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2023, 44(2):58-67.
DOI: 10.19663/j.issn2095-9869.20210930003
Abstract:
Environmental conditions regulate the growth and reproduction of fish. The increase in sea temperature during winter may have adverse effects on Takifugu rubripes. To study the mechanism of low-temperature tolerance of T. rubripes, the expression of antifreeze protein (AFP) gene, cold-induced RNA binding protein (CIRP) gene, high mobility group protein box-1 (HMGB1) gene, and Y-box binding protein (YB-1) gene in the liver, spleen, kidney, brain, heart, intestine, muscle, gonad, and skin tissues of T. rubripes obtained from different temperatures (18℃, 13℃, 8℃, and 5℃) was analyzed by quantitative real-time PCR. The results showed that the AFP gene was widely expressed in tissues, with the highest expression in the muscle (P<0.05). With the decrease in temperature, the expression of the AFP gene in each tissue showed a significant increasing trend, reaching the highest value in the 5℃ group. The expression of the CIRP gene was the highest in the muscle (P<0.05). With a decrease in temperature, the trend of CIRP gene expression in various tissues was different. The CIRP gene expression levels of liver, kidney, brain, heart, intestine, and skin showed a trend of initial increase, followed by a decrease, and then an increase. The expression levels in the spleen, muscle, and gonads showed an upward trend, reaching the highest value in the 5℃ group. The expression of the HMGB1 gene was the highest in muscle (P<0.05), followed by that in the brain, liver, heart and skin. As the temperature decreased, the expression of the HMGB1 gene in all tissues except the liver increased first and then decreased, and reached the maximum value in the 8℃ group, which was significantly higher than that of the other groups (P<0.05). The expression of the YB-1 gene was the highest in the muscle (P<0.05), with the lowest expression level in other tissues. As the temperature decreased, the expression level of most tissues (brain, heart, intestine, kidney, liver, muscle, and spleen) increased first, then decreased, and then increased, reaching the minimum value in the 8℃ group (P<0.05). These results show that the expression levels of the four genes are different at different temperature, reflecting the functional specificity of these four genes. Under low-temperature stress, these genes responded positively. Their expression changed to varying degrees, suggesting that the four genes may have potentially important roles in the adaptation of T. rubripes to low temperatures. In addition, by analyzing the law of gene expression, 8℃ may be the key regulatory point for T. rubripes to deal with low-temperature stress. Too low temperature may cause its regulation disorder. The results of this study can provide a relevant basis for studying the regulation mechanism of the low-temperature response of T. rubripes.
Environmental conditions regulate the growth and reproduction of fish. The increase in sea temperature during winter may have adverse effects on Takifugu rubripes. To study the mechanism of low-temperature tolerance of T. rubripes, the expression of antifreeze protein (AFP) gene, cold-induced RNA binding protein (CIRP) gene, high mobility group protein box-1 (HMGB1) gene, and Y-box binding protein (YB-1) gene in the liver, spleen, kidney, brain, heart, intestine, muscle, gonad, and skin tissues of T. rubripes obtained from different temperatures (18℃, 13℃, 8℃, and 5℃) was analyzed by quantitative real-time PCR. The results showed that the AFP gene was widely expressed in tissues, with the highest expression in the muscle (P<0.05). With the decrease in temperature, the expression of the AFP gene in each tissue showed a significant increasing trend, reaching the highest value in the 5℃ group. The expression of the CIRP gene was the highest in the muscle (P<0.05). With a decrease in temperature, the trend of CIRP gene expression in various tissues was different. The CIRP gene expression levels of liver, kidney, brain, heart, intestine, and skin showed a trend of initial increase, followed by a decrease, and then an increase. The expression levels in the spleen, muscle, and gonads showed an upward trend, reaching the highest value in the 5℃ group. The expression of the HMGB1 gene was the highest in muscle (P<0.05), followed by that in the brain, liver, heart and skin. As the temperature decreased, the expression of the HMGB1 gene in all tissues except the liver increased first and then decreased, and reached the maximum value in the 8℃ group, which was significantly higher than that of the other groups (P<0.05). The expression of the YB-1 gene was the highest in the muscle (P<0.05), with the lowest expression level in other tissues. As the temperature decreased, the expression level of most tissues (brain, heart, intestine, kidney, liver, muscle, and spleen) increased first, then decreased, and then increased, reaching the minimum value in the 8℃ group (P<0.05). These results show that the expression levels of the four genes are different at different temperature, reflecting the functional specificity of these four genes. Under low-temperature stress, these genes responded positively. Their expression changed to varying degrees, suggesting that the four genes may have potentially important roles in the adaptation of T. rubripes to low temperatures. In addition, by analyzing the law of gene expression, 8℃ may be the key regulatory point for T. rubripes to deal with low-temperature stress. Too low temperature may cause its regulation disorder. The results of this study can provide a relevant basis for studying the regulation mechanism of the low-temperature response of T. rubripes.
ZHU Liguang
,
LIU Zhifeng
,
MA Aijun
,
WANG Xin´an
,
SUN Zhibin
,
CHANG Haowen
,
LIU Shengcong
,
BAO Yulong
,
MA Deyou
2023, 44(6):74-82.
