Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apopt...Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apoptotic cell death.Emerging evidence suggests that mitochondrial autophagy(mitophagy)is the most effective method for eliminating damaged mitochondria and ROS,with choline dehydrogenase(CHDH)identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3(LC3)in vertebrates.However,the functional role of CHDH in invertebrates is largely unknown.In this study,we observed a significant increase in the mRNA and protein expression levels of A.japonicus CHDH(AjCHDH)in response to V.splendidus infection and lipopolysaccharide(LPS)challenge,consistent with changes in mitophagy under the same conditions.Notably,AjCHDH was localized to the mitochondria rather than the cytosol following V.splendidus infection.Moreover,AjCHDH knockdown using si RNA transfection significantly reduced mitophagy levels,as observed through transmission electron microscopy and confocal microscopy.Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region(LIR)for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1.The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis.Furthermore,laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells.In contrast,AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria,a critical step in damaged mitochondrial degradation.Thus,AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS,followed by increased apoptosis and decreased coelomocyte survival.Collectively,these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A.japonicus following V.splendidus infection.展开更多
The sea cucumber Apostichopus japonicus plays important roles in marine benthic ecosystem as environmental cleaners,and it is the important aquaculture species in China.High water temperature poses critical threat for...The sea cucumber Apostichopus japonicus plays important roles in marine benthic ecosystem as environmental cleaners,and it is the important aquaculture species in China.High water temperature poses critical threat for the survival of A.japonicus,which has resulted in extensive death in summer.To explore the genes expression profiles under different levels of heat stress,the high-throughput RNA-seq was applied in this study.Our results revealed a total of 1371,1225 and 1408 differentially expressed genes(DEGs)in 26℃for 6 h,26℃for 48 h and 30℃for 6 h respectively in comparison with Control group.The pathway analysis suggested“Protein processing in endoplasmic reticulum(ER)”was significantly enriched in all these heat stress(HS)treatment groups.The expression results of key DEGs in this pathway(Hsp70,Derlin,NEF,PDI,GPR94 and ERP57)by qRT-PCR was in accordance with the RNA-seq data.The subcluster analysis of DEGs revealed that a variety of heat shock proteins(HSPs)and calcium ion binding proteins had an obvious up-regulated expression in 26℃for 6 h,comparatively low expression in 26℃for 48 h,and the highest expression in 30℃for 6 h.The other DEGs subcluster,consisting of critical components of extracellular matrix(ECM)and a subset of peptidases and proteases,showed significantly rising tendency in 30℃for 6 h.Additionally,the expression of matrix metalloproteases(MMP1,MMP16 and MMP19)was prominently affected by HS,and peaked in 30℃for 6 h.This study provides a series of candidate genes for further study about heat shock response in A.japonicus,especially genes associated with protein processing in ER and regulation of ECM,which also offers new insights into cellular homeostasis under stressful conditions in marine invertebrates.展开更多
Sea cucumber(Apostichopus japonicus)is an excellent model for investigating effects of bottom-dwellers on carbon mig-ration and transformation.However,the molecular mechanism of respiratory metabolism process variatio...Sea cucumber(Apostichopus japonicus)is an excellent model for investigating effects of bottom-dwellers on carbon mig-ration and transformation.However,the molecular mechanism of respiratory metabolism process variation caused by feeding rations is poorly understood.In this study,treatment groups set as 1%(about 0.63g),3%,and 7%of total body weight(named F1,F3 and F7 groups respectively).The potential molecular mechanisms behind the functions of respiratory tree and body wall were investigated by RNA-Seq.A total of 52411 expressed genes were identified from 89342 expressed transcripts.The results showed 759,254 and 334 genes were up-regulated,and 334,445 and 992 genes were down-regulated in respiratory tree of F1 vs.F3,F1 vs.F7 and F3 vs.F7,respectively.Meanwhile,2070,1601 and 896 genes were up-regulated,and 1303,1337 and 1144 genes were down-regulated in body wall between F1 vs.F3,F1 vs.F7 and F3 vs.F7,respectively.Differentially expressed genes were enriched in salivary secretion and ECM-receptor interaction pathways in respiratory tree,and in various types of N-glycan biosynthesis,ribosome and sphingolipid metabolism pathways in body wall.These results suggested respiratory tree and body wall were involved in activation of respiratory metabolisms in response to different feeding rations.Our research provided valuable knowledge for physiological differences in res-piratory metabolism.展开更多
In order to control the spawning quality and improve the economic benefit of Apostichopus japonicus Selenka, the indoor temperature and light control methods were employed for artificial maturation, spawning and hatch...In order to control the spawning quality and improve the economic benefit of Apostichopus japonicus Selenka, the indoor temperature and light control methods were employed for artificial maturation, spawning and hatching of A. japonicus, and the larval survival rate, disease incidence rate and economic benefit were analyzed. The results showed that compared with those of conventional temperature-reared larvae, the survival rate of raised temperature-reared larvae was increased by 15.8%, the disease incidence rate was reduced by 10.2%, and the economic benefit was improved by 21.6%. These results will provide theoretical basis and technical support for the artificial breeding of A. japonicus.展开更多
