Mitochondria undergo morphological changes during spermatogenesis in some animals.The mechanism and role of mitochondrial morphology regulation,however,remain somewhat unclear.In this study,we analyzed the molecular c...Mitochondria undergo morphological changes during spermatogenesis in some animals.The mechanism and role of mitochondrial morphology regulation,however,remain somewhat unclear.In this study,we analyzed the molecular characteristics,expression dynamics and subcellular localization of optic atrophy protein 1(OPA1),a mitochondrial fusion and cristae maintenance-related protein,to reveal the possible regulatory mechanisms underlying mitochondrial morphology in Phascolosoma esculenta spermiogenesis.The full-length cDNA of the P.esculenta opa1 gene(Pe-opa1)is 3743 bp in length and encodes 975 amino acids.The Pe-OPA1 protein is highly conservative and includes a transmembrane domain,a GTPase domain,two helical bundle domains,and a lipid-interacting stalk.Gene and protein expression was higher in the coelomic fluid(a site of spermatid development)of male P.esculenta and increased first and then decreased from March to December.Moreover,their expression during the breeding stage was significantly higher than during the non-breeding stage,suggesting that Pe-OPA1 is involved in P.esculenta reproduction.The Pe-OPA1 protein was more abundant in components consisting of many spermatids than in components without,indicating that Pe-OPA1 mainly plays a role in the spermatid in coelomic fluid.Moreover,Pe-OPA1 was mainly detected in the spermatid mitochondria.Immunofluorescence experiments showed that the Pe-OPA1 are constitutively expressed and co-localized with mitochondria during spermiogenesis,suggesting its involvement in P.esculenta spermiogenesis.These results provide evidence for Pe-OPA1's involvement in the regulation of mitochondrial morphology during spermiogenesis.展开更多
Phascolosoma esculenta is an intertidal organism that has recently attracted attention because of its ability to survive at relatively low temperatures.However,the gene regulation in P.esculenta in relation to its res...Phascolosoma esculenta is an intertidal organism that has recently attracted attention because of its ability to survive at relatively low temperatures.However,the gene regulation in P.esculenta in relation to its response to low temperatures is unclear.To explore the low temperature adaptability of P.esculenta,this study analyzed the changes in the morphology and hsp70 and hsp90 gene expression of P.esculenta exposed to a low temperature gradient.At 5℃,P.esculenta stretched and softened,and some individuals moved apart from the group.Histological analysis revealed cuticle breaches,myofiber scattering,disruption of the body wall,and epithelial layer dispersion and muscle fiber rupturing in the nephridium.Furthermore,the mRNA expression levels of hsp70 and hsp90 increased under acute low temperature stress,suggesting that these genes function in low temperature tolerance.Overall,low temperature stress causes morphological changes and histological damage in P.esculenta,and hsp70 and hsp90 potentially function in the low temperature adaptability of P.esculenta.Our results provide new insights into the adaptive strategies of P.esculenta under low temperature environments.展开更多
The microtubule(MT)-associated proteins KIF3A and KIF3B are ubiquitously expressed in a wide range of taxa.This study investigated the functions of these proteins in spermiogenesis,which involves various MT-dependent ...The microtubule(MT)-associated proteins KIF3A and KIF3B are ubiquitously expressed in a wide range of taxa.This study investigated the functions of these proteins in spermiogenesis,which involves various MT-dependent processes,in Phasco-losoma esculenta.We cloned the complete cDNA of Pe-KIF3A/3B.Structural predictions showed that Pe-KIF3A/3B are composed of a highly conserved motor domain,a coiled-coil domain,and a tail domain.Real-time quantitative PCR showed that Pe-kif3a/3b are expressed in all tissues evaluated,with the highest levels in sperm masses.Fluorescence in situ hybridization and immunofluo-rescence were employed to analyze the dynamic expression patterns of KIF3A/3B during spermiogenesis.Pe-KIF3A/3B consistently co-localized with MTs at all stages of spermiogenesis,indicating their potential functions in cargo trafficking.Pe-KIF3A/3B co-localized with mitochondria,suggesting that they may mediate mitochondrial movement.In late-stage spermatids and mature sperm,co-localization was detected in the midpiece,suggesting that Pe-KIF3A/3B could facilitate midpiece formation.The co-localization of Pe-KIF3A and Pe-KIF3B at all stages of spermiogenesis suggests their function as the heterodimeric KIF3AB.Basing on the ob-served temporal and spatial expression patterns of Pe-KIF3A/3B,MTs,and mitochondria in our study,we suggest that heterodimer KIF3AB has a potential role in acrosome biogenesis,sperm head remodeling,enflagellation,and mitochondrial migration during spermiogenesis in P.esculenta.In addition,our study on the morphological characteristics of spermatogenic cells provided fundamental data on the reproductive biology of P.esculenta.展开更多
