Transcriptome Analysis of Crassostrea sikamea (♀) × Crassostrea gigas (♂) Hybrids Under and After Thermal Stress

Crossbreeding is an effective approach to manage the genetic decline in aquaculture.One-way hybrids of Crassostrea sikamea(♀)and Crassostrea gigas(♂)have advantages in growth traits and adaptation to high temperature.Here,we used high-throughput sequencing to analyze the molecular processes in the hybrids under and after thermal stress.The hybrids were cultured in the seawater with an increasing temperature from 25℃to 40℃during 10 hours,which is regarded as the thermal stress stage.Then the temperature decreased from 40℃to 25℃within 2 h,which is regarded as the recovery stage.In this study,1293 significant diffe-rentially expressed genes(DEGs)were obtained under thermal stress,of which 576 were upregulated and 717 were downregulated,and 740 significant differentially expressed genes(DEGs)were obtained in the recovery stage,of which the number of upregulated and downregulated genes was 409 and 331,respectively.The antigen processing and presentation,NOD-like,and NF-kappa B path-ways were significantly enriched during the thermal stress stage.The MAPK and PPAR signaling pathways were significantly enrich-ed during the recovery stage.The HSP70,HSP90,and CANX genes were strongly and rapidly upregulated in the control/thermal stress groups but were slightly less upregulated in the thermal stress/recovery group.These results indicate that the innate immune system or nonspecific immunity was deployed to protect interior tissues from thermal stress.In addition,85%of the mutual DEGs were involved in bidirectional regulation(up/down or down/up)when the oysters were removed from the thermal stress to recover.This study provides preliminary insight into the molecular response of C.sikamea(♀)and C.gigas(♂)hybrids to thermal stress and provides a basis for future studies on temperature-adaptation and the possible expansion of hybrid breeding.     

波吉卵囊藻胞外滤液对铜绿微囊藻转录水平的影响

【目的】探索波吉卵囊藻胞外滤液对铜绿微囊藻的抑制机理。【方法】采用Illumina平台,对使用BG11培养基(对照组)和波吉卵囊藻胞外滤液(实验组)培养的铜绿微囊藻(Microcystis aeruginosa)进行转录组测序,筛选差异表达基因并进行通路富集分析,并对部分差异表达基因进行qRT-PCR验证。【结果】与对照组相比,波吉卵囊藻胞外滤液处理后的铜绿微囊藻组中筛选1483个表达差异显著的基因,其中上调表达基因788个,下调表达基因695个。GO功能分析将差异基因划分为35个子类别,包括催化活性、结合、代谢过程、细胞过程、膜、膜部分等。KEGG通路分析中,所有差异基因聚集在108个通路,其中84个差异基因注释在代谢功能类别中,而氧化磷酸化通路富集最显著。氧化磷酸化通路中,相关基因表达以上调为主(26个DEGs,20个上调,6个下调)。qRT-PCR验证结果与转录组测序结果的表达变化趋势一致。【结论】波吉卵囊藻滤液引起铜绿微囊藻氧化磷酸化途径上基因表达上调,ATP合成效率提高。     

Domestication of marine microalga Nannochloropsis oceanica to freshwater medium and the physiological responses

Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated(-73 0 generations,-two days each)to adapt freshwater BGll medium.A number of freshwater medium adapted colonyderived strains were obtained.The strains were verified phylogenetically to be N.oceanica LAMB0001 based on the 18 S ribosomal RNA gene.Freshwater-medium adapted strain(FA1)grew faster in the BG11 medium prepared with freshwater than wild-type N.oceanica grew in f/2 medium prepared with seawater.We assumed that(1)the expression patterns of the genes that expressed differentially between FA1 and the wild-type N.oceanica exposing to the BG11 medium(WT-F)have been reprogrammed;(2)the physiological processes in which these genes involved have been modified;and(3)a Gene Ontology(GO)term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up-or down-regulated if it was enriched simultaneously by up-or down-regulated DEGs between FA1 and WT-F,respectively.Under these assumptions,we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion,membrane and membrane integrity,material transportation,cell movement,and cellular signaling network.These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium.It is feasible to domesticate marine microalgae to a freshwater habitat,which may aid to modify their cultivation performances.     