The effect of water temperature on gut mass and digestive enzyme activity in sea cucumber Apostichopusjaponicus, including relative gut mass （RGM）, amylase, lipase, pepsin and trypsin activities were studied at temp...The effect of water temperature on gut mass and digestive enzyme activity in sea cucumber Apostichopusjaponicus, including relative gut mass （RGM）, amylase, lipase, pepsin and trypsin activities were studied at temperatures of 7, 14, 21, and 28℃ over a period of 40 days. Results show that RGM significantly decreased after 40 days at 21 ℃ and markedly decreased over the whole experiment period at 28℃; however, no significant effect of duration was observed at 7 or 14℃. At 14℃, trypsin activity significantly decreased over 10 and 20 days, then increased; amylase and trypsin activity significantly decreased after 40 days at 28℃. However, no significant effect of duration was found on amylase, pepsin or trypsin activities in the other temperature treatment groups. At 28℃, lipase activity peaked in 20 days and then markedly decreased to a minimum at the end of the experiment. On the other hand, pepsin activity at 28℃ continuously increased over the whole experimental period. Principle component analysis showed that sea cucumbers on day 40 in the 21℃ group and in the previous 20 days in the 28℃ group were in the prophase of aestivation. At 28℃, sea cucumbers aestivated at 30-40 days after the start of the experiment. It is concluded that the effect of temperature on the digestion ofA. japonicus is comparatively weak within a specific range of water temperatures and aestivation behavior is accompanied by significant changes in RGM and digestive enzyme activities.展开更多
Activities of hexokinase （HK）, pyruvate kinase （PK）, superoxide dismutase （SOD） and catalase （CAT） and Hsp70 level were measured to evaluate the response of the commercially important sea cucumber （Apostichop...Activities of hexokinase （HK）, pyruvate kinase （PK）, superoxide dismutase （SOD） and catalase （CAT） and Hsp70 level were measured to evaluate the response of the commercially important sea cucumber （Apostichopus japonicus Selenka） to rapid temperature changes in laboratory. Animals were subjected to a higher temperature （from 10 to 20℃） （Tinc treatment） or to a lower temperature （from 20 to 10℃） （Tddec treatment） for 72 h. At 1, 3, 12, 24, 72 h of exposure, animals were removed and prepared for further analysis. Results showed that the effect of acute temperature changes on enzyme activities was significant. In Tinc treatment, activities of SOD and CAT increased immediately. The significant enhancement of SOD and CAT activities suggested that oxidative stress increases significantly when ambient temperature increasing from 10 to 20℃. The up-regulation of Flsp70 in Tinc and Tdec treatments indicated that Hsp70 was a bioindicator of thermal stress in the sea cucumber, and the expression pattern depended on the thermal treatment.展开更多
Sea cucumbers, Apostichopus japonicus Selenka, were fed diets containing non-immunostimulant (basal diet), 0.2% β-glucan and 0.02% glycyrrhizin in a recirculatory water system for 45 days, and subsequently challenged...Sea cucumbers, Apostichopus japonicus Selenka, were fed diets containing non-immunostimulant (basal diet), 0.2% β-glucan and 0.02% glycyrrhizin in a recirculatory water system for 45 days, and subsequently challenged with Vibrio splendidus by injection at 1.0×108 cfu/sea cucumber for 15 days.Phagocytic capacity (PC), intracellular superoxide anion production (ISAP), ly-sozyme (LSZ) activity and superoxide dismutase (SOD) activity in the coelomic fluid were analyzed on the 0th, 5th, 10th and 15th days after injection.Results showed that after the 45-day feeding period, PC, ISAP, LSZ activity and SOD activity in sea cucumbers fed with dietary β-glucan or glycyrrhizin were significantly higher than in those fed with the basal diet.On the 5th day after infection, all the immune parameters examined in the sea cucumbers injected with V.splendidus decreased in value significantly.On the 15th day, PC, ISAP and LSZ activity returned to levels similar to those on the 0th day.For the sea cucumbers injected with saline, there were no significant differences in all the immune parameters examined and in the cumulative morbidity during the 15-day challenging trial.After injecting with V.splendidus, the cumulative morbidity of sea cucumbers fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin when challenged with V.splendidus challenged sea cucumber fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin.There was no significant difference in cumulative morbidity between the dietary β-glucan and glycyrrhizin treatments over time.展开更多
This study investigated the seasonal changes in carbon (C) and nitrogen (N) stable isotope values of several typical food sources of Apostichopus japonicus in a farm pond, including particulate organic matter (POM), m...This study investigated the seasonal changes in carbon (C) and nitrogen (N) stable isotope values of several typical food sources of Apostichopus japonicus in a farm pond, including particulate organic matter (POM), macroalgae, benthic microalgae and animals such as nematode and copepod. The stable isotope technique was used to quantify relative contributions of various sources to the food uptake by A. japonicus. The results showed that significant changes occurred in the C and N stable isotope values of sea cucumber food sources due to the seasonality of micro-or macroalgae prosperity and the fluctuation of environmental conditions. The sea cucumber A. japonicus exhibited corresponding alterations in feeding strategy in response to the changes in food conditions. Calculation with a stable isotope mixing model showed that macroalgae was the principal food source for A. japonicus throughout the 1-yr investigation, with the relative contribution averaging 28.1% - 63.2%. The relative contributions of other food sources such as copepod and nematode, POM, benthic microalgae to the total food uptake by sea cucumber averaged 22.6% - 39.1%, 6.3% - 22.2%, 2.8% - 6.5%, and 2.8% - 4.2%, respectively. Together these results indicated that the seasonal changes in food sources led to the obvious temporal differences in the relative contribution of various food sources utilized by A. japonicus. Such findings provide the basic scientific information for improving the aquaculture techniques of A. japonicus, particularly for optimizing the food environment of A. japonicus culture in farm ponds.展开更多