Salinity is an important parameter that influences the developmental progress of aquatic organisms.The larvae of Phascolosoma esculenta developed into the early stage trochosphere,the late stage trochosphere and the e...Salinity is an important parameter that influences the developmental progress of aquatic organisms.The larvae of Phascolosoma esculenta developed into the early stage trochosphere,the late stage trochosphere and the early stage pelagosphere in the low-salinity group(15),the middle group(22.5)and the high-salinity group(30),respectively,after being incubated for 50 h,according to morphological observation.The results showed that high salinity could accelerate the cellular development of P.esculenta.To elucidate the molecular mechanism of larval development of P.esculenta under different salinities,we performed transcriptome analysis of the larvae in P.esculenta.Nine transcriptome libraries of P.esculenta larvae were constructed,i.e.,the low-salinity group(A1,A2 and A3),the middle group(B1,B2 and B3)and the high-salinity group(C1,C2 and C3),and each group with three repeats.These nine paired-end libraries were sequenced using the Illumina HiSeq 2500 platform.In total,57.63 Gb clean data were obtained and de novo assembled into 320,527 unigenes with mean lengths of 482.62 bp and N50 of 529 bp.A total of 249,162 unigenes were significantly matched to known unique proteins.The gene ontology(GO)annotation and the Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to determine the regulation pathways and candidate genes that are associated with the process of larval development.A total number of 12,811,16,991 and 1735 transcripts were identified as differentially expressed genes(DEGs)from the comparison of A vs.B,A vs.C,and B vs.C,respectively.Of these differentially expressed genes,many DEGs that were upregulated in the high-salinity group were involved in the function that controls the larval development of P.esculenta,such as the neuroactive ligand-receptor interaction and ECM-receptor interaction.In addition,the results of the transcriptome analysis were verified by quantitative real-time PCR(qRT-PCR).After morphological observation and transcriptome analysis,we think high salinity(30)is more suitable than medium(22.5)or low(15)salinity for the development of P.esculenta larvae.This information provides guidance for artificial breeding and paves the way to the further research on the individual development of P.esculenta under different salinities.展开更多
为分离鉴定可口革囊星虫(Phascolosoma esculenta)肠道细菌,获得产蛋白酶菌株,以野生和养殖可口革囊星虫为材料,通过常规细菌培养获得部分肠道菌群,利用脱脂乳蛋白培养基筛选产蛋白酶的菌株,采用16S r RNA对其种属进行鉴定,并测定其蛋...为分离鉴定可口革囊星虫(Phascolosoma esculenta)肠道细菌,获得产蛋白酶菌株,以野生和养殖可口革囊星虫为材料,通过常规细菌培养获得部分肠道菌群,利用脱脂乳蛋白培养基筛选产蛋白酶的菌株,采用16S r RNA对其种属进行鉴定,并测定其蛋白酶活力。结果表明,从野生可口革囊星虫肠道分离得到5株产蛋白酶菌株,其中WE6属于弧菌属(Vibrio),WE16属于盐芽孢杆菌属(Halobacillus),WG4和WP1属于芽孢杆菌属(Bacillus),WP2属于微球菌属(Micrococcus);从养殖可口革囊星虫肠道分离得到6株产蛋白酶菌株,其中CE4属于微球菌属,CE5、CE11及CP6属于芽孢杆菌属,CE17属于希瓦氏菌属(Shevagella),CP7属于寡养单胞菌属(Stenotrophomonas)。各菌株蛋白酶活力测定结果显示,水解圈直径与其相关性最大,pearson相关系数为0.628。野生和养殖可口革囊星虫肠道中占据优势的产蛋白酶细菌均为芽孢杆菌属;其中微球菌属和芽孢杆菌属产蛋白酶活力较高。展开更多
基金the Ningbo Science and Technology Plan Projects(Nos.2019B10016,2016C10004)the Major Science and Technology Projects in Zhejiang Province(No.2011C12013)+1 种基金the Natural Science Foundation of Zhejiang Province(No.LY18C190007)the Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture,the K.C.Wong Magna Fund in Ningbo University。
