Mulberry Leaf Polysaccharides Attenuate Oxidative Stress Injury in Peripheral Blood Leukocytes by Regulating Endoplasmic Reticulum Stress.

The present study assessed the protective effects and underlying mechanisms of mulberry leaf polysaccharides (MLPs) against hydrogen peroxide (H2O2)-induced oxidative stress injury in the peripheral blood leukocytes (PBLs) of Megalobrama amblycephala. Five treatment groups were established in vitro: the NC group (PBLs incubated in an RPMI-1640 complete medium for 4 h), the HP group (PBLs incubated in an RPMI-1640 complete medium for 3 h, and then stimulated with 100 µM of H2O2 for 1 h), and the 50/100/200-MLP pre-treatment groups (PBLs were pre-treated with MLPs (50, 100, and 200 µg/mL) for 3 h, and then stimulated with 100 µM of H2O2 for 1 h). The results showed that MLP pre-treatment dose-dependently enhanced PBLs' antioxidant capacities. The 200 µg/mL MLP pre-treatment effectively protected the antioxidant system of PBLs from H2O2-induced oxidative damage by reducing the malondialdehyde content and lactic dehydrogenase cytotoxicity, and increasing catalase and superoxide dismutase activities (p < 0.05). The over-production of reactive oxygen species, depletion of nicotinamide adenine dinucleotide phosphate, and collapse of the mitochondrial membrane potential were significantly inhibited in the 200-MLP pre-treatment group (p < 0.05). The expressions of endoplasmic reticulum stress-related genes (forkhead box O1α (foxO1α), binding immunoglobulin protein (bip), activating transcription factor 6 (atf6), and C/EBP-homologous protein (chop)), Ca2+ transport-related genes (voltage-dependent anion-selective channel 1 (vdac1), mitofusin 2 (mfn2), and mitochondrial Ca2+ uniporter (mcu)), and interleukin 6 (il-6) and bcl2-associated x (bax) were significantly lower in the 200-MLP pre-treatment group than in the HP group (p < 0.05), which rebounded to normal levels in the NC group (p > 0.05). These results indicated that MLP pre-treatment attenuated H2O2-induced PBL oxidative damage in the M. amblycephala by inhibiting endoplasmic reticulum stress and maintaining mitochondrial function. These findings also support the possibility that MLPs can be exploited as a natural dietary supplement for M. amblycephala, as they protect against oxidative damage.

Integrative Analysis of Transcriptome and Metabolome Reveals Molecular Responses in Eriocheir sinensis with Hepatopancreatic Necrosis Disease.

Hepatopancreatic necrosis disease (HPND) is a highly lethal disease that first emerged in 2015 in Jiangsu Province, China. So far, most researchers believe that this disease is caused by abiotic factors. However, its true pathogenic mechanism remains unknown. In this study, the effects of HPND on the metabolism and other biological indicators of the Chinese mitten crab (Eriocheir sinensis) were evaluated by integrating transcriptomics and metabolomics. Our findings demonstrate that the innate immunity, antioxidant activity, detoxification ability, and nervous system of the diseased crabs were affected. Additionally, metabolic pathways such as lipid metabolism, nucleotide metabolism, and protein metabolism were dysregulated, and energy production was slightly increased. Moreover, the IL-17 signaling pathway was activated and high levels of autophagy and apoptosis occurred in diseased crabs, which may be related to hepatopancreas damage. The abnormal mitochondrial function and possible anaerobic metabolism observed in our study suggested that functional hypoxia may be involved in HPND progression. Furthermore, the activities of carboxylesterase and acetylcholinesterase were significantly inhibited, indicating that the diseased crabs were likely stressed by pesticides such as pyrethroids. Collectively, our findings provide new insights into the molecular mechanisms altered in diseased crabs, as well as the etiology and pathogenic mechanisms of HPND.

Mulberry leaf meal: A potential feed supplement for juvenile Megalobrama amblycephala "Huahai No. 1".