In laboratory conditions,effects of rearing temperature and stocking density were examined on hatching of fertilized egg and growth of auricularia larvae of Apostichopus japonicus respectively.Data series like larval ...In laboratory conditions,effects of rearing temperature and stocking density were examined on hatching of fertilized egg and growth of auricularia larvae of Apostichopus japonicus respectively.Data series like larval length and density,metamorphic time,and survival rate of the larvae were recorded.Statistics showed that for A.japonicus,survival rate(from fertilized egg to late auricularia) decreased significantly with the increasing rearing temperature(P<0.05).At different temperatures SGR was statistically significant as well(P<0.05) from day 1,and maximal SGR was found on day 9 at 24℃(159.26±3.28).This study clearly indicated that at low temperature(<24℃),metamorphic rate was remarkably higher than at higher temperature(>26℃).Hatching rate was significantly different between 0.2-5 ind./ml groups and 20-50 ind./ml groups.Rearing larvae at the higher density had the smaller maximal-length,whereas needed longer time to complete metamorphosis.This study suggested that 21℃ and 0.4 ind./ml can be used as the most suitable rearing temperature and stocking density for large-scale artificial breeding of A.japonicus's larvae.展开更多
The growth, metabolism and physiological response of the sea cucumber, Apostichopus japonicus, were investigated during periods of inactivity. The body weight, oxygen consumption rate (OCR), activities of acidic phosp...The growth, metabolism and physiological response of the sea cucumber, Apostichopus japonicus, were investigated during periods of inactivity. The body weight, oxygen consumption rate (OCR), activities of acidic phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT) and superoxide dismutase (SOD), and content of heat shock protein 70 (Hsp70) in the body wall and coelomic fluid of A. japonicus were measured during starvation, experimental aestivation and aestivation. The results showed that the body weight of sea cucumber in the three treatments decreased significantly during the experimental period (P < 0.05). The OCR of sea cucumber reduced in starvation and experimental aestivation treatments, but increased gradually in natural aestivation treatment. The activities of ACP and AKP of sea cucumber decreased gradually in all treatments, whereas those of SOD and CAT as well as Hsp70 content decreased in the starvation and experimental aestivation treatments and increased in natural aestivation treatment. The sea cucumber entered a state of aestivation at 24℃. To some extent, the animals in experimental aestivation were different from those in natural aestivation in metabolism and physiological response. These findings suggested that the aestivation mechanism of A. japonicus is complex and may not be attributed to the elevated temperature only.展开更多
The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. ...The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.展开更多
Different amounts of vitamin C were added to diets fed to juveniles （2.5＋0.15 g） of sea cucumber Apostichopus japonicus （Selenka） in an attempt to reduce the stress response of specimens exposed to nitrite stress...Different amounts of vitamin C were added to diets fed to juveniles （2.5＋0.15 g） of sea cucumber Apostichopus japonicus （Selenka） in an attempt to reduce the stress response of specimens exposed to nitrite stress. A commercial feed was used as the control diet and three experimental diets were made by supplementing 1 000, 1 500, or 2 000 mg vitamin C/kg diet to control diet separately in a 45-day experiment. Sea cucumbers were exposed to three different levels （0.5, 1.0, and 1.5 mg/L） of nitrite stress for 4, 8, and 12 h at four time intervals （0, 15, 30, and 45 d）. Growth of the animals was recorded during the experiment. Reactive oxygen species （ROS） （i.e. hydroxyl free radical （-OH）, malondialdehyde （MDA） and total antioxidant capacity （T-AOC）） and antioxidant enzyme activities （i.e., superoxide dismutase （SOD） and eatalase （CAT）） were measured. Response surface methodology （RSM） was used to analyze the effect of multiple factors on ROS indices and enzyme activities. Weight gain （WG） and special growth rate （SGR） of vitamin C supplementation groups were significantly higher than those of control group （P〈0.05）. The levels of-OH and MDA increased under exposure time extending and nitrite concentration increasing, whereas T-AOC level decreased. SOD and CAT activities increased at 4 h and 8 h and decreased at 12 h. During the days in which the animal consumed experimental diets, the levels of-OH and MDA decreased and that of T-AOC increased. This result suggests that diets containing vitamin C could reduce the nitrite stress response in the animals and increase their antioxidant capacity. The multifactor regression equation of growth performance, ROS indices, and duration of feeding results suggest that vitamin C supplementation of 1 400-2 000 mg/kg diet for 29-35 days could reduce effectively the effects of nitrite exposure.展开更多
Microflora in the intestinal tract and on the intestinal wall of both cultured and wild Apostichopus japonicus was studied in this paper. The screening for probiotics was performed based on enzyme producing and hemoly...Microflora in the intestinal tract and on the intestinal wall of both cultured and wild Apostichopus japonicus was studied in this paper. The screening for probiotics was performed based on enzyme producing and hemolytic analysis. The results showed that the number of bacteria in the intestinal wall and tract of wild Apostichopus japonicus was（3.30 ± 0.41） ×107cfu/g and（6.39 ± 0.32） ×10~7cfu/g, respectively. The number of bacteria in the intestinal wall and tract of cultured group was（2.83 ± 0.31） ×10~7cfu/g and（5.67 ± 0.53） ×10~7cfu/g, respectively. The dominant species in the intestinal tract of wild group was Vibrio and the Pseudomonas and Shewanella were the secondary dominant species. The dominant species in the cultured group was Vibrio and Pseudomonas. In 224 strains of bacteria, a total of160 strains of bacteria produced enzyme with a ratio of 71.43%. Among these bacteria, 114 strains could produce protease, 114 strains could produce amylase, and108 strains could produce lipase. The percentages were 50.89%, 50.89%, and48.21%, respectively. A total of 23 strains of bacteria could produce hemolytic toxin in 99 strains of bacteria, which accounts for 23.23% of the total bacterial population.Through the comprehensive analysis of test data, we selected 6 strains of bacteria as intestinal potential probiotic strains of Apostichopus japonicus, which were HS1（Pseudomonas）, HS5（Bacillus）, HS7（Shewanella）, HS8（Vibrio）, HS10（Vibrio）, and HS11（Vibrio） respectively.展开更多
Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal ...Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.展开更多
The effect of a potential probiotic on the growth performance and immune response of sea cucumber(Apostichopus japonicus) was investigated. Bacillus baekryungensis YD13 isolated from sea cucumber culturing ponds was a...The effect of a potential probiotic on the growth performance and immune response of sea cucumber(Apostichopus japonicus) was investigated. Bacillus baekryungensis YD13 isolated from sea cucumber culturing ponds was added to sea cucumber basal feed as a probiotic in different doses(0, the control; 1×104(YD134), 1×106(YD136) and 1×108(YD138) CFU g-1 of diet), and administered orally to A. japonicus(initial mean wet weight 5.44 g ± 0.17 g). The sea cucumbers were fed in 20 aquaria, 5 each treatment, for 60 d. At the end of growth trial, 20 sea cucumbers from each treatment were challenged with Vibrio splendidus. A. japonicus in YD134 and YD136 exhibited significantly better growth performance than control(P < 0.05). Five non-specific immune parameters including lysozyme, acid phosphatase, alkaline phosphatase, superoxide dismutase and catalase in coelomic fluid were measured to evaluate the immune response of A. japonicus to the probiotics. Results showed that all parameters were significantly improved when YD11 was supplemented in the dose of 1×106 CFU g-1(P < 0.05). The cumulative incidence and mortality after the Vibrio splendidus challenge decreased significantly in sea cucumbers of YD136. Accordingly, 1×106 CFU g-1 of YD13 in diet was recommended for the growth promotion and immune enhancement of A. japonicus.展开更多
Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopusjaponicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation o...Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopusjaponicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation or hibernation in the sea cucumber. The present study provided insight into the physiological adaptations ofA. japonicus during the three types of inactivity （hibernation, estivation, and starvation） by measuring the oxygen consumption rates （Vo2） and biochemical compositions under laboratory conditions of low （3℃）, normal （17℃） and high （24℃） temperature. The results show that the characteristics of A. japonicus in dormancy （hibernation and estivation） states were quite different from higher animals, such as fishes, amphibians, reptiles, and mammals, but more closely resembled a semi-dormant state. It was observed that the shift in the A. japonicus physiological state from normal to dormancy was a chronic rather than acute process, indicated by the gradual depression of metabolic rate. While metabolic rates declined 44.9% for the estivation group and 71.7% for the hibernation group, relative to initial rates, during the 36 d culture period, metabolic rates were not maintained at constant levels during these states. The metabolic depression processes for sea cucumbers in hibernation and estivation appeared to be a passive and an active metabolic suppression, respectively. In contrast, the metabolic rates （128.904-11.70 μg/g h） of estivating sea cucumbers were notably higher （107.85±6.31 μg/gh） than in starving sea cucumbers at 17℃, which indicated that the dormancy mechanism here, as a physiological inhibition, was not as efficient as in higher animals. Finally, the principle metabolic substrate or energy source of sea cucumbers in hibernation was lipid, whereas in estivation they mainly consumed protein in the early times and both protein and lipid thereafter.展开更多
Albino Apostichopus japonicus occur both in the wild and in captivity. The offspring of albino A. japonicus also suffer from albinism. The formation of melanin in the melanocytes is dependant on microphthalmia-associa...Albino Apostichopus japonicus occur both in the wild and in captivity. The offspring of albino A. japonicus also suffer from albinism. The formation of melanin in the melanocytes is dependant on microphthalmia-associated transcription factor (MITF). To investigate the role of MITF in controlling albinism, we cloned the full-length MITF cDNA from A. japonicus and compared MITF mRNA expression in albino and normal A. japonicus. In addition, we used light and electron microscopy to compare histological samples of normal and albino A. japonicus. The body wall of albino adults was characterized by significantly lower levels of MITF expression and lower numbers of epidermal melanocytes, which also contained less melanin. In albino juvenile offspring, MITF expression levels were significantly lower 32 d after fertilization and there were fewer, and less developed, epidermal melanocytes. Thus, we conclude that albino A. japonicus have fewer melanocytes and a reduced ability to synthesize melanin, likely because of lower expression of MITF.展开更多
基金supported by the National Natural Science Foundation of China (32102825)Natural Science Foundation of Zhejiang Province (LQ22C190003)。
文摘Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apoptotic cell death.Emerging evidence suggests that mitochondrial autophagy(mitophagy)is the most effective method for eliminating damaged mitochondria and ROS,with choline dehydrogenase(CHDH)identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3(LC3)in vertebrates.However,the functional role of CHDH in invertebrates is largely unknown.In this study,we observed a significant increase in the mRNA and protein expression levels of A.japonicus CHDH(AjCHDH)in response to V.splendidus infection and lipopolysaccharide(LPS)challenge,consistent with changes in mitophagy under the same conditions.Notably,AjCHDH was localized to the mitochondria rather than the cytosol following V.splendidus infection.Moreover,AjCHDH knockdown using si RNA transfection significantly reduced mitophagy levels,as observed through transmission electron microscopy and confocal microscopy.Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region(LIR)for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1.The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis.Furthermore,laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells.In contrast,AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria,a critical step in damaged mitochondrial degradation.Thus,AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS,followed by increased apoptosis and decreased coelomocyte survival.Collectively,these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A.japonicus following V.splendidus infection.