文摘Mitochondria undergo morphological changes during spermatogenesis in some animals.The mechanism and role of mitochondrial morphology regulation,however,remain somewhat unclear.In this study,we analyzed the molecular characteristics,expression dynamics and subcellular localization of optic atrophy protein 1(OPA1),a mitochondrial fusion and cristae maintenance-related protein,to reveal the possible regulatory mechanisms underlying mitochondrial morphology in Phascolosoma esculenta spermiogenesis.The full-length cDNA of the P.esculenta opa1 gene(Pe-opa1)is 3743 bp in length and encodes 975 amino acids.The Pe-OPA1 protein is highly conservative and includes a transmembrane domain,a GTPase domain,two helical bundle domains,and a lipid-interacting stalk.Gene and protein expression was higher in the coelomic fluid(a site of spermatid development)of male P.esculenta and increased first and then decreased from March to December.Moreover,their expression during the breeding stage was significantly higher than during the non-breeding stage,suggesting that Pe-OPA1 is involved in P.esculenta reproduction.The Pe-OPA1 protein was more abundant in components consisting of many spermatids than in components without,indicating that Pe-OPA1 mainly plays a role in the spermatid in coelomic fluid.Moreover,Pe-OPA1 was mainly detected in the spermatid mitochondria.Immunofluorescence experiments showed that the Pe-OPA1 are constitutively expressed and co-localized with mitochondria during spermiogenesis,suggesting its involvement in P.esculenta spermiogenesis.These results provide evidence for Pe-OPA1's involvement in the regulation of mitochondrial morphology during spermiogenesis.
基金supported by the Ningbo Science and Technology Plan Projects(Nos.2019B10016,2016C10004)the Collaborative Innovation Center for Zhejiang Marine High-Efficiency and Healthy Aquaculture,the K.C.Wong Magna Fund in Ningbo University。
文摘Phascolosoma esculenta is an intertidal organism that has recently attracted attention because of its ability to survive at relatively low temperatures.However,the gene regulation in P.esculenta in relation to its response to low temperatures is unclear.To explore the low temperature adaptability of P.esculenta,this study analyzed the changes in the morphology and hsp70 and hsp90 gene expression of P.esculenta exposed to a low temperature gradient.At 5℃,P.esculenta stretched and softened,and some individuals moved apart from the group.Histological analysis revealed cuticle breaches,myofiber scattering,disruption of the body wall,and epithelial layer dispersion and muscle fiber rupturing in the nephridium.Furthermore,the mRNA expression levels of hsp70 and hsp90 increased under acute low temperature stress,suggesting that these genes function in low temperature tolerance.Overall,low temperature stress causes morphological changes and histological damage in P.esculenta,and hsp70 and hsp90 potentially function in the low temperature adaptability of P.esculenta.Our results provide new insights into the adaptive strategies of P.esculenta under low temperature environments.
基金supported by the Ningbo Science and Technology Plan Projects (Nos. 2019B10016, 2016C 10004)the Major Science and Technology Projects in Zhejiang Province (No. 2011C12013)+2 种基金the Natural Science Foundation of Zhejiang Province (No. LY18C190007)the National Natural Science Foundation of China (No. 312 72642)the Collaborative Innovation Center for Marine High-efficiency and Healthy Aquaculture of Zhejiang Province, and K.C.Wong Magna Fund in Ningbo University
文摘The microtubule(MT)-associated proteins KIF3A and KIF3B are ubiquitously expressed in a wide range of taxa.This study investigated the functions of these proteins in spermiogenesis,which involves various MT-dependent processes,in Phasco-losoma esculenta.We cloned the complete cDNA of Pe-KIF3A/3B.Structural predictions showed that Pe-KIF3A/3B are composed of a highly conserved motor domain,a coiled-coil domain,and a tail domain.Real-time quantitative PCR showed that Pe-kif3a/3b are expressed in all tissues evaluated,with the highest levels in sperm masses.Fluorescence in situ hybridization and immunofluo-rescence were employed to analyze the dynamic expression patterns of KIF3A/3B during spermiogenesis.Pe-KIF3A/3B consistently co-localized with MTs at all stages of spermiogenesis,indicating their potential functions in cargo trafficking.Pe-KIF3A/3B co-localized with mitochondria,suggesting that they may mediate mitochondrial movement.In late-stage spermatids and mature sperm,co-localization was detected in the midpiece,suggesting that Pe-KIF3A/3B could facilitate midpiece formation.The co-localization of Pe-KIF3A and Pe-KIF3B at all stages of spermiogenesis suggests their function as the heterodimeric KIF3AB.Basing on the ob-served temporal and spatial expression patterns of Pe-KIF3A/3B,MTs,and mitochondria in our study,we suggest that heterodimer KIF3AB has a potential role in acrosome biogenesis,sperm head remodeling,enflagellation,and mitochondrial migration during spermiogenesis in P.esculenta.In addition,our study on the morphological characteristics of spermatogenic cells provided fundamental data on the reproductive biology of P.esculenta.