This study was performed to evaluate the potential application of mulberry leaf meal (ML) and fermented mulberry leaf meal (FML) as feed supplements in aquatic animals for developing varieties of practical and economical feed ingredients. Juveniles Megalobrama amblycephala were fed a basal diet (35.7% crude protein, 10.4% crude lipid; control group) supplemented with 2.22% and 4.44% mulberry leaf meals (ML2, ML4) and fermented mulberry leaf meals (FML2, FML4) for 8 weeks. Generally, the two-way ANOVA showed the supplementation level exhibited a prominent effect on the growth performance and physiological status of fish. Furthermore, the two-way ANOVA showed the supplementary fermented mulberry leaf meal increased plasma complement 4 (C4) content (P < 0.05). The weight gain rate (WGR, 145.87%) and the specific growth rate (SGR, 1.63%) were significantly increased in FML2 group compared with the control group (P < 0.05). The muscle crude lipid content and hepatosomatic index (HSI) were higher in FML2 group than that in ML2 group (P < 0.05). The hepatic GSH content in ML4 group and CAT, T-SOD activities in FML4 group were significantly increased compared with the control group (P < 0.05). The hepatic MDA content in FML4 group was significantly decreased compared with the FML2 group (P < 0.05). Total cholesterol (TC) contents showed a significant decrease in ML4 and FML4 groups compared with the control group (P < 0.05). Regarding the gene expression, sirtiun 1 (Sirt1) gene expression was elevated in FML2 group compared with the ML2 group (P < 0.05). Compare to the control group, FML2 diet significantly increased the expression of i-kappa-B alpha (IKBα) gene in liver, and decreased the expression of forkhead box O1 α (FoxO1α), toll-like receptors 4 (TLR4) and nuclear factor-kappa B (NF-κB) genes (P < 0.05). In conclusion, 2.22% FML promoted the growth performance of M. amblycephala and enhanced the anti-inflammatory responses by inhibiting TLR4/NF-κB signaling pathway. On the other hand, 4.44% FML reduced plasma lipid content (hypolipedemic effect) and improved the hepatic antioxidant capacity of M. amblycephala.

Molecular characterization and expression of the autophagy-related gene Atg14 in WSSV-infected Procambarus clarkii.

Atg14 (autophagy-related gene 14), also known as Atg14L or Barkor (Beclin-1 associated autophagy-related key regulator), plays an important role in a variety of biological processes including immunity, development, tumor inhibition, longevity, and protection against some cardiac and neurodegenerative diseases. However, very few studies have characterized Atg14 expression in invertebrates, particularly crustaceans. Here, a novel Atg14 gene from Procambarus clarkii (named PcAtg14) was characterized via RACE technology. Bioinformatics analysis showed that the total length of the PcAtg14 gene sequence was 2,880 bp, and it was predicted to encode 488 amino acids. The results of homology comparison showed that PcAtg14 exhibited the highest homology with crustacean the American lobster (Homarus americanus). Quantitative real-time PCR expression analysis showed that PcAtg14 was expressed in all tissues of P. clarkii, with the hepatopancreas having the highest expression and the eyestalk exhibiting the lowest expression. Upon white spot syndrome virus (WSSV) infection, the relative expression of PcAtg14 in the hepatopancreas, muscle, hemocyte, gill, heart and epidermis were significantly up-regulated at different time periods. After PcAtg14 gene silencing via RNA interference (RNAi), the proliferation of WSSV in P. clarkii was significantly inhibited, which coincided with a significant increase in P. clarkii mortality and an increase in the expression of autophagy-related genes (ATGs). Transmission electron microscopy analysis demonstrated an increase in the number of autophagosomes in the hepatopancreas of the PcAtg14 gene silencing group compared to the control group after WSSV infection. Collectively, these results indicated that PcAtg14 suppressed autophagy by reduce the fusion of autophagosomes and lysosomes, thereby promoting WSSV replication in P. clarkii. The findings here therefore provide novel insights into the immune mechanisms through which P. clarkii responds to WSSV infection.

16S rRNA gene sequencing analysis reveals an imbalance in the intestinal flora of Eriocheir sinensis with hepatopancreatic necrosis disease.