基金The National Natural Science Foundation of China under contract Nos 42276143 and 31902360the Shandong Provincial Natural Science Foundation under contract Nos ZR2022MC050 and ZR2022QD003the“First Class Fishery Discipline”Programme[(2020)3]in Shandong Province。
文摘The sea cucumber Apostichopus japonicus plays important roles in marine benthic ecosystem as environmental cleaners,and it is the important aquaculture species in China.High water temperature poses critical threat for the survival of A.japonicus,which has resulted in extensive death in summer.To explore the genes expression profiles under different levels of heat stress,the high-throughput RNA-seq was applied in this study.Our results revealed a total of 1371,1225 and 1408 differentially expressed genes(DEGs)in 26℃for 6 h,26℃for 48 h and 30℃for 6 h respectively in comparison with Control group.The pathway analysis suggested“Protein processing in endoplasmic reticulum(ER)”was significantly enriched in all these heat stress(HS)treatment groups.The expression results of key DEGs in this pathway(Hsp70,Derlin,NEF,PDI,GPR94 and ERP57)by qRT-PCR was in accordance with the RNA-seq data.The subcluster analysis of DEGs revealed that a variety of heat shock proteins(HSPs)and calcium ion binding proteins had an obvious up-regulated expression in 26℃for 6 h,comparatively low expression in 26℃for 48 h,and the highest expression in 30℃for 6 h.The other DEGs subcluster,consisting of critical components of extracellular matrix(ECM)and a subset of peptidases and proteases,showed significantly rising tendency in 30℃for 6 h.Additionally,the expression of matrix metalloproteases(MMP1,MMP16 and MMP19)was prominently affected by HS,and peaked in 30℃for 6 h.This study provides a series of candidate genes for further study about heat shock response in A.japonicus,especially genes associated with protein processing in ER and regulation of ECM,which also offers new insights into cellular homeostasis under stressful conditions in marine invertebrates.
基金supported by the National Natural Science Foundation of China(No.31672657).
文摘Sea cucumber(Apostichopus japonicus)is an excellent model for investigating effects of bottom-dwellers on carbon mig-ration and transformation.However,the molecular mechanism of respiratory metabolism process variation caused by feeding rations is poorly understood.In this study,treatment groups set as 1%(about 0.63g),3%,and 7%of total body weight(named F1,F3 and F7 groups respectively).The potential molecular mechanisms behind the functions of respiratory tree and body wall were investigated by RNA-Seq.A total of 52411 expressed genes were identified from 89342 expressed transcripts.The results showed 759,254 and 334 genes were up-regulated,and 334,445 and 992 genes were down-regulated in respiratory tree of F1 vs.F3,F1 vs.F7 and F3 vs.F7,respectively.Meanwhile,2070,1601 and 896 genes were up-regulated,and 1303,1337 and 1144 genes were down-regulated in body wall between F1 vs.F3,F1 vs.F7 and F3 vs.F7,respectively.Differentially expressed genes were enriched in salivary secretion and ECM-receptor interaction pathways in respiratory tree,and in various types of N-glycan biosynthesis,ribosome and sphingolipid metabolism pathways in body wall.These results suggested respiratory tree and body wall were involved in activation of respiratory metabolisms in response to different feeding rations.Our research provided valuable knowledge for physiological differences in res-piratory metabolism.
文摘In order to control the spawning quality and improve the economic benefit of Apostichopus japonicus Selenka, the indoor temperature and light control methods were employed for artificial maturation, spawning and hatching of A. japonicus, and the larval survival rate, disease incidence rate and economic benefit were analyzed. The results showed that compared with those of conventional temperature-reared larvae, the survival rate of raised temperature-reared larvae was increased by 15.8%, the disease incidence rate was reduced by 10.2%, and the economic benefit was improved by 21.6%. These results will provide theoretical basis and technical support for the artificial breeding of A. japonicus.