DOI: 10.19663/j.issn2095-9869.20220608001
Abstract:
Takifugu rubripes are warm temperate fish, suggesting that the reduced seawater temperatures in winter are likely to have a substantial impact on their survival. Considering this, there is likely to be some industrial value in breeding extremely low-temperature tolerant varieties of this fish. Here, we evaluate the expression changes in six major QTL candidate genes (tacc2, fsip1, exoc4, arhgap44a, pde10a, and unc5b) in response to reduced temperature in an effort to understand cold tolerance in T. rubripes. The expression changes of these six genes in the liver, heart, and kidney were detected using real-time quantitative PCR. This study used three groups of 8-month-old fish, all from the same family established by our research group, exposed to three different temperature gradients, where 8 ℃ and 13 ℃ acted as the minimum in the low temperature groups and 18 ℃ acted as the minimum in the control group. Our results showed that all six genes were expressed at different levels across each of these three tissues at different temperatures. The relative expression of pde10a first increased and then decreased in all three tissues, whereas the relative expression of tacc2 and exoc4 were distinctly different in the liver, kidney, and heart at 8 ℃. In this case, these transcripts first decreased and then stabilized in the liver, increased and then stabilized in the kidneys, and increased and then decreased in the heart. The relative expression of unc5b was low in the liver and heart, but high in the kidney following a second week of low-temperature growth, whereas arhgap44a expression was slightly upregulated in the liver and stable in the kidney and heart. fsip1 expression demonstrated a downward trend in the liver but seemed to first increase and then decrease in the heart and kidney. Taken together, these results demonstrate that all six of these genes are differentially expressed in different tissues of T. rubripes, with these differences exhibiting dynamic changes with respect to tissue origin and temperature. In addition, this data clearly revealed a positive correlation between cold stress and the expression of these QTL candidate genes. Thus, we can conclude that these six QTL candidate genes may play a substantial role in the low temperature adaptation of T. rubripes. This is significant because although low temperature is known to be an important factor limiting the development of the industrial utility of T. rubripes, there are still relatively few reports describing their cold stress response. This study provides a theoretical basis for the study of signaling pathways related to the low temperature tolerance response of T. rubripes and the development of low temperature tolerant varieties.
Takifugu rubripes are warm temperate fish, suggesting that the reduced seawater temperatures in winter are likely to have a substantial impact on their survival. Considering this, there is likely to be some industrial value in breeding extremely low-temperature tolerant varieties of this fish. Here, we evaluate the expression changes in six major QTL candidate genes (tacc2, fsip1, exoc4, arhgap44a, pde10a, and unc5b) in response to reduced temperature in an effort to understand cold tolerance in T. rubripes. The expression changes of these six genes in the liver, heart, and kidney were detected using real-time quantitative PCR. This study used three groups of 8-month-old fish, all from the same family established by our research group, exposed to three different temperature gradients, where 8 ℃ and 13 ℃ acted as the minimum in the low temperature groups and 18 ℃ acted as the minimum in the control group. Our results showed that all six genes were expressed at different levels across each of these three tissues at different temperatures. The relative expression of pde10a first increased and then decreased in all three tissues, whereas the relative expression of tacc2 and exoc4 were distinctly different in the liver, kidney, and heart at 8 ℃. In this case, these transcripts first decreased and then stabilized in the liver, increased and then stabilized in the kidneys, and increased and then decreased in the heart. The relative expression of unc5b was low in the liver and heart, but high in the kidney following a second week of low-temperature growth, whereas arhgap44a expression was slightly upregulated in the liver and stable in the kidney and heart. fsip1 expression demonstrated a downward trend in the liver but seemed to first increase and then decrease in the heart and kidney. Taken together, these results demonstrate that all six of these genes are differentially expressed in different tissues of T. rubripes, with these differences exhibiting dynamic changes with respect to tissue origin and temperature. In addition, this data clearly revealed a positive correlation between cold stress and the expression of these QTL candidate genes. Thus, we can conclude that these six QTL candidate genes may play a substantial role in the low temperature adaptation of T. rubripes. This is significant because although low temperature is known to be an important factor limiting the development of the industrial utility of T. rubripes, there are still relatively few reports describing their cold stress response. This study provides a theoretical basis for the study of signaling pathways related to the low temperature tolerance response of T. rubripes and the development of low temperature tolerant varieties.
2023, 44(6):97-106.
DOI: 10.19663/j.issn2095-9869.20230418001
Abstract:
Variation in fish temperature is determined by changes in the external aquatic environment, and temperature serves as a crucial regulatory factor for the behavior, physiology, and molecular processes of fish. A rapid decline in water temperature can elicit a strong stress response in fish, potentially inhibiting growth and even leading to mortality. Low temperature stress significantly impacts various life processes of organisms, including growth and development, energy metabolism, and reproductive development. In recent years, there has been increasing research on the effects of low-temperature stress on organisms, with a greater focus at the molecular level. Low temperatures induce a decrease in cellular activity and even growth arrest, resulting in apoptosis. Apoptosis is a form of programmed cell death ubiquitous in the biological world, and it plays a vital role in maintaining the normal physiological function of an organism. Once apoptosis occurs, it is irreversible and cannot be stopped. Temperature regulation is crucial for fish, and current knowledge of the role of leptin in fish primarily focuses on the regulation of feeding, lipid energy metabolism, and reproduction, with limited reports on its role in temperature regulation. Leptin, a protein hormone produced by adipocytes, has long been recognized as a product of the obese gene, which regulates organismal metabolism, neuroendocrine function, and other physiological processes. In animals, it exerts diverse physiological functions related to cell growth, proliferation, apoptosis, and metabolism. The homology of the leptin gene in fish is relatively low compared to that in mammals, and multiple leptin proteins encoded by multiple leptin genes can be produced due to genomic duplication events. Regarding the role of leptin in temperature regulation, more research has been conducted on mammals, mainly related to the regulation of energy metabolism and the influence on temperature adaptation evolution. There has been limited research on the regulatory mechanism of leptin in fish during cold stress. Dissostichus mawsoni belongs to the group of Antarctic vertebrates and has lived in the cold and isolated environment of the Southern Ocean at a temperature of –1.9 ℃ for ~30 million years. It has adapted to the extremely low temperatures of the surrounding Antarctic waters and is an excellent material and living fossil for studying temperature adaptation mechanisms in extreme environments. Compared with other fish species at the same depth, it has a large amount of lipid deposition in its subcutaneous and muscle tissues, mainly triglycerides (TGs). Predictions based on 3D structural models suggest