopancreas, but not in the posterior gill. The crabs did not modify water metabolism or permeability. We conclude that MO2 was maintained but not enhanced during air exposure, while ammonia and urea-N excretion were impaired. As a result, all of these parameters increase greatly during re-immersion recovery, and oxidative stress also occurs. Clearly, emersion is not without physiological costs.

Copper exposure does not alter the ability of intertidal sea cucumber Cucumaria miniata to tolerate emersion during low tide.

Intertidal animals experience cycles of tidal emersion from water and are vulnerable to copper (Cu) exposure due to anthropogenic toxicant input into marine waters. Both emersion and Cu toxicity can cause damage to physiological processes like aerobic metabolism, ammonia excretion, and osmoregulation, but the interactions of the combination of these two stressors on marine invertebrates are understudied. Mixed effects of 96 h of low and high Cu exposure (20 and 200 µg/L) followed by 6 h of tidal emersion were evaluated on the intertidal sea cucumber Cucumaria miniata. The respiratory tree accumulated the highest concentrations of Cu, followed by the introvert retractor muscle, body wall, and coelomic fluid. Emersion affected accumulation of Cu, perhaps by inhibiting excretion. 200 µg/L of Cu increased lactate production in the respiratory tree, indicative of damaged aerobic metabolism. Cu diminished ammonia excretion, but emersion increased oxygen uptake and ammonia excretion upon re-immersion. The combination of the two stressors did not have any interactive effects on metabolism or ammonia excretion. Neither Cu exposure nor emersion altered ion (sodium, potassium, calcium, magnesium) content of the coelomic fluid. Overall, results of this study suggest that Cu exposure does not alter C. miniata's high tolerance to emersion, and some potential strategies that this species uses to overcome environmental stress are illuminated.

Effect of Air Exposure and Re-Submersion on the Histological Structure, Antioxidant Response, and Gene Expression of Procambarus Clarkii.

Air exposure is an important environmental stressor during the transportation and cultivation of Procambarus clarkii. We evaluated the effect of re-submersion for 24 h after dry transportation for 24 h on the histological structure, antioxidant activity, and gene expression of crayfish. The antioxidant parameters of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH), and the relative expression of CAT, SOD, HSP70, and ferritin genes were subsequently measured in the hepatopancreas and gills at both stages. Histopathology found that air exposure led to vacuolation of the hepatopancreas and disorderly arrangement of respiratory epithelial cells (REC) in the gills. The activities of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and lactic dehydrogenase (LDH) in the hepatopancreas and gills increased with short-term air exposure. The relative expression of genes (CAT, SOD, HSP70, and Ferritin) were induced after short-term air exposure. During re-submersion, MDA content and CAT and SOD activities in the hepatopancreas and gills were restored after 24 h, however, LDH activity and hepatopancreatic tissue damage were not repaired. Our results indicate that air exposure can cause oxidative damage to P. clarkii, and CAT and SOD can be used to determine the response of crayfish exposed to air, in addition to some damage that can be eliminated after re-submersion to a limited degree. This study provides foundational data that re-submersion can improve crayfish performance under hypoxic stress to a certain extent and will lead to the development of more effective transportation strategies and decrease economic losses in the future.

The effect of air exposure and re-water on gill microstructure and molecular regulation of Pacific white shrimp Penaeus vannamei.

The Penaeus vannamei is an important shrimp species with enormous commercial and ecological values. In production process, the air exposure resistance is vital for live transportation without water. We tested the air exposure resistant ability of P. vannamei, and carried out gill histological observation and gene expression analysis. The physiology and molecular response to the air exposure stress of P. vannamei was revealed. We found that body weight could affect the air exposure tolerance. Air exposure caused epithelial cell of gill filament shrinking and tissue fluid exudation within half of hour, and triggered oxidative stress response. After retrieved to water, epithelial cell shrinking and tissue fluid exudation recovered gradually, but oxidative and antioxidant response is still going on. Organisms reduced oxidative stress by regulating levels of antioxidants and antioxidant enzymes that remove reactive oxygen species (ROS) and RNA and DNA processing to repair tissue damage, and expression of apoptosis associated-genes altered. Furthermore, the survive shrimps could live steadily more than 5 days, and their gill filament recovered to normal state, proving that the damage of air exposure is reversible. These findings could be considered in the waterless live transportation of P. vannamei.