基金This work was partially supported by the Scientific Research Program of Fujian Province under contract No.2015R1003-14the Open Research Fund Program of Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-environment under contract No.fjmfre2019005.
文摘Salinity is an important parameter that influences the developmental progress of aquatic organisms.The larvae of Phascolosoma esculenta developed into the early stage trochosphere,the late stage trochosphere and the early stage pelagosphere in the low-salinity group(15),the middle group(22.5)and the high-salinity group(30),respectively,after being incubated for 50 h,according to morphological observation.The results showed that high salinity could accelerate the cellular development of P.esculenta.To elucidate the molecular mechanism of larval development of P.esculenta under different salinities,we performed transcriptome analysis of the larvae in P.esculenta.Nine transcriptome libraries of P.esculenta larvae were constructed,i.e.,the low-salinity group(A1,A2 and A3),the middle group(B1,B2 and B3)and the high-salinity group(C1,C2 and C3),and each group with three repeats.These nine paired-end libraries were sequenced using the Illumina HiSeq 2500 platform.In total,57.63 Gb clean data were obtained and de novo assembled into 320,527 unigenes with mean lengths of 482.62 bp and N50 of 529 bp.A total of 249,162 unigenes were significantly matched to known unique proteins.The gene ontology(GO)annotation and the Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to determine the regulation pathways and candidate genes that are associated with the process of larval development.A total number of 12,811,16,991 and 1735 transcripts were identified as differentially expressed genes(DEGs)from the comparison of A vs.B,A vs.C,and B vs.C,respectively.Of these differentially expressed genes,many DEGs that were upregulated in the high-salinity group were involved in the function that controls the larval development of P.esculenta,such as the neuroactive ligand-receptor interaction and ECM-receptor interaction.In addition,the results of the transcriptome analysis were verified by quantitative real-time PCR(qRT-PCR).After morphological observation and transcriptome analysis,we think high salinity(30)is more suitable than medium(22.5)or low(15)salinity for the development of P.esculenta larvae.This information provides guidance for artificial breeding and paves the way to the further research on the individual development of P.esculenta under different salinities.
文摘为分离鉴定可口革囊星虫(Phascolosoma esculenta)肠道细菌,获得产蛋白酶菌株,以野生和养殖可口革囊星虫为材料,通过常规细菌培养获得部分肠道菌群,利用脱脂乳蛋白培养基筛选产蛋白酶的菌株,采用16S r RNA对其种属进行鉴定,并测定其蛋白酶活力。结果表明,从野生可口革囊星虫肠道分离得到5株产蛋白酶菌株,其中WE6属于弧菌属(Vibrio),WE16属于盐芽孢杆菌属(Halobacillus),WG4和WP1属于芽孢杆菌属(Bacillus),WP2属于微球菌属(Micrococcus);从养殖可口革囊星虫肠道分离得到6株产蛋白酶菌株,其中CE4属于微球菌属,CE5、CE11及CP6属于芽孢杆菌属,CE17属于希瓦氏菌属(Shevagella),CP7属于寡养单胞菌属(Stenotrophomonas)。各菌株蛋白酶活力测定结果显示,水解圈直径与其相关性最大,pearson相关系数为0.628。野生和养殖可口革囊星虫肠道中占据优势的产蛋白酶细菌均为芽孢杆菌属;其中微球菌属和芽孢杆菌属产蛋白酶活力较高。