that the partial absence of the lepB structure in D. mawsoni leads to only three α-helices, and lepA has four α-helices and a short and twisted E-helix as well as several irregular turns, forming a hollow barrel-like structure, which differs from the protein structure of all other known leptins. Moreover, previous studies have found a close correlation between lepA and temperature evolution, leading to speculation that Antarctic fish lepB may play an essential role in low-temperature adaptation. In this study, a eukaryotic expression vector of the Antarctic toothfish lepb gene was constructed and transfected into ZFL cells to establish a model of overexpression of the D. mawsoni lepb (DMLB) gene in the ZFL cells. After conducting a cell culture temperature experiment, 10 ℃ was selected as the significant difference temperature. Following 2 weeks of cold stimulation, the DMLB experimental group cells remained viable, whereas the control group cells died. The results indicated that the overexpression of the DMLB gene provided strong resistance to low-temperature conditions. By detecting changes in cell proliferation, apoptosis, reactive oxygen species (ROS) level, and ATP content under low-temperature stress, it was discovered that DMLB maintained cell growth and proliferation, reduced ROS production, and inhibited cell apoptosis. DMLB effectively maintained ATP levels in cells under low-temperature stress, which helps maintain the mitochondrial status and reduce the effects of apoptosis and necrosis caused by low-temperature stress, thereby protecting cells from cold stress. Additionally, the results of Oil red O staining and TG detection suggested that the DMLB gene may slow down the depletion of cell lipids under low-temperature stress, perhaps by lowering lipid metabolism to preserve lipids to cope with low-temperature damage. Consequently, the DMLB gene may be functional in the cold resistance of D. mawsoni by protecting cells from damage at low temperatures, but its specific molecular regulatory mechanism needs to be further explored. In this study, 10 ℃ was used as the key temperature, providing a basis for understanding the regulation of energy homeostasis by the lepb gene in D. mawsoni under low-temperature stress. This study explored the role of the lepB gene in the adaptation of D. mawsoni to extremely low-temperature environments and provided fundamental data for further study of the evolution of similar species in the Antarctic region. Furthermore, this study enriched the basic data related to low-temperature tolerance and provided reference materials for further investigation of the low-temperature tolerance mechanism of leptin in Notothenioids, as well as laid a foundation for further study of the mechanism of leptin in temperature regulation.
Variation in fish temperature is determined by changes in the external aquatic environment, and temperature serves as a crucial regulatory factor for the behavior, physiology, and molecular processes of fish. A rapid decline in water temperature can elicit a strong stress response in fish, potentially inhibiting growth and even leading to mortality. Low temperature stress significantly impacts various life processes of organisms, including growth and development, energy metabolism, and reproductive development. In recent years, there has been increasing research on the effects of low-temperature stress on organisms, with a greater focus at the molecular level. Low temperatures induce a decrease in cellular activity and even growth arrest, resulting in apoptosis. Apoptosis is a form of programmed cell death ubiquitous in the biological world, and it plays a vital role in maintaining the normal physiological function of an organism. Once apoptosis occurs, it is irreversible and cannot be stopped. Temperature regulation is crucial for fish, and current knowledge of the role of leptin in fish primarily focuses on the regulation of feeding, lipid energy metabolism, and reproduction, with limited reports on its role in temperature regulation. Leptin, a protein hormone produced by adipocytes, has long been recognized as a product of the obese gene, which regulates organismal metabolism, neuroendocrine function, and other physiological processes. In animals, it exerts diverse physiological functions related to cell growth, proliferation, apoptosis, and metabolism. The homology of the leptin gene in fish is relatively low compared to that in mammals, and multiple leptin proteins encoded by multiple leptin genes can be produced due to genomic duplication events. Regarding the role of leptin in temperature regulation, more research has been conducted on mammals, mainly related to the regulation of energy metabolism and the influence on temperature adaptation evolution. There has been limited research on the regulatory mechanism of leptin in fish during cold stress. Dissostichus mawsoni belongs to the group of Antarctic vertebrates and has lived in the cold and isolated environment of the Southern Ocean at a temperature of –1.9 ℃ for ~30 million years. It has adapted to the extremely low temperatures of the surrounding Antarctic waters and is an excellent material and living fossil for studying temperature adaptation mechanisms in extreme environments. Compared with other fish species at the same depth, it has a large amount of lipid deposition in its subcutaneous and muscle tissues, mainly triglycerides (TGs). Predictions based on 3D structural models suggest that the partial absence of the lepB structure in D. mawsoni leads to only three α-helices, and lepA has four α-helices and a short and twisted E-helix as well as several irregular turns, forming a hollow barrel-like structure, which differs from the protein structure of all other known leptins. Moreover, previous studies have found a close correlation between lepA and temperature evolution, leading to speculation that Antarctic fish lepB may play an essential role in low-temperature adaptation. In this study, a eukaryotic expression vector of the Antarctic toothfish lepb gene was constructed and transfected into ZFL cells to establish a model of overexpression of the D. mawsoni lepb (DMLB) gene in the ZFL cells. After conducting a cell culture temperature experiment, 10 ℃ was selected as the significant difference temperature. Following 2 weeks of cold stimulation, the DMLB experimental group cells remained viable, whereas the control group cells died. The results indicated that the overexpression of the DMLB gene provided strong resistance to low-temperature conditions. By detecting changes in cell proliferation, apoptosis, reactive oxygen species (ROS) level, and ATP content under low-temperature stress, it was discovered that DMLB maintained cell growth and proliferation, reduced ROS production, and inhibited cell apoptosis. DMLB effectively maintained ATP levels in cells under low-temperature stress, which helps maintain the mitochondrial status and reduce the effects of apoptosis and necrosis caused by low-temperature stress, thereby protecting cells from cold stress. Additionally, the results of Oil red O staining and TG detection suggested that the DMLB gene may slow down the depletion of cell lipids under low-temperature stress, perhaps by lowering lipid metabolism to preserve lipids to cope with low-temperature damage. Consequently, the DMLB gene may be functional in the cold resistance of D. mawsoni by protecting cells from damage at low temperatures, but its specific molecular regulatory mechanism needs to be further explored. In this study, 10 ℃ was used as the key temperature, providing a basis for understanding the regulation of energy homeostasis by the lepb gene in D. mawsoni under low-temperature stress. This study explored the role of the lepB gene in the adaptation of D. mawsoni to extremely low-temperature environments and provided fundamental data for further study of the evolution of similar species in the Antarctic region. Furthermore, this study enriched the basic data related to low-temperature tolerance and provided reference materials for further investigation of the low-temperature tolerance mechanism of leptin in Notothenioids, as well as laid a foundation for further study of the mechanism of leptin in temperature regulation.