Hepatopancreas necrosis disease (HPND) is a highly fatal disease that first appeared in Jiangsu Province, China, in 2015, and later spread to many other provinces, which had a severe impact on the culture of Chinese mitten crab (Eriocheir sinensis). Here, changes in the intestinal flora of healthy and HPND-affected Chinese mitten crabs were compared via 16S rRNA sequencing. Our findings indicated that Firmicutes, Bacteroidota, and Proteobacteria were the three dominant phyla in both healthy and HPND-affected crabs and exhibited no significant differences in α-diversity (richness p = 0.0892; evenness and diversity p = 0.0630). Furthermore, there were no significant changes in the abundance of Proteobacteria between the experimental groups. However, the abundance of Bacteroidota in the HPND group was significantly higher than that of the control group (HPND: 30.12%, Control: 16.60%), whereas the abundance of Firmicutes was significantly lower (HPND: 29.90%, Control: 50.55%). At the genus level, the abundance of Candidatus Bacilloplasma, Desulfovibrio, Bacteroides, and Aeromonas also differed significantly between groups (P < 0.05). Collectively, our study confirms an imbalance in the gut microbiota of Chinese mitten crabs with HPND and we speculate that this alteration may affect the metabolism and immune function of these organisms. Furthermore, we suspect that the structural changes in the intestinal flora of sick crabs observed in our study may be related to HPND.

PcVDAC promotes WSSV infection by regulating the apoptotic activity of haemocytes in Procambarus clarkii.

Apoptosis is programmed cell death that is strictly regulated by a series of related genes and is of great importance in resisting pathogen invasion and maintaining cell environment homeostasis. Among apoptotic proteins, the voltage-dependent anion channel protein (VDAC) plays a key role in the mitochondrial apoptosis pathway because of its close connection with changes in mitochondrial membrane potential. However, the role of VDAC in apoptosis and immune regulation in Procambarus clarkii is poorly understood. In this study, the VDAC gene in P. clarkii (PcVDAC) was cloned by rapid amplification of cDNA ends (RACE) technology. The gene was found to have a total length of 2277 bp, including a 194-bp 5'-UTR, 1234-bp 3'-UTR and 849-bp open reading frame (ORF), and to encode 282 amino acids. PcVDAC was expressed in all tissues tested, and its expression was upregulated after white spot syndrome virus (WSSV) infection (P < 0.05). The RNA interference (RNAi) method was used to explore the role of PcVDAC in WSSV infection. The results showed that the number of WSSV copies in haemocytes was significantly reduced after RNAi (P < 0.05), and the survival rate was significantly increased. In addition, after RNAi, the apoptosis rate was significantly increased (P < 0.05), the mitochondrial membrane potential was reduced (P < 0.01), and the expression of caspase-3 and other genes was upregulated (P < 0.05). These results indicate that PcVDAC promotes the replication of WSSV in P. clarkii by inhibiting haemocytes apoptosis. Therefore, the results presented in this paper provide new insights into the immune response of P. clarkii infected with WSSV.

Transcriptome analysis reveals the molecular mechanism of long-term exposure of Eriocheir sinensis to low concentration of trichlorfon.

Trichlorphon, a common organophosphorus pesticide (OPs), is widely used in aquaculture to prevent aquatic insects from infecting cultured objects as well as to control the excessive proliferation of plankton in water bodies. However, its repeated use time can contaminate water bodies and impart direct/indirect toxicity to beneficial aquatic species. However, the underlying mechanism regarding toxicity and cellular metabolism remains unclear. Understanding the mechanism would enable the standardized use and management of OPs and their use in the aquatic environment. Here, low concentration of trichlorphon (5 × 10-5 g/L) was used to construct a hepatopancreatic transcriptional library 30 d, 60 d and 90 d after exposure using RNA-Seq. We detected 649, 148, and 2949 DEGs in the hepatopancreas of E. sinensis for the Tri01 vs. Ctr01, Tri02 vs. Ctr02 and Tri03 vs. Ctr03 library, respectively. The results of KEGG pathway enrichment analysis showed that DEGs were mainly enriched in signal transduction, carbohydrate metabolism, transport and catabolism, endocrine system, and digestive system. Also, under trichlorfon stress, DEGs of E. sinensis were enriched in thyroid hormone signaling pathways, protein digestion and absorption, cancer pathways, etc. The significant DEGs were mainly related to metabolism and the apoptosis and autophagy pathways. This study lays a foundation for further revealing the effects of long-term trichlorfon stress on E. sinensis as well as the potential physiological toxicity. The relevant transcriptome data could provide a reference for the molecular toxicological evaluation of trichlorfon in aquaculture.