基金Supported by the Science Fund for Creative Research Groups (No.40821004)National Natural Science Foundation of China (No.40576073)+1 种基金Breeding Project of Shandong Province (China),National Key Technology Research and Development Program of China (No. 2006BAD09A02)the National High Technology Research and Development Program of China (863 Program) (No.2006AA100304 /2006AA10A411)
文摘The effect of water temperature on gut mass and digestive enzyme activity in sea cucumber Apostichopusjaponicus, including relative gut mass （RGM）, amylase, lipase, pepsin and trypsin activities were studied at temperatures of 7, 14, 21, and 28℃ over a period of 40 days. Results show that RGM significantly decreased after 40 days at 21 ℃ and markedly decreased over the whole experiment period at 28℃; however, no significant effect of duration was observed at 7 or 14℃. At 14℃, trypsin activity significantly decreased over 10 and 20 days, then increased; amylase and trypsin activity significantly decreased after 40 days at 28℃. However, no significant effect of duration was found on amylase, pepsin or trypsin activities in the other temperature treatment groups. At 28℃, lipase activity peaked in 20 days and then markedly decreased to a minimum at the end of the experiment. On the other hand, pepsin activity at 28℃ continuously increased over the whole experimental period. Principle component analysis showed that sea cucumbers on day 40 in the 21℃ group and in the previous 20 days in the 28℃ group were in the prophase of aestivation. At 28℃, sea cucumbers aestivated at 30-40 days after the start of the experiment. It is concluded that the effect of temperature on the digestion ofA. japonicus is comparatively weak within a specific range of water temperatures and aestivation behavior is accompanied by significant changes in RGM and digestive enzyme activities.
文摘Activities of hexokinase （HK）, pyruvate kinase （PK）, superoxide dismutase （SOD） and catalase （CAT） and Hsp70 level were measured to evaluate the response of the commercially important sea cucumber （Apostichopus japonicus Selenka） to rapid temperature changes in laboratory. Animals were subjected to a higher temperature （from 10 to 20℃） （Tinc treatment） or to a lower temperature （from 20 to 10℃） （Tddec treatment） for 72 h. At 1, 3, 12, 24, 72 h of exposure, animals were removed and prepared for further analysis. Results showed that the effect of acute temperature changes on enzyme activities was significant. In Tinc treatment, activities of SOD and CAT increased immediately. The significant enhancement of SOD and CAT activities suggested that oxidative stress increases significantly when ambient temperature increasing from 10 to 20℃. The up-regulation of Flsp70 in Tinc and Tdec treatments indicated that Hsp70 was a bioindicator of thermal stress in the sea cucumber, and the expression pattern depended on the thermal treatment.
基金supported by grant No.2006AA100313 from the National High Technology Research and Development Project of P.R. China (863 Project)
文摘Sea cucumbers, Apostichopus japonicus Selenka, were fed diets containing non-immunostimulant (basal diet), 0.2% β-glucan and 0.02% glycyrrhizin in a recirculatory water system for 45 days, and subsequently challenged with Vibrio splendidus by injection at 1.0×108 cfu/sea cucumber for 15 days.Phagocytic capacity (PC), intracellular superoxide anion production (ISAP), ly-sozyme (LSZ) activity and superoxide dismutase (SOD) activity in the coelomic fluid were analyzed on the 0th, 5th, 10th and 15th days after injection.Results showed that after the 45-day feeding period, PC, ISAP, LSZ activity and SOD activity in sea cucumbers fed with dietary β-glucan or glycyrrhizin were significantly higher than in those fed with the basal diet.On the 5th day after infection, all the immune parameters examined in the sea cucumbers injected with V.splendidus decreased in value significantly.On the 15th day, PC, ISAP and LSZ activity returned to levels similar to those on the 0th day.For the sea cucumbers injected with saline, there were no significant differences in all the immune parameters examined and in the cumulative morbidity during the 15-day challenging trial.After injecting with V.splendidus, the cumulative morbidity of sea cucumbers fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin when challenged with V.splendidus challenged sea cucumber fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin.There was no significant difference in cumulative morbidity between the dietary β-glucan and glycyrrhizin treatments over time.
基金funded by the National Natural Science Foundation of China (Grant Nos.31172426 and 30871931)the Ministry of Science and Technology of China (Grant Nos.2011BAD13B03 and JQ201009)+1 种基金the State Oceanic Administration of China (Grant No.200905020)supported by the Program for New Century Excellent Talents in University
文摘This study investigated the seasonal changes in carbon (C) and nitrogen (N) stable isotope values of several typical food sources of Apostichopus japonicus in a farm pond, including particulate organic matter (POM), macroalgae, benthic microalgae and animals such as nematode and copepod. The stable isotope technique was used to quantify relative contributions of various sources to the food uptake by A. japonicus. The results showed that significant changes occurred in the C and N stable isotope values of sea cucumber food sources due to the seasonality of micro-or macroalgae prosperity and the fluctuation of environmental conditions. The sea cucumber A. japonicus exhibited corresponding alterations in feeding strategy in response to the changes in food conditions. Calculation with a stable isotope mixing model showed that macroalgae was the principal food source for A. japonicus throughout the 1-yr investigation, with the relative contribution averaging 28.1% - 63.2%. The relative contributions of other food sources such as copepod and nematode, POM, benthic microalgae to the total food uptake by sea cucumber averaged 22.6% - 39.1%, 6.3% - 22.2%, 2.8% - 6.5%, and 2.8% - 4.2%, respectively. Together these results indicated that the seasonal changes in food sources led to the obvious temporal differences in the relative contribution of various food sources utilized by A. japonicus. Such findings provide the basic scientific information for improving the aquaculture techniques of A. japonicus, particularly for optimizing the food environment of A. japonicus culture in farm ponds.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2006AA10A411)Shandong Agriculture Seed Stock Projects
文摘In laboratory conditions,effects of rearing temperature and stocking density were examined on hatching of fertilized egg and growth of auricularia larvae of Apostichopus japonicus respectively.Data series like larval length and density,metamorphic time,and survival rate of the larvae were recorded.Statistics showed that for A.japonicus,survival rate(from fertilized egg to late auricularia) decreased significantly with the increasing rearing temperature(P<0.05).At different temperatures SGR was statistically significant as well(P<0.05) from day 1,and maximal SGR was found on day 9 at 24℃(159.26±3.28).This study clearly indicated that at low temperature(<24℃),metamorphic rate was remarkably higher than at higher temperature(>26℃).Hatching rate was significantly different between 0.2-5 ind./ml groups and 20-50 ind./ml groups.Rearing larvae at the higher density had the smaller maximal-length,whereas needed longer time to complete metamorphosis.This study suggested that 21℃ and 0.4 ind./ml can be used as the most suitable rearing temperature and stocking density for large-scale artificial breeding of A.japonicus's larvae.