2023, 44(6):155-165.
DOI: 10.19663/j.issn2095-9869.20220713001
Abstract:
The Ras-related proteins in brain (Rab) subfamily is the largest group of small molecule guanine nucleotide-binding regulatory proteins (GTP-binding proteins). These proteins are widely distributed across various eukaryotes and consist of approximately 200 amino acids, producing a molecular weight of approximately 20~25 kDa. Rab is known to be an important regulator in the transport of membrane vesicles and is embedded in almost all membrane-related proteins. Rab is primarily regulated through GTP binding and hydrolysis within the organelles and is known to perform several important functions across various stages of vesicle transport. Current research on Rab proteins produced by aquatic animals has revealed that the Rab gene plays an important role in immune response. The Pearl oyster, Pinctada fucata martensii, is the most common species used in the cultivation of seawater pearls, making them economically important. These oysters are primarily distributed along the coastal areas of Guangdong, Guangxi, and Hainan provinces in China. However, they display weak tolerance to low temperatures, which severely limits their cultivation area and the overall production of seawater pearls. Given the desire to expand their cultivation northward, we designed this study to help create low-temperature resistant P. f. martensii breeding lines F3 (R). We used genome resequencing technology to compare and analyze the R and Beibu Gulf wildtype populations (W), and identified a group of candidate genes, including the Rab gene, that were strongly positively selected during the breeding process. In addition, the full-length sequence of Rab7 of P. f. martensii (Pm-Rab7) was cloned using RACE technology, and the expression levels of Pm-Rab7 in adductor muscle, gill, gonad, hepatopancreas, foot, and mantle, and their expression patterns under temperature stress (17 ℃ in the low temperature group, 22 ℃ in the control group, and 32 ℃ in the high temperature group) were detected using bioinformatics analysis. Furthermore, single nucleotide polymorphisms (SNPs) in the exon regions of Pm-Rab7 in the R and W were screened, and this information was used to determine the genetic polymorphism, haplotype, and frequency rates for each mutation. Sequence analysis also revealed that the Pm-Rab7 gene displayed a full length of 1 153 bp, with the 5' and 3' UTR adding 30 bp and 505 bp, respectively. This gene was also shown to encode a single open reading frame (ORF) of 618 bp and 205 amino acids, producing a theoretical protein of 23.04 kDa with the isoelectric point at 5.40. This was later confirmed using the cloned Pm-Rab7 gene, which produced 5 conserved amino acid sequences, including a RAB conserved domain. Similarity evaluations revealed a high degree of overlap with C. gigas (92.79%), whereas phylogenetic tree construction revealed that the Rab7 gene produced a tree with three branches, one each for protostomes, echinoderms, and vertebrates. Closer inspection of the protostomes branch revealed that Pm-Rab7 first clusters with other mollusks, and then with arthropods to produce a large clade, suggesting that this protein is relatively well conserved during the evolutionary process. Pm-Rab7 was also shown to be expressed in all the tested tissues, with the most Pm-Rab7 expression recorded in the gonad and gill (P<0.05). Time-course results from the gill tissues of temperature stressed samples revealed that Pm-Rab7 expression first increased and then decreased in response to low temperature (17 ℃) exposure, with all time points showing a significant increase in Pm-Rab7 expression when compared to that in the 22 ℃ control from 6 h to 3 d (P<0.05). In addition, we noted an expression peak at 1 d. The expression of Pm-Rab7 was generally stable in response to growth at 32 ℃ (high temperature group), but was also significantly increased when compared to that in the control group following 12 h of exposure (P<0.05). This suggests that Pm-Rab7 is most likely linked to the low temperature response process in these shellfish. We also identified a total of seven SNPs in the exon region of Pm-Rab7, three (g.112712470, g.112712503, and g.11271477) of which demonstrated significant differences in occurrence rate between the R and W populations (P<0.05). Genetic evaluations of all seven SNPs revealed that three could be classified as low polymorphism loci (PIC<0.25) and four could be classified as moderate polymorphism loci (0.25
The Ras-related proteins in brain (Rab) subfamily is the largest group of small molecule guanine nucleotide-binding regulatory proteins (GTP-binding proteins). These proteins are widely distributed across various eukaryotes and consist of approximately 200 amino acids, producing a molecular weight of approximately 20~25 kDa. Rab is known to be an important regulator in the transport of membrane vesicles and is embedded in almost all membrane-related proteins. Rab is primarily regulated through GTP binding and hydrolysis within the organelles and is known to perform several important functions across various stages of vesicle transport. Current research on Rab proteins produced by aquatic animals has revealed that the Rab gene plays an important role in immune response. The Pearl oyster, Pinctada fucata martensii, is the most common species used in the cultivation of seawater pearls, making them economically important. These oysters are primarily distributed along the coastal areas of Guangdong, Guangxi, and Hainan provinces in China. However, they display weak tolerance to low temperatures, which severely limits their cultivation area and the overall production of seawater pearls. Given the desire to expand their cultivation northward, we designed this study to help create low-temperature resistant P. f. martensii breeding lines F3 (R). We used genome resequencing technology to compare and analyze the R and Beibu Gulf wildtype populations (W), and identified a group of candidate genes, including the Rab gene, that were strongly positively selected during the breeding process. In addition, the full-length sequence of Rab7 of P. f. martensii (Pm-Rab7) was cloned using RACE technology, and the expression levels of Pm-Rab7 in adductor muscle, gill, gonad, hepatopancreas, foot, and mantle, and their expression patterns under temperature stress (17 ℃ in the low temperature group, 22 ℃ in the control group, and 32 ℃ in the high temperature group) were detected using bioinformatics analysis. Furthermore, single nucleotide polymorphisms (SNPs) in the exon regions of Pm-Rab7 in the R and W were screened, and this information was used to determine the genetic polymorphism, haplotype, and frequency rates for each mutation. Sequence analysis also revealed