A novel Dicistro-like virus discovered in Procambarus clarkii with "Black May" disease.

The peak period of morbidity and death in cultured Procambarus clarkii is around May each year and is called the "Black May" disease. The pathogen causing "Black May" disease is believed to be a white spot syndrome virus (WSSV). In 2018, a significant number of P. clarkii died in the pond culture of Xinglong Township, Xuyi County. Two sampling tests on the affected pond showed that, in addition to WSSV, a novel Dicistro-like virus (PcDV) was present. Genomic sequence analysis indicated that this new virus belongs to the Dicistroviridae family, Picornaviridaes order. A high number of spherical particles were detected in gill tissues of P. clarkii with "Black May" disease by electron microscopy, a finding consistent with viruses from the Picornaviridaes order. From October 2018 to September 2019, we took monthly samples from Hubei, Jiangsu and Anhui provinces, and tested for the presence of PcDV and WSSV in P. clarkii. The detection rates of PcDV in P. clarkii peaked from April to June, consistent with the onset of the "Black May" disease. In conclusion, we believe that the discovery of PcDV will provide new research directions for investigating the pathogens causing "Black May" disease in P. clarkii.

Transcriptomic analysis of Procambarus clarkii affected by "Black May" disease.

Each year from April to May, high mortality rates are reported in red swamp crayfish (Procambarus clarkii) cultured in Jiangsu and other regions, in China, and this phenomenon has come to be known as "Black May" disease (BMD). Therefore, in order to investigate the possible causes of this disease, this study gathered BMD-affected P. clarkii samples and performed transcriptome analysis on hepatopancreas, gill, and muscle tissues. A total of 19,995,164, 149,212,804, and 222,053,848 clean reads were respectively obtained from the gills, muscle, and hepatopancreas of BMD-affected P. clarkii, and 114,024 unigenes were identified. The number of differentially expressed genes (DEGs) in gill, muscle, and hepatopancreas was 1703, 964, and 476, respectively. GO and KEGG enrichment analyses of the DEGs were then conducted. Based on KEGG pathway enrichment analysis, the most significantly differentially expressed pathways were mainly those involved with metabolism, human disease, and cellular processes. Further analysis of the significantly DEGs revealed that they were mainly related to the mitochondrial-mediated apoptosis pathway and that the expression of these DEGs was mostly down-regulated. Moreover, the expression of genes related to immune and metabolism-related pathways was also significantly down-regulated, and these significantly-inhibited pathways were the likely causes of P. clarkii death. Therefore, our results provide a basis for the identification of BMD causes.

Molecular characterization, expression and enzyme activity of three glutathione S-transferase genes from Eriocheir sinensis under pesticide stresses.

Glutathione S-transferases (GSTs) are a multifunctional protein superfamily that can catalyze the detoxification processes in an organism. In the present study, we determined the structure and function of GSTs in Chinese mitten crab (Eriocheir sinensis) by gene cloning, expression, and enzyme activity in order to investigate the metabolic detoxification of GSTs in the hepatopancreas and muscles under three pesticide (trichlorfon, ß-cypermethrin and avermectin) stresses. Multiple sequence alignment analysis showed that all the three Es-GST genes possessed N-terminal, and C-terminal domain as well as G-binding sites, while Es-GST2 and Es-GST3 contained Mu-type GST-specific Mu-loop structures. Phylogenetic tree analysis revealed that the three Es-GSTs belonged to the Mu-type GST of crustaceans. The quantitative real-time PCR revealed that the three Es-GSTS were expressed in 9 tissues of Eriocheir sinensis, with highest expression in hepatopancreas and muscle. The expression of the three Es-GSTS significantly increased in the hepatopancreas and muscle under the three pesticide stresses compared to the control group, and a steady increase in GST activity was observed. The study showed that the three Es-GSTs belong to the Mu-type GST of the crustaceans and might play an important role in the metabolic detoxification in Eriocheir sinensis.

