Integrated transcriptome analysis of immune-related mRNAs and microRNAs in Macrobrachium rosenbergii infected with Spiroplasma eriocheiris.

Macrobrachium rosenbergii (M. rosenbergii), is a major aquaculture species in China and Southeast Asia. However, infection with Spiroplasma eriocheiris (S. eriocheiris) has caused huge economic losses to the cultivation of M. rosenbergii. Currently, there are few reports on the immune response mechanism of M. rosenbergii that are infected with S. eriocheiris. To clarify the immune response mechanism of M. rosenbergii infected with S. eriocheiris, the key immune genes which respond to the infection with the pathogen and the regulation of related microRNAs (miRNAs) on them were identified. In this study, the mRNA and miRNA transcriptome of hepatopancreas of M. rosenbergii at different infection stages were analyzed using high-throughput sequencing and qRT-PCR. In the mRNA transcriptome, 27,703 and 33,402 genes were expressed in healthy and susceptible M. rosenbergii, respectively. By digital gene-expression profiling analysis, 23,929 and 24,325 genes were expressed, and 223 and 373 genes were significantly up-regulated and down-regulated, respectively. A total of 145 key genes related to Toll, IMD, JAK/STAT and MAPK were excavated from the transcriptome. In the miRNA transcriptome, 549 miRNAs (Conserved: 41, PN-type: 83, PC-type: 425) were sequenced, of which 87 were significantly up-regulated and 23 were significantly down-regulated. Among the related immune pathways, there are 259 miRNAs involved in the regulation of target genes in the Toll and IMD pathways, 231 JAK/STAT pathways and 122 MAPK pathways. qRT-PCR differential detection of immune-related miRNAs and mRNAs showed that 22 miRNAs with significant differences (P < 0.05) such as mro-miR-100, PC-mro-3p-27 and PN-mro-miR-316 had corresponding regulatory relationships with 22 important immune genes such as TLR2, TLR3, TLR4, TLR5, MyD88, Pelle and Relish in different stages after infection. In this study, the immune genes and related regulatory miRNAs of M. rosenbergii in response to S. eriocheiris infection were obtained. The results can provide basic data to further reveal the immune defense mechanism of M. rosenbergii against S. eriocheiris infection.

Full-length transcriptome and long non-coding RNA profiling of whiteleg shrimp Penaeus vannamei hemocytes in response to Spiroplasma eriocheiris infection.

Spiroplasma eriocheiris (S. eriocheiris) infection causes a significant economic loss in Penaeus vannamei (P. vannamei) culture industry. However, the response of P. vannamei hemocytes to S. eriocheiris infection has not been extensively studied. In this study, we conducted full-length transcriptome and long non-coding RNA (lncRNA) analyses of P. vannamei hemocytes by a challenge test with S. eriocheiris. Following assembly and annotation, there were 8077 high-quality unigenes. A total of 1168 differentially expressed genes (DEGs) were obtained, including 792 up-regulated and 376 down-regulated genes by differential expression analysis. Gene ontology (GO) enrichment analysis showed that the up-regulated DEGs were mainly clustered into immune system process, defense response, cell cycle and organelle organization. On the other hand, the down-regulated DEGs included that genes that were mainly clustered into metabolic processes related to organic compounds, metabolic process and cellular metabolic process. Protein-protein interaction (PPI) network analysis of DEGs indicated that the pivotal gene interactions were connected to stress response, immune system process and cell cycle. The lncRNA analysis identified multiple lncRNAs, which were highly co-expressed with the immune-related genes, such as lncRNA transcript-12631 and transcript-12631, suggesting that lncRNAs may be involved in the regulation of immune defense in shrimp hemocytes. Additionally, 20 hub unigenes and putative lncRNAs related to immune system were validated by quantitative real-time PCR (qRT-PCR), validating the reliability of RNA-Seq. This study revealed a close connection between the immune and metabolic systems of S. eriocheiris infected P. vannamei.