that the Pm-Rab7 gene displayed a full length of 1 153 bp, with the 5' and 3' UTR adding 30 bp and 505 bp, respectively. This gene was also shown to encode a single open reading frame (ORF) of 618 bp and 205 amino acids, producing a theoretical protein of 23.04 kDa with the isoelectric point at 5.40. This was later confirmed using the cloned Pm-Rab7 gene, which produced 5 conserved amino acid sequences, including a RAB conserved domain. Similarity evaluations revealed a high degree of overlap with C. gigas (92.79%), whereas phylogenetic tree construction revealed that the Rab7 gene produced a tree with three branches, one each for protostomes, echinoderms, and vertebrates. Closer inspection of the protostomes branch revealed that Pm-Rab7 first clusters with other mollusks, and then with arthropods to produce a large clade, suggesting that this protein is relatively well conserved during the evolutionary process. Pm-Rab7 was also shown to be expressed in all the tested tissues, with the most Pm-Rab7 expression recorded in the gonad and gill (P<0.05). Time-course results from the gill tissues of temperature stressed samples revealed that Pm-Rab7 expression first increased and then decreased in response to low temperature (17 ℃) exposure, with all time points showing a significant increase in Pm-Rab7 expression when compared to that in the 22 ℃ control from 6 h to 3 d (P<0.05). In addition, we noted an expression peak at 1 d. The expression of Pm-Rab7 was generally stable in response to growth at 32 ℃ (high temperature group), but was also significantly increased when compared to that in the control group following 12 h of exposure (P<0.05). This suggests that Pm-Rab7 is most likely linked to the low temperature response process in these shellfish. We also identified a total of seven SNPs in the exon region of Pm-Rab7, three (g.112712470, g.112712503, and g.11271477) of which demonstrated significant differences in occurrence rate between the R and W populations (P<0.05). Genetic evaluations of all seven SNPs revealed that three could be classified as low polymorphism loci (PIC<0.25) and four could be classified as moderate polymorphism loci (0.25
2017, 38(2):70-76.
DOI: 10.11758/yykxjz.20160103001
Abstract:
Low temperature tolerance of Epinephelus moara♀× E. lanceolatus♂(hereinafter Yunlong) was investigated in this study. The initial water temperature (20℃) was served as control, and then was dropped at a rate of 1℃/d, the changes in serum biochemical indices and metabolic enzyme activities of Yunlong were determined when the water temperatures reached 16℃, 15℃, 13℃ and 10℃. During the cold stress period, survival rates and the semi-lethal temperatures of Yunlong, E. fuscoguttatus♀× E.lanceolatus♂ (Pearl Gentian), and E. coioides♀ × E. lanceolatus♂(Qinglong) were observed and recorded. The semi-lethal temperature was recorded when the survival rate reached 50%. We found that the semi-lethal temperatures for Pearl Gentian, Qinglong and Yunlong were 11℃, 9.5℃, 9℃, and 8℃ respectively. The levels of serum creatinine (CREA) and total cholesterol (T-CHO) were first increased and then decreased along with increase of the intensity and duration of the low temperature stress. The level of serum triglycerides (TG) fluctuated at different temperatures, and the values at 15℃ and 10℃ were significantly different from that of the control group (P<0.05). The content of serum glucose (GLU) also varied with the temperatures, the levels at 16℃ and 13℃ were different from that of the control group (P<0.05). The activity of serum alkaline phosphatase (AKP) at 16℃ was slightly and insignificantly increased (P>0.05). The activity of serum catalase (CAT) fluctuated and showed significant differences between the control group and low temperatures at 16℃, 15℃ and 10℃ (P<0.05). The activity of serum glutamic-oxaloacetic transaminase (GOT) was slightly higher than that of the control group (P>0.05). The activity of serum glutamate pyruvate transaminase (GPT) was elevated followed by a decline along with the increase in the intensity and duration of low temperature stress. At 16℃ and 15℃, the activity of GPT was different from that of the control group (P<0.05). In conclusion, Yunlong was the most tolerant to low temperature stress among the three hybrids. Our data also suggested that low temperature could impair the immunity and the antioxidant capacity of juvenile fish, therefore the intensity and duration of the low temperature condition should be limited during practice.
Low temperature tolerance of Epinephelus moara♀× E. lanceolatus♂(hereinafter Yunlong) was investigated in this study. The initial water temperature (20℃) was served as control, and then was dropped at a rate of 1℃/d, the changes in serum biochemical indices and metabolic enzyme activities of Yunlong were determined when the water temperatures reached 16℃, 15℃, 13℃ and 10℃. During the cold stress period, survival rates and the semi-lethal temperatures of Yunlong, E. fuscoguttatus♀× E.lanceolatus♂ (Pearl Gentian), and E. coioides♀ × E. lanceolatus♂(Qinglong) were observed and recorded. The semi-lethal temperature was recorded when the survival rate reached 50%. We found that the semi-lethal temperatures for Pearl Gentian, Qinglong and Yunlong were 11℃, 9.5℃, 9℃, and 8℃ respectively. The levels of serum creatinine (CREA) and total cholesterol (T-CHO) were first increased and then decreased along with increase of the intensity and duration of the low temperature stress. The level of serum triglycerides (TG) fluctuated at different temperatures, and the values at 15℃ and 10℃ were significantly different from that of the control group (P<0.05). The content of serum glucose (GLU) also varied with the temperatures, the levels at 16℃ and 13℃ were different from that of the control group (P<0.05). The activity of serum alkaline phosphatase (AKP) at 16℃ was slightly and insignificantly increased (P>0.05). The activity of serum catalase (CAT) fluctuated and showed significant differences between the control group and low temperatures at 16℃, 15℃ and 10℃ (P<0.05). The activity of serum glutamic-oxaloacetic transaminase (GOT) was slightly higher than that of the control group (P>0.05). The activity of serum glutamate pyruvate transaminase (GPT) was elevated followed by a decline along with the increase in the intensity and duration of low temperature stress. At 16℃ and 15℃, the activity of GPT was different from that of the control group (P<0.05). In conclusion, Yunlong was the most tolerant to low temperature stress among the three hybrids. Our data also suggested that low temperature could impair the immunity and the antioxidant capacity of juvenile fish, therefore the intensity and duration of the low temperature condition should be limited during practice.