New insights into the regulation mechanism of red claw crayfish (Cherax quadricarinatus) hepatopancreas under air exposure using transcriptome analysis.

Red claw crayfish (Cherax quadricarinatus) is an important freshwater shrimp species worldwide with enormous economic value. Waterless transportation is an inherent feature of red claw crayfish transportation. However, the high mortality of red claw crayfish is a severe problem in the aquaculture of crayfish after waterless transportation. In this study, we investigated the responses of the hepatopancreas from the red claw crayfish undergoing air exposure stress and normal conditions on transcriptome levels. We used Illumina-based RNA sequencing (RNA-Seq) to perform a transcriptome analysis from the hepatopancreas of red claw crayfish challenged by air exposure. An average of 57,148,800 clean reads per library was obtained, and 33,567 unigenes could be predicted and classified according to their homology with matches in the National Center for Biotechnology Information (NCBI) non-redundant protein sequences (Nr), Gene Ontology (GO), a manually annotated and reviewed protein sequence database (Swiss-Prot), protein families (Pfam), Clusters of Orthologous Groups (COG) of proteins, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. 690 and 3407 differentially expressed genes (DEGs) were identified between the two stress stages of the red claw crayfish. More DEGs were identified in 12 h, indicating that gene expressions were largely changed at 12 h. Some immune-related pathways and genes were identified according to KEGG and GO enrichment analysis. A total of 12 DEGs involved in immune response and trehalose mechanism were verified by quantitative real-time-polymerase chain reaction (qRT-PCR). The results indicated that the red claw crayfish might counteract the stress of air exposure at the transcriptomic level by increasing expression levels of antioxidant-, immune-, and trehalose metabolism-related genes. These transcriptome results from the hepatopancreas provide significant insights into the influence mechanism of air exposure to the trehalose mechanism and immune response in the red claw crayfish.

Metabolic adaptation of the clam Ruditapes philippinarum during air exposure and the positive effects of sodium nitroprusside pretreatment.

The Manila clam (Ruditapes philippinarum), as one of the shellfish living in the intertidal zone, is known for its strong ability to withstand air exposure. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been shown to be useful for antioxidant and immune regulation in aquatic animals. In this study, an untargeted metabolomics (LC-MS/MS) technique was employed for the first time in Manila clam to analyze the metabolic and histological impacts after air exposure and the positive effects of SNP pretreatment. During air exposure, a significant increase in taurine, L-glutamate, and several polyunsaturated fatty acids in clams was detected, which indicates that clams may experience inflammatory reactions, oxidative stress, and an increase in blood ammonia content. When clams were exposed to SNP for 6 h, arginine, spermine, L-glutamic acid, and glutathione content were all upregulated, indicating that the SNP exposure induced NO production and improved antioxidant capacity in clams. When the clams were exposed to air after SNP pretreatment, there were no significant differences in the levels of taurine, L-glutamate, or aliphatic acids between the experimental and control groups. Gill tissue was more severely damaged in clams directly exposed to air than in those that experienced air exposure after SNP pretreatment, especially in clams exposed to air for a long time (72 h). Both metabolomics and tissue section structure indicated that SNP pretreatment decreased the stress responses caused by air exposure in R. philippinarum. These findings provided fresh insights and a theoretical foundation for understanding the tolerance to air exposure and physiological functions of SNP (or NO) in R. philippinarum.

Identifying thresholds in air exposure, water temperature and fish size that determine reflex impairment in brook trout exposed to catch-and-release angling.