Effects of ammonia-N exposure on the growth, metabolizing enzymes, and metabolome of Macrobrachium rosenbergii.

Ammonia nitrogen elevated is one of the commonest problem in the aquatic system, which caused a great threat to the survival and growth of prawn. However, little is know about the ammonia metabolism and detoxification strategy of prawn. In this study, the effects of ammonia-N (0, 0.108, 0.216, 0.324, or 0.54 mg L-1) on growth and metabolizing enzymes in hepatopancreas of Macrobrachium rosenbergii, including glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamate dehydrogenase (GDH), were investigated. The metabolome of its muscle was also analyzed after exposure to ammonia-N (0, 0.108, 0.324, or 0.54 mg L-1) for 20 days. The survival rate of M. rosenbergii decreased significantly after treatment with 0.54 mg L-1 ammonia-N compared with that in the other groups. However, ammonia-N had no significant effect on the growth of the river prawn after exposure for 20 days. GS activity increased significantly after exposure to 0.108 mg L-1 ammonia-N compared with the control and other ammonia-N-treated groups. Hepatopancreatic GDH activity was lower in the prawns treated with 0.216, 0.324, or 0.54 mg L-1 ammonia-N than in the control by 34.70%, 38.80%, or 41.94%, respectively. Ammonia-N had no significant effect on hepatopancreatic AST or ALT activity. Urea nitrogen was higher in the prawns treated with 0.216 mg L-1 ammonia-N than in the control or those treated with 0.54 mg L-1 ammonia-N. Ammonia-N had significant effects on the lipid, carbohydrate. and protein metabolism of M. rosenbergii, including purine metabolism, amino sugar and nucleotide sugar metabolism, α-linolenic acid metabolism, arginine and proline metabolism, glutathione metabolism, and phosphonate and phosphate metabolism, and on the terpenoid biosynthesis, lysine degradation, and lysine biosynthesis pathways. High concentrations of ammonia-N stress increased the content of glutamate and arginine, which may participate in the urea cycle, which synthesizes glutamine or urea to eliminate ammonia toxicity.

Habitual feeding patterns impact polystyrene microplastic abundance and potential toxicity in edible benthic mollusks.

That increasing microplastics (MPs, <5 mm) eventually end up in the sediment which may become a growing menace to diverse benthic lives is worthy of attention. In this experiment, three edible mollusks including one deposit-feeding gastropod (Bullacta exarate) and two filter-feeding bivalves (Cyclina sinensis and Mactra veneriformis) were exposed to polystyrene microplastic (PS-MP) for 7 days and depurated for 3 days. PS-MP numbers in the digestive system and non-digestive system, digestive enzymes, oxidative stress indexes, and a neurotoxicity index of three mollusks were determined at day 0, 3, 7, 8 and 10. After seven-day exposure, the PS-MP were found in all three mollusks' digestive and non-digestive systems. And PS-MP in M. veneriformis (9.57 ± 2.19 items/individual) was significantly higher than those in C. sinensis (3.00 ± 2.16 items/individual) and B. exarate (0.83 ± 1.07 items/individual) at day 7. Three-day depuration could remove most of the PS-MP in the mollusks, and higher PS-MP clearance rates were found in filter-feeding C. sinensis (77.78 %) and M. veneriformis (82.59 %) compared to surface deposit-feeding B. exarate (50.00 %). The digestive enzymes of B. exarate significantly reacted to PS-MP exposure, while oxidative responses were found in C. sinensis. After three-day depuration, the changes of digestive enzymes and the oxidative states were fixed, but neurotoxicity induced by PS-MP was not recoverable. Besides, it is noteworthy that changes of digestive enzymes and acetylcholinesterase are related to feeding patterns.

Molecular cloning and characterization of an estrogen receptor gene in the marine polychaete Perinereis aibuhitensis.