2018, 39(3):96-102.
Abstract:
In the present study, a preliminary evaluation was conducted on the cold tolerance and growth traits of 69 Fenneropenaeus chinensis by indoor artificial cooling. A linear mixed model with average information restricted maximum likelihood was used to evaluate the genetic parameters. Two animal models were used to evaluate the heritability of body weight (BW) and body length (BL) under low-temperature stress in F. chinensis. The heritability of BW and BL observed in model 1 were (0.206±0.177) and (0.187±0.179), respectively, and for model 2, these were (0.317±0.065) and (0.298±0.063), respectively, which ranged between medium to low. The differences between the two models were tested by likelihood ratio test, and the likelihood ratio values for Model 1 and Model 2 were 1.640 and 1.764, respectively, which were not significantly different (P>0.05). The results showed that the two models were not significantly different, and that model 2 was the optimal model. The heritability of the survival status at half lethal time (SS50) among cold tolerant traits was (0.169±0.078). The phenotypic and genetic correlation coefficients between BW and BL under low-temperature stress were (0.823±0.010) and (0.969±0.018), respectively, which were relatively high. The phenotypic correlation coefficients between BW and SS50; and BL and SS50 were comparatively low with values (0.187±0.030) and (0.218±0.030), respectively. The genetic correlation coefficient between BW and SS50; and BL and SS50 were comparatively high with values (0.517±0.205) and (0.538±0.203), respectively. The results showed that during breeding of F. chinensis, the cold tolerant variety can be selected together with growth traits.
In the present study, a preliminary evaluation was conducted on the cold tolerance and growth traits of 69 Fenneropenaeus chinensis by indoor artificial cooling. A linear mixed model with average information restricted maximum likelihood was used to evaluate the genetic parameters. Two animal models were used to evaluate the heritability of body weight (BW) and body length (BL) under low-temperature stress in F. chinensis. The heritability of BW and BL observed in model 1 were (0.206±0.177) and (0.187±0.179), respectively, and for model 2, these were (0.317±0.065) and (0.298±0.063), respectively, which ranged between medium to low. The differences between the two models were tested by likelihood ratio test, and the likelihood ratio values for Model 1 and Model 2 were 1.640 and 1.764, respectively, which were not significantly different (P>0.05). The results showed that the two models were not significantly different, and that model 2 was the optimal model. The heritability of the survival status at half lethal time (SS50) among cold tolerant traits was (0.169±0.078). The phenotypic and genetic correlation coefficients between BW and BL under low-temperature stress were (0.823±0.010) and (0.969±0.018), respectively, which were relatively high. The phenotypic correlation coefficients between BW and SS50; and BL and SS50 were comparatively low with values (0.187±0.030) and (0.218±0.030), respectively. The genetic correlation coefficient between BW and SS50; and BL and SS50 were comparatively high with values (0.517±0.205) and (0.538±0.203), respectively. The results showed that during breeding of F. chinensis, the cold tolerant variety can be selected together with growth traits.
2020, 41(3):88-93.
DOI: 10.19663/j.issn2095-9869.20190226001
Abstract:
The long-spread nuclear element-1 (LINE1) retrotransposon is a mobile element in genome. Previous comparative genomic studies found that Antarctic Notothenioid fish underwent a long low-temperature adaptation evolution, and compared with Notothenioid fish outside the Antarctic circle, LINE1 genes were duplicated by 8~300 fold. The link between this augmentation and the resistance of fish to cold is not known. In this study, zebrafish (Danio rerio) embryonic fibroblasts ZF4 were exposed to a low temperature (18℃) for 5 days and 30 days, and adult zebrafish were exposed to a low temperature (10℃) for 3 h, 6 h, 1 d, 3 d, and 5 d. The mRNA expression of LINE1 was examined using RT-qPCR. The promoter regions of zebrafish LINE1 gene were cloned and the biological activity of LINE1 5'UTR at low temperature were verified in ZF4 cells by using the dual-luciferase reporter system. The following results were obtained: (1) In ZF4 cells, LINE1 mRNA expression was decreased by short-term low temperature treatment, but was significantly increased by long-term low temperature treatment. (2) In adult fish, LINE1 mRNA expression was decreased by short-term low temperature treatment, but was significantly increased in long-term low temperature treatment. (3) The LINE1 5'UTR was found to be biologically active in ZF4 cells. (4) It was found that during low temperature treatment (18℃, 3 d), the reporter gene signal was weakened, which indirectly indicated that the LINE1 promoter activity was weakened. The results showed that low temperature stress affected LINE1 expression in fish, which presents a foundation for further study on the mechanism of action of LINE1 in fish adaptation to a low temperature environment.