A Meta-Transcriptomics Survey Reveals Changes in the Microbiota of the Chinese Mitten Crab Eriocheir sinensis Infected with Hepatopancreatic Necrosis Disease.

Infection of the freshwater Chinese mitten crab Eriocheir sinensis with hepatopancreatic necrosis disease (HPND) has been a major problem in the crab-cultivated Chinese Province of Jiangsu since 2015. To explore the etiology of HPND, meta-transcriptomic libraries of the hepatopancreata from crabs with and without HPND were constructed. Comparison analyses showed that there were no statistically significant differences in viral and microsporidial communities in the hepatopancreata of diseased and healthy crabs. Bacteroidetes, Proteobacteria, and Firmicutes were the most dominant bacterial phyla in the hepatopancreata of healthy crabs, with a combined prevalence of 93%. However, a decrease in bacterial diversity and a striking shift in the microbial composition were found in the hepatopancreata of crabs infected with HPND. Tenericutes was the most prevalent bacterial phylum in diseased crabs (31.82%), whereas its prevalence was low in healthy crabs (0.02%). By contrast, the prevalence of Bacteroidetes was significantly lower in crabs with HPND (3.49%) than in crabs without HPND (41.04%). We also found that the prevalence of Actinobacteria was higher in crabs with HPND (16.70%) than in crabs without the disease (4.03%). The major bacterial family within the Tenericutes phylum in crabs with HPND was detected by polymerase chain reaction and determined to be Mycoplasmataceae. In conclusion, there were striking changes in the microbiota of diseased and healthy crabs. Specifically, the prevalence of bacteria belonging to Tenericutes and Actinobacteria phyla increased, whereas the prevalence of bacteria belonging to the Bacteroidetes phylum decreased in crabs with HPND, clearly pointing to an association with HPND.

Cloning and Expression of Ecdysone Receptor and Retinoid X Receptor from Procambarus clarkii: Induction by Eyestalk Ablation.

Ecdysone receptor and retinoid X receptor are key regulators in molting. Here, full length ecdysone receptor (PcEcR) and retinoid X receptor (PcRXR) cDNAs from Procambarus clarkii were cloned. Full length cDNA of PcEcR has 2500 bp, encoding 576 amino acid proteins, and full length cDNA of PcRXR has 2593 bp, in which a 15 bp and a 204 bp insert/deletion splice variant regions in DNA binding domain and hinge domain were identified. The two splice variant regions in PcRXR result four isoforms: PcRXR1-4, encoding 525, 520, 457 and 452 amino acids respectively. PcEcR was highly expressed in the hepatopancreas and eyestalk and PcRXR was highly expressed in the eyestalk among eight examined tissues. Both PcEcR and PcRXR had induced expression after eyestalk ablation (ESA) in the three examined tissues. In muscle, PcEcR and PcRXR were upregulated after ESA, PcEcR reached the highest level on day 3 after ESA and increased 33.5-fold relative to day 0, and PcRXR reached highest the level on day 1 after ESA and increased 2.7-fold relative to day 0. In the hepatopancreas, PcEcR and PcRXR dEcReased continuously after ESA, and the expression levels of PcEcR and PcRXR were only 0.7% and 1.7% on day 7 after ESA relative to day 0, respectively. In the ovaries, PcEcR was upregulated after ESA, reached the highest level on day 3 after ESA, increased 3.0-fold relative to day 0, and the expression level of PcRXR changed insignificantly after ESA (p > 0.05). The different responses of PcEcR and PcRXR after ESA indicates that different tissues play different roles (and coordinates their functions) in molting.

Near-Full-Length Genome Sequence of a Novel Reovirus from the Chinese Mitten Crab, Eriocheir sinensis.