基金supported by the Natural Science Funds for Distinguished Young Scientists of Shandong Province (No.JQ201009)the National Great Project of Scientific and Technical Supporting Programs (No.2011-BAD13B03)+2 种基金the National Natural Science Foundation of China (No.30771661)the Public Science and Technology Research Funds Projects of Ocean, State Oceanic Administration of China (No.200905020)the 111 Project of China Ministry of Education (No.B08049)
文摘The growth, metabolism and physiological response of the sea cucumber, Apostichopus japonicus, were investigated during periods of inactivity. The body weight, oxygen consumption rate (OCR), activities of acidic phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT) and superoxide dismutase (SOD), and content of heat shock protein 70 (Hsp70) in the body wall and coelomic fluid of A. japonicus were measured during starvation, experimental aestivation and aestivation. The results showed that the body weight of sea cucumber in the three treatments decreased significantly during the experimental period (P < 0.05). The OCR of sea cucumber reduced in starvation and experimental aestivation treatments, but increased gradually in natural aestivation treatment. The activities of ACP and AKP of sea cucumber decreased gradually in all treatments, whereas those of SOD and CAT as well as Hsp70 content decreased in the starvation and experimental aestivation treatments and increased in natural aestivation treatment. The sea cucumber entered a state of aestivation at 24℃. To some extent, the animals in experimental aestivation were different from those in natural aestivation in metabolism and physiological response. These findings suggested that the aestivation mechanism of A. japonicus is complex and may not be attributed to the elevated temperature only.
基金Supported by the National Key Technology Research and Development Program(No.2006AA10A411)the Agricultural Seed Project of Shandong Province
文摘The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.
基金Supported by the Main Direction Program of Knowledge Innovation of Chinese Academy of Sciences(No.KZCX2-EWQ215)
文摘Different amounts of vitamin C were added to diets fed to juveniles （2.5＋0.15 g） of sea cucumber Apostichopus japonicus （Selenka） in an attempt to reduce the stress response of specimens exposed to nitrite stress. A commercial feed was used as the control diet and three experimental diets were made by supplementing 1 000, 1 500, or 2 000 mg vitamin C/kg diet to control diet separately in a 45-day experiment. Sea cucumbers were exposed to three different levels （0.5, 1.0, and 1.5 mg/L） of nitrite stress for 4, 8, and 12 h at four time intervals （0, 15, 30, and 45 d）. Growth of the animals was recorded during the experiment. Reactive oxygen species （ROS） （i.e. hydroxyl free radical （-OH）, malondialdehyde （MDA） and total antioxidant capacity （T-AOC）） and antioxidant enzyme activities （i.e., superoxide dismutase （SOD） and eatalase （CAT）） were measured. Response surface methodology （RSM） was used to analyze the effect of multiple factors on ROS indices and enzyme activities. Weight gain （WG） and special growth rate （SGR） of vitamin C supplementation groups were significantly higher than those of control group （P〈0.05）. The levels of-OH and MDA increased under exposure time extending and nitrite concentration increasing, whereas T-AOC level decreased. SOD and CAT activities increased at 4 h and 8 h and decreased at 12 h. During the days in which the animal consumed experimental diets, the levels of-OH and MDA decreased and that of T-AOC increased. This result suggests that diets containing vitamin C could reduce the nitrite stress response in the animals and increase their antioxidant capacity. The multifactor regression equation of growth performance, ROS indices, and duration of feeding results suggest that vitamin C supplementation of 1 400-2 000 mg/kg diet for 29-35 days could reduce effectively the effects of nitrite exposure.