The long-spread nuclear element-1 (LINE1) retrotransposon is a mobile element in genome. Previous comparative genomic studies found that Antarctic Notothenioid fish underwent a long low-temperature adaptation evolution, and compared with Notothenioid fish outside the Antarctic circle, LINE1 genes were duplicated by 8~300 fold. The link between this augmentation and the resistance of fish to cold is not known. In this study, zebrafish (Danio rerio) embryonic fibroblasts ZF4 were exposed to a low temperature (18℃) for 5 days and 30 days, and adult zebrafish were exposed to a low temperature (10℃) for 3 h, 6 h, 1 d, 3 d, and 5 d. The mRNA expression of LINE1 was examined using RT-qPCR. The promoter regions of zebrafish LINE1 gene were cloned and the biological activity of LINE1 5'UTR at low temperature were verified in ZF4 cells by using the dual-luciferase reporter system. The following results were obtained: (1) In ZF4 cells, LINE1 mRNA expression was decreased by short-term low temperature treatment, but was significantly increased by long-term low temperature treatment. (2) In adult fish, LINE1 mRNA expression was decreased by short-term low temperature treatment, but was significantly increased in long-term low temperature treatment. (3) The LINE1 5'UTR was found to be biologically active in ZF4 cells. (4) It was found that during low temperature treatment (18℃, 3 d), the reporter gene signal was weakened, which indirectly indicated that the LINE1 promoter activity was weakened. The results showed that low temperature stress affected LINE1 expression in fish, which presents a foundation for further study on the mechanism of action of LINE1 in fish adaptation to a low temperature environment.
2022, 43(2):157-166.
DOI: 10.19663/j.issn2095-9869.20210108001
Abstract:
Marsupenaeus japonicus is an important cultured species of marine shrimp in China. Its characteristics include a varied diet, fast growth, and tolerating periods out of water, as well as being suitable for live shrimp marketing and long-distance transportation. The suitable growth temperature of M. japonicus is 23℃~32℃. The culture cycle of M. japonicus is seriously affected by low winter temperatures in northern China, which increases the costs of parent shrimp overwintering. Therefore, it is necessary to breed new varieties with low temperature tolerances. M. japonicus was exposed to water at 10℃, 16℃, and 22℃ for 72 h, with M. japonicus exposed to water at 28℃ used as the control group. The results showed that the total antioxidant capacity (T-AOC), superoxide dismutase (SOD) enzyme activity, and GSH content in the gill and hepatopancreas decreased first, then increased and then decreased, while the MDA content and caspase-3 enzyme activity increased over time. The relative expression of the SOD and Caspase-3 genes decreased first, then increased, then decreased, and continued to increase, which was similar to their corresponding changes in enzyme activity. In order to prove that low temperature stress caused damage to the body of M. japonicus, a TUNEL test was performed. It was found that the apoptosis rate increased significantly with the extension of the stress time. The apoptotic rates of the gill control group, 3 h at 10℃, and 72 h at 10℃ were 2.03%, 6.20%, and 26.27%, respectively. The apoptotic rates of the hepatopancreas cells were 1.06%, 25.65%, and 42.33% for these exposure times, respectively. In conclusion, low temperature stress reduced the antioxidant capacity of M. japonicus, destroyed the original balance of the internal environment, and then led to oxidative damage. In this study, the changes in cell apoptosis and the antioxidant status of M. japonicus under low temperature conditions provided data that support the need to cultivate new varieties with low temperature tolerances.
Marsupenaeus japonicus is an important cultured species of marine shrimp in China. Its characteristics include a varied diet, fast growth, and tolerating periods out of water, as well as being suitable for live shrimp marketing and long-distance transportation. The suitable growth temperature of M. japonicus is 23℃~32℃. The culture cycle of M. japonicus is seriously affected by low winter temperatures in northern China, which increases the costs of parent shrimp overwintering. Therefore, it is necessary to breed new varieties with low temperature tolerances. M. japonicus was exposed to water at 10℃, 16℃, and 22℃ for 72 h, with M. japonicus exposed to water at 28℃ used as the control group. The results showed that the total antioxidant capacity (T-AOC), superoxide dismutase (SOD) enzyme activity, and GSH content in the gill and hepatopancreas decreased first, then increased and then decreased, while the MDA content and caspase-3 enzyme activity increased over time. The relative expression of the SOD and Caspase-3 genes decreased first, then increased, then decreased, and continued to increase, which was similar to their corresponding changes in enzyme activity. In order to prove that low temperature stress caused damage to the body of M. japonicus, a TUNEL test was performed. It was found that the apoptosis rate increased significantly with the extension of the stress time. The apoptotic rates of the gill control group, 3 h at 10℃, and 72 h at 10℃ were 2.03%, 6.20%, and 26.27%, respectively. The apoptotic rates of the hepatopancreas cells were 1.06%, 25.65%, and 42.33% for these exposure times, respectively. In conclusion, low temperature stress reduced the antioxidant capacity of M. japonicus, destroyed the original balance of the internal environment, and then led to oxidative damage. In this study, the changes in cell apoptosis and the antioxidant status of M. japonicus under low temperature conditions provided data that support the need to cultivate new varieties with low temperature tolerances.
WANG Yingpu
,
LI Jiaqi
,
XUE Suyan
,
MA Zhanfei
,
CHANG Lirong
,
LU Longfei
,
ZHANG Yitao
,
MAO Yuze
2023, 44(5):202-210.