Primary molecular basis of androgenic gland endocrine sex regulation revealed by transcriptome analysis in Eriocheir sinensis

In crustaceans, the male sexual differentiation and maintenance are specially regulated by androgenic gland (AG). However, little is known about the genes involved in the regulation process. RNA-Seq was performed on AG with ejaculatory duct (AG_ED) and ejaculatory duct (ED) as control in Eriocheir sinensis, one of the most important economic and fishery crabs with typically sex dimorphism. A total of 925 unigenes were identified as differentially expressed genes (DEGs) and the expression of nine genes randomly selected was confirmed by qRT-PCR. 667 unigenes were up-regulated in AG_ED, being supposed to be AG preferential genes. Among them, the full length of insulin-like androgenic gland factor (IAG) cDNA named as Es-IAG was obtained as a logo gene of AG, which together with the genes insulin-like receptor (INR), and single insulin binding domain protein (SIBD), might constitute the sex regulation pathway. Several sex related genes were identified, and their function will have to be investigated. Also, the identification of juvenile hormone epoxide hydrolase 1 (JHEH1), ecdysteroid 22-hydroxylase (DIB) and ecdysone receptor (ECR) preliminarily clarified the molecular regulation mechanism of eyestalk-AG-testis axis, which plays important roles in molting and reproduction. The results will enhance our understanding for the molecular basis of the AG involved in male sex regulation in crabs.

     

The reaction time of organ-forming substance in goldfish EGG and its relationship with mesodermal formation

Fertilized goldfish eggs were dechorionated with a pair of forceps and were cut off along or a little above the equator into animal and vegetative parts at desired stages with a glass needle or ligated into two connected fragments before cleavage with baby hair loop. Some of the ligated eggs were detached by further fastening soon after ligation, and some released later at different stages (2-cell, 16-cell, 128-cell, 512-cell, mid-blastula) to let the organ-forming substance (OFS) enter the blastoderm. The cholinesterase (ChE) in the resulting embryos was assayed. The results are as follows. 1. All the 142 embryos developed from the animal hemispheres cut off or ligated off before cleavage gave rise to hyperblastula in which no ChE activity was observed. 2. All 50 embryos obtained from animal halves isolated at the 8-cell stage produced ChE. 3. Embryos developed from the eggs released before the 512-cell stage formed ChE, but the later the releasing of the hair knots, the smaller the number of ChE-producing embryos. 4. After the 512-cell stage (excluding this stage), neither ChE nor tissue differentiation occurred in the embryos developed from the unfastened eggs though their OFS flow was set free. Since ChE is thought to be a muscle-specific enzyme in the early developmental stage, it is concluded that the OFS in goldfish egg appears to be indispensable for the establishment of the mesoderm. Contribution No. 1507 from the Institute of Oceanology, Academia Sinica     

Using iTRAQ-Based Quantitative Proteomics Analysis to Identify Differentially Expressed Proteins Related to Larval Development of Portunus trituberculatus

The swimming crab Portunus trituberculatus is an economically important marine crustacean species. Here isobaric tags for relative and absolute quantitation(iTRAQ) analysis were used to identify proteins that are differentially expressed during larval development of P. trituberculatus to elucidate the underlying mechanisms. In comparison with the first zoea stage(Z1), 3980 proteins were identified from 32789 peptides, which were matched with 115497 spectrums. A total of 241 proteins were screened with significantly differential expressions in all development stages. These 241 proteins are involved in various biological processes, such as cytoskeleton organization, protein synthesis, energy production and substance metabolism, physiological activities, and transport.Cluster analyses of the 241 differentially expressed proteins led to the generation of four protein clusters based on the overall similarity in protein expression patterns. Exactly 54, 70, 36, and 45 proteins clustered in profiles 10(0, 0, 0,-1, 0, 0), 15(0, 1, 0, 1, 0, 1), 18(0, 1, 2, 2, 1, 0), and 19(0, 1, 2, 3, 4, 5), respectively. Muscle development and exoskeleton renewal were important processes throughout the development stages. In addition, protein synthesis, degradation, and digestion actively occurred, especially at the Z4 stage. These results provide novel insights into the mechanisms underlying larval development of swimming crab and can assist in larval rearing.     

Molecular characterization and expression profiles of four transformer-2 isoforms in the Chinese mitten crab Eriocheir sinensis

The transformer-2(tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster.In this study,sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized.Four tra-2 isoforms,designated as Estra-2a,Estra-2b,Estra-2c,and Estra-2d,were isolated.They all contained an RNA-recognition motif(RRM) and a linker region,which shared high similarity with other reported tra-2s.Sequence analysis revealed that Estra-2a,Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA.Compared with the other three isoforms,Estra-2d lacks the RS2 domain.Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg,and in the 2-4 cell and blastula stages compared with larval stages(P0.01),suggesting their maternal origin in early embryonic developmental stages.Notably,Estra-2a was highly expressed in male somatic tissues,while Estra-2c was significantly highly expressed in the ovary.These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab.Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.