Understanding the factors that contribute to fish impairment and survival from angling events is essential to guide best angling practices for catch-and-release (C&R) recreational fisheries. Complex interactions often exist between angler behaviour, environmental conditions, and fish characteristics that ultimately determine biological outcomes for fish. Yet, few studies focus on identifying biologically relevant thresholds. We therefore examined the effects of water temperature, air exposure and fish size on reflex impairment and mortality in brook trout Salvelinus fontinalis exposed to experimental and simulated angling stressors (n = 337). Using conditional inference trees, we identified interactions among these factors as well as threshold values within them that determine brook trout reflex impairment as an indicator of whole animal stress. Specifically, longer air exposure times (>30 sec) and warmer temperatures (>19.5°C) had a synergistic effect leading to higher reflex impairment scores. Further, larger fish (>328 mm) were more sensitive to air exposure durations >10 sec. Of the reflex impairment measures, loss of equilibrium and time to regain equilibrium were strongly and moderately associated with brook trout mortality (18-24 h monitoring), although mortality rates were generally low (6%). These findings support previous research that has established strong links between these reflex impairment measures and fish health outcomes in other species. They also highlight the important interactions among air exposure duration, water temperature and fish size that determine impairment in brook trout, providing specific thresholds to guide best angling practices for C&R fisheries. This approach may be widely applicable to generate similar thresholds that can be encouraged by regulators and adopted by anglers for other common C&R fishes.

Human cold habituation: Physiology, timeline, and modifiers.

Habituation is an adaptation seen in many organisms, defined by a reduction in the response to repeated stimuli. Evolutionarily, habituation is thought to benefit the organism by allowing conservation of metabolic resources otherwise spent on sub-lethal provocations including repeated cold exposure. Hypermetabolic and/or insulative adaptations may occur after prolonged and severe cold exposures, resulting in enhanced cold defense mechanisms such as increased thermogenesis and peripheral vasoconstriction, respectively. Habituation occurs prior to these adaptations in response to short duration mild cold exposures, and, perhaps counterintuitively, elicits a reduction in cold defense mechanisms demonstrated through higher skin temperatures, attenuated shivering, and reduced cold sensations. These habituated responses likely serve to preserve peripheral tissue temperature and conserve energy during non-life threatening cold stress. The purpose of this review is to define habituation in general terms, present evidence for the response in non-human species, and provide an up-to-date, critical examination of past studies and the potential physiological mechanisms underlying human cold habituation. Our aim is to stimulate interest in this area of study and promote further experiments to understand this physiological adaptation.

Skin ionocyte density of amphibious killifishes is shaped by phenotypic plasticity and constitutive interspecific differences.

When amphibious fishes are on land, gill function is reduced or eliminated and the skin is hypothesized to act as a surrogate site of ionoregulation. Skin ionocytes are present in many fishes, particularly those with amphibious life histories. We used nine closely related killifishes spanning a range of amphibiousness to first test the hypothesis that amphibious killifishes have evolved constitutively increased skin ionocyte density to promote ionoregulation on land. We found that skin ionocyte densities were constitutively higher in five of seven amphibious species examined relative to exclusively water-breathing species when fish were prevented from leaving water, strongly supporting our hypothesis. Next, to examine the scope for plasticity, we tested the hypothesis that skin ionocyte density in amphibious fishes would respond plastically to air-exposure to promote ionoregulation in terrestrial environments. We found that air-exposure induced plasticity in skin ionocyte density only in the two species classified as highly amphibious, but not in moderately amphibious species. Specifically, skin ionocyte density significantly increased in Anablepsoides hartii (168%) and Kryptolebias marmoratus (37%) following a continuous air-exposure, and only in K. marmoratus (43%) following fluctuating air-exposure. Collectively, our data suggest that highly amphibious killifishes have evolved both increased skin ionocyte density as well as skin that is more responsive to air-exposure compared to exclusively water-breathing and less amphibious species. Our findings are consistent with the idea that gaining the capacity for cutaneous ionoregulation is a key evolutionary step that enables amphibious fishes to survive on land.

An accidental discovery of mannan-oligosaccharide's protection effect against air exposure and its potential mechanism in hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀).