A novel Eriocheir sinensis reovirus (EsRV) was identified using deep-sequencing techniques in crabs afflicted with trembling disease (TD). Near-full-length genome sequences of 12 segments of EsRV were obtained. The genome of EsRV will facilitate further studies on the causative agent of TD.

In-depth transcriptome analysis of Coilia ectenes, an important fish resource in the Yangtze River: de novo assembly, gene annotation.

Coilia ectenes is an important teleost species in the Yangtze River and a model organism that can be used to study the protection of fish resources. In this report, we performed de novo transcriptome sequencing of ten cDNA libraries from the brain, gill, heart, intestine, kidney, liver, muscle, stomach, ovary, and testis tissues. A total of 352 million raw reads of 100 base pairs were generated, and 130,113 transcripts, corresponding to 65,350 non-redundant transcripts, with a mean length of 1520 bp, were assembled. BLASTx-based gene annotation (E-value<1 × 10(-5)) allowed the identification of 73,900 transcripts against at least one of four databases, including the NCBI non-redundant database, the GO database, the COG database, and the KEGG database. Our study provides a valuable resource for C. ectenes genomic and transcriptomic data that will facilitate future functional studies of C. ectenes.

In-depth transcriptome analysis of the red swamp crayfish Procambarus clarkii.

The red swamp crayfish Procambarus clarkii is a highly adaptable, tolerant, and fecund freshwater crayfish that inhabits a wide range of aquatic environments. It is an important crustacean model organism that is used in many research fields, including animal behavior, environmental stress and toxicity, and studies of viral infection. Despite its widespread use, knowledge of the crayfish genome is very limited and insufficient for meaningful research. This is the use of next-generation sequencing techniques to analyze the crayfish transcriptome. A total of 324.97 million raw reads of 100 base pairs were generated, and a total of 88,463 transcripts were assembled de novo using Trinity software, producing 55,278 non-redundant transcripts. Comparison of digital gene expression between four different tissues revealed differentially expressed genes, in which more overexpressed genes were found in the hepatopancreas than in other tissues, and more underexpressed genes were found in the testis and the ovary than in other tissues. Gene ontology (GO) and KEGG enrichment analysis of differentially expressed genes revealed that metabolite- and immune-related pathway genes were enriched in the hepatopancreas, and DNA replication-related pathway genes were enriched in the ovary and the testis, which is consistent with the important role of the hepatopancreas in metabolism, immunity, and the stress response, and with that of the ovary and the testis in reproduction. It was also found that 14 vitellogenin transcripts were highly expressed specifically in the hepatopancreas, and 6 transcripts were highly expressed specifically in the ovary, but no vitellogenin transcripts were highly expressed in both the hepatopancreas and the ovary. These results provide new insight into the role of vitellogenin in crustaceans. In addition, 243,764 SNP sites and 43,205 microsatellite sequences were identified in the sequencing data. We believe that our results provide an important genome resource for the crayfish.

Sex-lethal gene of the Chinese mitten crab Eriocheir sinensis: cDNA cloning, induction by eyestalk ablation, and expression of two splice variants in males and females.

Sex-lethal (Sxl) plays an important role in sex determination in insects. In this study, the full-length complementary DNA (cDNA) of Sxl of the Chinese mitten crab Eriocheir sinensis (EsSxl) was cloned. EsSxl is expressed during different developmental stages of embryos, and its expression level in the cleavage stage is lower than that in other stages of embryonic development, such as the original zoea stage when it reaches the highest level. The expression level of EsSxl in eight different tissues was investigated. EsSxl was expressed in seven examined tissues, excluding eyestalk, and the highest expression levels were observed in testis and hepatopancreas. EsSxl expression in testis was 13-fold higher than that in ovary. After induction by eyestalk ablation, changes in EsSxl expression were also tissue-specific, with EsSxl expression increasing by 2.6-fold and 11.5-fold in ovary and muscle, respectively, between 0 and 7 days after eyestalk ablation and decreasing by 2.0-fold in testis between 0 and 3 days after eyestalk ablation and by 265-fold in hepatopancreas between 0 and 7 days after eyestalk ablation. Two splice variants of EsSxl were identified, and the only difference between them was a 77-bp alternatively spliced intron that is transcripted in EsSxl1 and skipped in EsSxl2. Both variants were expressed in males and females, demonstrating the lack of a sex-specific expression pattern for Sxl in Chinese mitten crab.