基金Supported by Spark Program(2012GA651002)the 863 Program(2012-AA10A412)Liaonning Key Program for Science and Technology Development(2012003003)~~
文摘Microflora in the intestinal tract and on the intestinal wall of both cultured and wild Apostichopus japonicus was studied in this paper. The screening for probiotics was performed based on enzyme producing and hemolytic analysis. The results showed that the number of bacteria in the intestinal wall and tract of wild Apostichopus japonicus was（3.30 ± 0.41） ×107cfu/g and（6.39 ± 0.32） ×10~7cfu/g, respectively. The number of bacteria in the intestinal wall and tract of cultured group was（2.83 ± 0.31） ×10~7cfu/g and（5.67 ± 0.53） ×10~7cfu/g, respectively. The dominant species in the intestinal tract of wild group was Vibrio and the Pseudomonas and Shewanella were the secondary dominant species. The dominant species in the cultured group was Vibrio and Pseudomonas. In 224 strains of bacteria, a total of160 strains of bacteria produced enzyme with a ratio of 71.43%. Among these bacteria, 114 strains could produce protease, 114 strains could produce amylase, and108 strains could produce lipase. The percentages were 50.89%, 50.89%, and48.21%, respectively. A total of 23 strains of bacteria could produce hemolytic toxin in 99 strains of bacteria, which accounts for 23.23% of the total bacterial population.Through the comprehensive analysis of test data, we selected 6 strains of bacteria as intestinal potential probiotic strains of Apostichopus japonicus, which were HS1（Pseudomonas）, HS5（Bacillus）, HS7（Shewanella）, HS8（Vibrio）, HS10（Vibrio）, and HS11（Vibrio） respectively.
基金Supported by the National Nature Science Foundation of China(Nos.41176139,31201972)
文摘Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.
基金supported by the National Programs for High Technology Research and Development of China (Grant No. 2006AA10Z409)the program for Excellent Youth Foundation of Shandong Province (Grant No. JQ201009)the program for New Century Excellent Talents in University (Grant No. NCET-08-0503)
文摘The effect of a potential probiotic on the growth performance and immune response of sea cucumber(Apostichopus japonicus) was investigated. Bacillus baekryungensis YD13 isolated from sea cucumber culturing ponds was added to sea cucumber basal feed as a probiotic in different doses(0, the control; 1×104(YD134), 1×106(YD136) and 1×108(YD138) CFU g-1 of diet), and administered orally to A. japonicus(initial mean wet weight 5.44 g ± 0.17 g). The sea cucumbers were fed in 20 aquaria, 5 each treatment, for 60 d. At the end of growth trial, 20 sea cucumbers from each treatment were challenged with Vibrio splendidus. A. japonicus in YD134 and YD136 exhibited significantly better growth performance than control(P < 0.05). Five non-specific immune parameters including lysozyme, acid phosphatase, alkaline phosphatase, superoxide dismutase and catalase in coelomic fluid were measured to evaluate the immune response of A. japonicus to the probiotics. Results showed that all parameters were significantly improved when YD11 was supplemented in the dose of 1×106 CFU g-1(P < 0.05). The cumulative incidence and mortality after the Vibrio splendidus challenge decreased significantly in sea cucumbers of YD136. Accordingly, 1×106 CFU g-1 of YD13 in diet was recommended for the growth promotion and immune enhancement of A. japonicus.
基金Supported by the National Natural Science Foundation of China (No.30400333)the National Key Program of Science and Technology of China (2006BAD09A01)
文摘Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopusjaponicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation or hibernation in the sea cucumber. The present study provided insight into the physiological adaptations ofA. japonicus during the three types of inactivity （hibernation, estivation, and starvation） by measuring the oxygen consumption rates （Vo2） and biochemical compositions under laboratory conditions of low （3℃）, normal （17℃） and high （24℃） temperature. The results show that the characteristics of A. japonicus in dormancy （hibernation and estivation） states were quite different from higher animals, such as fishes, amphibians, reptiles, and mammals, but more closely resembled a semi-dormant state. It was observed that the shift in the A. japonicus physiological state from normal to dormancy was a chronic rather than acute process, indicated by the gradual depression of metabolic rate. While metabolic rates declined 44.9% for the estivation group and 71.7% for the hibernation group, relative to initial rates, during the 36 d culture period, metabolic rates were not maintained at constant levels during these states. The metabolic depression processes for sea cucumbers in hibernation and estivation appeared to be a passive and an active metabolic suppression, respectively. In contrast, the metabolic rates （128.904-11.70 μg/g h） of estivating sea cucumbers were notably higher （107.85±6.31 μg/gh） than in starving sea cucumbers at 17℃, which indicated that the dormancy mechanism here, as a physiological inhibition, was not as efficient as in higher animals. Finally, the principle metabolic substrate or energy source of sea cucumbers in hibernation was lipid, whereas in estivation they mainly consumed protein in the early times and both protein and lipid thereafter.
基金Supported by the National Natural Science Foundation of China(No. 40976089)the National Marine Public Welfare Research Project(No. 200805069)+1 种基金the National Science and Technology Support Program of China (No. 2011BAD13B02)the Breeding Project of Shandong Province (China)
文摘Albino Apostichopus japonicus occur both in the wild and in captivity. The offspring of albino A. japonicus also suffer from albinism. The formation of melanin in the melanocytes is dependant on microphthalmia-associated transcription factor (MITF). To investigate the role of MITF in controlling albinism, we cloned the full-length MITF cDNA from A. japonicus and compared MITF mRNA expression in albino and normal A. japonicus. In addition, we used light and electron microscopy to compare histological samples of normal and albino A. japonicus. The body wall of albino adults was characterized by significantly lower levels of MITF expression and lower numbers of epidermal melanocytes, which also contained less melanin. In albino juvenile offspring, MITF expression levels were significantly lower 32 d after fertilization and there were fewer, and less developed, epidermal melanocytes. Thus, we conclude that albino A. japonicus have fewer melanocytes and a reduced ability to synthesize melanin, likely because of lower expression of MITF.