The original objective was to explore the potential benefiting effects of three prebiotics in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Therefore, three experimental diets (basal diet + 1% fructooligosaccharide, Diet F; basal diet + 1% inulin, Diet I; basal diet + 0.3% mannan-oligosaccharide, Diet M) and one basal diet (Diet C) were prepared and a feeding trial was conducted. However, at the end of the fourth week into the feeding experiment, a water-leaking accident occurred and fishes of all groups went through an unexpected air exposure event. Surprisingly, different prebiotic-supplemented groups showed significantly different air exposure tolerance: the mortality of M group was significantly lower (P ≤ 0.05) than all the other groups. Examination of antioxidant, non-specific immunity, and stress parameters revealed that comparing to control group, M group showed significantly increased catalase (CAT), acid phosphatase (ACP), and alkaline phosphatase (AKP) activities, decreased superoxide dismutase (SOD) activity, and similar cortisol level (P ≤ 0.05). Real-time PCR experiment revealed that M group significantly increased the expression of CAT, glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD) genes in head kidney (P ≤ 0.05). Overall, M exhibited the best anti-air exposure/antioxidative stress effects among the three prebiotics and could be considered a promising feed additive to relieve air exposure/oxidative stress in hybrid grouper culture.

Histological, physiological, and transcriptomic responses of hepatopancreas to air exposure in asian freshwater clam Corbicula fluminea.

Corbicula fluminea (C. fluminea) is an important freshwater economy shellfish in China, but it often suffers from air exposure during transportation. In this study, we investigated the histological, physiological (mainly including respiratory metabolism, antioxidant capacity, and immune function), and transcriptomic responses of hepatopancreas in C. fluminea to different times of air exposure. At histological level, air exposure caused vacuolation of digestive cells (24-96 h) and enlargement of digestive tubule lumen (6-96 h) in hepatopancreas. At physiological level, the activities of enzymes related to glycolysis (hexokinase and pyruvate kinase) and anaerobic respiration (lactate dehydrogenase) were increased first (6-24 h) of air exposure, then came back to normal level or even decreased. The activity of aerobic respiration-related enzyme (succinic dehydrogenase) began to reduce from 24 h of air exposure. The activities of antioxidant enzymes (superoxide dismutase and catalase) were enhanced during 6-48 h of air exposure and then returned to control level or even inhibited. The content of malondialdehyde (MDA) increased from 96 h of air exposure. The activities of immune-related enzymes (acid phosphatase and alkaline phosphatase) increased during 6-48 h, then returned to normal or began to decline. At transcriptome level, 44 differentially expressed genes (DEGs) in the hepatopancreas were identified after 96-h air exposure. Among these DEGs, 8 were associated with glycolysis, TCA cycle, immune, and antioxidant, and were downregulated after 96-h air exposure. Taken together, these findings illuminated the response of C. fluminea to air exposure at histological, physiological, and transcriptomic levels, which will be beneficial to the aquaculture and transportation of C. fluminea.

Effects of air exposure stress on crustaceans: Histopathological changes, antioxidant and immunity of the red swamp crayfish Procambarus clarkii.

Air exposure stress may result in oxidative damage and ultimately disease or death in crustaceans. Using the Procambarus clarkia, one of the main commercial aquaculture species in China, as a study model, the molecular mechanism including histopathological changes, antioxidant capacity and immunity response under the air exposure stress were firstly evaluated. Results showed that the surfaces of gill were wrinkled while the morphologies of the nuclei and mitochondria in the hepatopancreas were altered after 48 h of air exposure stress, and the damage of mitochondria was more serious after additional bacterial infection. Moreover, the activity of antioxidant enzymes increased at first and then decreased along with increasement of air exposure time. The concentration of malondialdehyde (MDA) in hepatopancreas was significantly increased under the air exposure stress, while the bacterial infection further aggravated such oxidative damage. The transcriptome analysis exhibited that the stress- and immunity-related genes in hepatopancreas altered when response to the air exposure stress. This study could help uncover the mechanisms of aerial exposure stress responses in Procambarus clarkii.