Calcium-calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting.

Calcium-calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium-calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3,262 bp in length and has an open reading frame (ORF) of 1,038 bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1 day after ESA in hepatopancreas, and 3 days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.

Ecdysone receptor gene from the freshwater prawn Macrobrachium nipponense: identification of different splice variants and sexually dimorphic expression, fluctuation of expression in the molt cycle and effect of eyestalk ablation.

The full-length cDNA of an ecdysone receptor gene (MnEcR) from Macrobrachium nipponense was cloned and the expression of the gene was investigated. MnEcR maintained a relatively low expression level in the early stages of embryos, but from nauplius stage, a steady increase in MnEcR expression was detected, it had the highest expression level in zoea stage. MnEcR was highly expressed in the hepatopancreas and gills among ten different tissues examined. MnEcR was rapidly upregulated in the premolt stage and rapidly downregulated in the postmolt stage. The expression of MnEcR was remarkably downregulated after eyestalk ablation in M. nipponense. An 18-amino-acid insertion/deletion and a 49-amino-acid substitution were found in the coding region of MnEcR, resulting in four splice variants: MnEcR-L1, -L2, -S1 and-S2. The expression of four splice variants of MnEcR in gonads was investigated using RT-PCR. Interestingly, the expression patterns of these splice variants differed between males and females. The dominant splice variants in testis were MnEcR-S1 and -S2, while in ovary they were MnEcR-L1 and -S2, indicating specific roles for these splice variants in male and female individuals.

OsWRKY30 is activated by MAP kinases to confer drought tolerance in rice.

Both the WRKY transcription factor (TF) and MAP kinases have been shown to regulate gene expression in response to biotic and abiotic stresses in plants. Several reports have shown that WRKY TFs may function downstream of MAPK cascades. Here, we have shown that OsWRKY30 interacted with OsMPK3, OsMPK4, OsMPK7, OsMPK14, OsMPK20-4, and OsMPK20-5, and could be phosphorylated by OsMPK3, OsMPK7, and OsMPK14. Overexpression of OsWRKY30 in rice dramatically increased drought tolerance. Overexpression of OsWRKY30AA, in which all SP (serine residue followed by proline residue) sites were replaced by AP (A, alanine), resulted in no improvement in drought tolerance. In addition, the function of transcriptional activation of OsWRKY30 was impaired after SP was replaced by AP. These results proved that the phosphorylation of OsWRKY30 by MAPKs was crucial in order for OsWRKY30 to perform its biological function.

Genome-wide analysis of DNA methylation and gene expression changes in two Arabidopsis ecotypes and their reciprocal hybrids.

Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid over its parents in many traits, but the underlying molecular basis remains elusive. To investigate whether DNA methylation plays a role in heterosis, we compared at single-base-pair resolution the DNA methylomes of Arabidopsis thaliana Landsberg erecta and C24 parental lines and their reciprocal F1 hybrids that exhibited heterosis. Both hybrids displayed increased DNA methylation across their entire genomes, especially in transposable elements. Interestingly, increased methylation of the hybrid genomes predominantly occurred in regions that were differentially methylated in the two parents and covered by small RNAs, implying that the RNA-directed DNA methylation (RdDM) pathway may direct DNA methylation in hybrids. In addition, we found that 77 genes sensitive to methylome remodeling were transcriptionally repressed in both reciprocal hybrids, including genes involved in flavonoid biosynthesis and two circadian oscillator genes circadian clock associated1 and late elongated hypocotyl. Moreover, growth vigor of F1 hybrids was compromised by treatment with an agent that demethylates DNA and by abolishing production of functional small RNAs due to mutations in Arabidopsis RNA methyltransferase HUA enhancer1. Together, our data suggest that genome-wide remodeling of DNA methylation directed by the RdDM pathway may play a role in heterosis.