DOI: 10.19663/j.issn2095-9869.20220310003
Abstract:
The Pacific abalone (Haliotis discus hannai) is naturally distributed in the Bohai Sea and Yellow Sea. In China, the Pacific abalone is an important living marine resource. Over the past 40 years, the abalone industry has gradually developed from wild harvesting to aquaculture. Currently, the main cultivation method is long-line culture, especially north-south relay aquaculture. The north-south relay involves transporting abalone cultivated in the East China Sea to the Yellow Sea and Bohai Sea over summer, to avoid extreme temperature stress. Due to the consistent favorable temperatures, this method achieves high survival with a shortened cultivation cycle. The rapid development of this efficient cultivation model has supported a substantial increase in domestic abalone production, exceeding 200 000 tons in 2020. However, the north-south relay aquaculture has several deficiencies, such as a large influx of abalone being supplied to the market over a very limited period with a homogenized flavor. This has led to a sharp decline in price. Abalone grow slowly in the bottom-sowing model in northern waters, however, the quality exceeds that of north-south relay cultured abalone. The optimum growing temperature of Pacific abalone is 10~22 ℃. Bottom-sowing cultivation in the northern waters has a lower seawater temperature, occasionally below 0 ℃. In addition, in long-term north-south relay cultivation, abalone are always in a suitable water temperature environment, reducing the low temperature tolerance of abalone. The increasing investment in recent years in marine ecological protection (such as marine pastures, habitat restoration and abalone habitat creation) and the technological breakthroughs in the cultivation of low-temperature resistant seedlings has enabled the optimization of bottom-sowing culture, reducing many issues, such as high mortality while overwintering, which has been partially solved. However, the impact of both cultivation methods on the nutrient contents and the physiological index of abalone is rarely reported. In this study, the north-south relay and the northern bottom-sowing abalone cultures were investigated. The total sugar, protein, total organic matter, and amino acid content characterized the nutritional value of individuals from both culture methods. The oxygen consumption rate and heart rate identified their low temperature tolerance. We explore the differences in body composition and physiological mechanisms in response to low temperature stress using specimens from both farming methods. The results showed that the total sugar content of the bottom-sowing culture individuals was (3.20±0.00)%, the total organic matter was (27.60±3.70)%, and the essential amino acid content was (4.19±0.09)%, which were significantly higher than those in the individuals from the north-south relay culture (P<0.05). At 24 ℃, the oxygen consumption rates of the bottom-sown abalone and relay cultured individuals were (0.077±0.024) mg/(g·h) and (0.082±0.012) mg/(g·h), respectively. The oxygen consumption rates of abalone in low temperature stress did not vary significantly, with (0.018±0.009) mg/(g·h) (bottom-sown abalone) and (0.017±0.006) mg/(g·h) (relay cultured abalone) (P>0.05). At 24 ℃, the heart rates of bottom-sown and relay-cultured abalone did not vary significantly, with (45.05±6.79) and (46.95±5.01) BPM, respectively (P>0.05). In low temperature, the heart rate of the bottom-sown abalone was (12.82±1.72) BPM, and the heart rate of the relay-cultured abalone was (18.11±2.79) BPM, statistically differing significantly (P<0.05). The results indicate variation in the abalone responses to external low temperature stress between individuals from the different farming models. The low heart rate level in low temperature conditions indicates a low metabolic level, which can reduce energy consumption, improving survival in the low temperature stress of a northern winter. Studies have revealed different farming models can significantly affect the nutritional value of abalone and the physiological responses to low temperature stress. Abalone cultured by bottom-sowing have higher nutritional value and a low temperature tolerance. In addition, abalone heart rate is a highly sensitive indicator for studying physiological responses to low temperature stress in abalone and other shellfish.
The Pacific abalone (Haliotis discus hannai) is naturally distributed in the Bohai Sea and Yellow Sea. In China, the Pacific abalone is an important living marine resource. Over the past 40 years, the abalone industry has gradually developed from wild harvesting to aquaculture. Currently, the main cultivation method is long-line culture, especially north-south relay aquaculture. The north-south relay involves transporting abalone cultivated in the East China Sea to the Yellow Sea and Bohai Sea over summer, to avoid extreme temperature stress. Due to the consistent favorable temperatures, this method achieves high survival with a shortened cultivation cycle. The rapid development of this efficient cultivation model has supported a substantial increase in domestic abalone production, exceeding 200 000 tons in 2020. However, the north-south relay aquaculture has several deficiencies, such as a large influx of abalone being supplied to the market over a very limited period with a homogenized flavor. This has led to a sharp decline in price. Abalone grow slowly in the bottom-sowing model in northern waters, however, the quality exceeds that of north-south relay cultured abalone. The optimum growing temperature of Pacific abalone is 10~22 ℃. Bottom-sowing cultivation in the northern waters has a lower seawater temperature, occasionally below 0 ℃. In addition, in long-term north-south relay cultivation, abalone are always in a suitable water temperature environment, reducing the low temperature tolerance of abalone. The increasing investment in recent years in marine ecological protection (such as marine pastures, habitat restoration and abalone habitat creation) and the technological breakthroughs in the cultivation of low-temperature resistant seedlings has enabled the optimization of bottom-sowing culture, reducing many issues, such as high mortality while overwintering, which has been partially solved. However, the impact of both cultivation methods on the nutrient contents and the physiological index of abalone is rarely reported. In this study, the north-south relay and the northern bottom-sowing abalone cultures were investigated. The total sugar, protein, total organic matter, and amino acid content characterized the nutritional value of individuals from both culture methods. The oxygen consumption rate and heart rate identified their low temperature tolerance. We explore the differences in body composition and physiological mechanisms in response to low temperature stress using specimens from both farming methods. The results showed that the total sugar content of the bottom-sowing culture individuals was (3.20±0.00)%, the total organic matter was (27.60±3.70)%, and the essential amino acid content was (4.19±0.09)%, which were significantly higher than those in the individuals from the north-south relay culture (P<0.05). At 24 ℃, the oxygen consumption rates of the bottom-sown abalone and relay cultured individuals were (0.077±0.024) mg/(g·h) and (0.082±0.012) mg/(g·h), respectively. The oxygen consumption rates of abalone in low temperature stress did not vary significantly, with (0.018±0.009) mg/(g·h) (bottom-sown abalone) and (0.017±0.006) mg/(g·h) (relay cultured abalone) (P>0.05). At 24 ℃, the heart rates of bottom-sown and relay-cultured abalone did not vary significantly, with (45.05±6.79) and (46.95±5.01) BPM, respectively (P>0.05). In low temperature, the heart rate of the bottom-sown abalone was (12.82±1.72) BPM, and the heart rate of the relay-cultured abalone was (18.11±2.79) BPM, statistically differing significantly (P<0.05). The results indicate variation in the abalone responses to external low temperature stress between individuals from the different farming models. The low heart rate level in low temperature conditions indicates a low metabolic level, which can reduce energy consumption, improving survival in the low temperature stress of a northern winter. Studies have revealed different farming models can significantly affect the nutritional value of abalone and the physiological responses to low temperature stress. Abalone cultured by bottom-sowing have higher nutritional value and a low temperature tolerance. In addition, abalone heart rate is a highly sensitive indicator for studying physiological responses to low temperature stress in abalone and other shellfish.