Estrogen receptors (ERs) are the primary mediators of estrogen signaling, and play crucial roles in the reproduction and development of vertebrates. The full-length cDNA of Perinereis aibuhitensis estrogen receptor (paER) was cloned and characterized for the first time. The positions of the cysteine residues and the residues around them, which constitute two zinc finger motifs and a P-box, are conserved in both vertebrates and invertebrates. A phylogenetic analysis revealed that paER is an orthologue of ER in the polychaete Platynereis dumerilii. A tissue distribution analysis of paER mRNA showed that it is expressed in various tissues, including the body wall, head, esophageal gland, esophagus, stomach, and most strongly in the intestines. Its expression was also measured in P. aibuhitensis after exposure to 17ß-estradiol (E2) for 48h. The paER mRNA levels in the body wall were measured after 6, 12, 24, and 48h in E2-exposed and control animals. However, no significant differences in paER expression were observed between them at any time point. This report describes the first molecular characterization of full-length paER and its tissue-specific expression in P. aibuhitensis.

[Effects of the environmental hormone cypermethrin on the reproduction of Brachionus calyciflorus].

The toxic effects of the antiandrogen cypermethrin on B. calyciflorus were investigated using the 2 d population growth rate (r) of B. calyciflorus. The effects of low-dose of cypermethrin (0.001-0.316 mg x L(-1)) on the 3 d population parameters, the 7 d resting egg production and the hatching rate of resting eggs of B. calyciflorus were also studied. The 2 d population parameters of B. calyciflorus were used to estimate the growth ability of larvae hatched from the resting eggs formed in cypermethrin. The 3 d population parameters were used to estimate the effect on the the reproduction of the offspring of B. calyciflorus, whose parent generation was preexposed in cypermethrin. The results indicated that the logarithmic concentration of cypermethrin had a linear negative correlation with the population growth rate. The half maximal effective concentration (EC50), the lowest observed effect concentration (LOEC) and no-observed effect concentration (NOEC) of cypermethrin were 14.22 mg x L(-1), 10 mg x L(-1) and 3.16 mg x L(-1), respectively. The resting egg production was decreased by 41.23% at 0.031 6 mg x L(-1) cypermethrin. The hatching rate of resting eggs was significantly decreased when formed in 0.031 6 mg x L(-1) cypermethrin. The population growth rate and mictic rate were significantly decreased for the individuals hatched from the resting eggs formed in cypermethrin. The 3 d population growth rate was significantly decreased by 15.96% compared to the control when their parent generation was pre-exposed in 0.316 mg x L(-1) cypermethrin. The results showed that the 2 d population growth rate of B. calyciflorus was less sensitive to cypermethrin. Low doses of cypermethrin reduced the resting egg production, the hatching rate of resting eggs and the population growth of hatched resting eggs, which would thus decrease the contribution of the offspring to the population growth.

[External morphological characteristics during the embryonic development of Portunus pelagicus].

The embryonic development of Portunus pelagicus was studied under laboratory conditions at a water temperature of 25-26 Degrees Celsius, salinity of 30, and pH of 7.8-8.4. The embryogenesis of Portunus pelagicus was divided into six stages: cleavage, blastula, gastrula, nauplius, metanauplius, and protozoea. Embryogenesis lasted about 300 h post spawning. Eggs began superficial cleavage about 28 h after spawning when the nucleus appeared at the surface of the egg till the egg divided into 16 cells. The blastula stage was observed about 40 h post spawning and gastrula stage appeared when the presumptive endoderm and other cells near them invaginated. The fourth-stage of embryogenesis, nauplius, was characterized by three pairs of appendages appearing about 90 h post spawning, while metanauplius, the fifth-stage of embryogenesis, was characterized by five pairs of appendages, which appeared about 110 h post spawning. The sixth stage of embryogenesis was protozoea, which was characterized by seven pairs of appendages appearing about 140 h post spawning. The compound eye, heart and pigment cells were also found in the protozoea stage. After the natatory seta formed on the top of maxilliped, the protozoea developed into the zoea at the time of hatching (about 300 h post spawning).