Identification and Profiling of MicroRNAs During Embryogenesis in the Red Claw Crayfish Cherax quadricarinatus.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that constitute a broad layer of gene regulation at both transcriptional and post-transcriptional levels from prokaryotes to eukaryotes. In embryonic development, they regulate the complex gene expression associated with the complexity of embryogenesis. There is little information about miRNAs in the red claw crayfish (Cherax quadricarinatus), an important commercial species and a potential biological model. In the present study, miRNAs and their target genes were identified during three embryonic developmental stages of C. quadricarinatus. Nineteen known miRNAs and 331 novel ones belonging to 50 miRNA families were obtained. A total of 113 differentially expressed miRNAs were identified, and 2,575 target genes were predicted, of which 1,257 were annotated. Additionally, 63 target genes of 9 miRNAs in C. quadricarinatus were found to be related to embryonic development. For example, miR-10 and its target genes may regulate the nervous system development and body segmentation and miR-2788 may regulate cell proliferation to impact embryonic development. Moreover, miR-28 (target gene tutl), miR-50 (target gene fbx5), and miR-1260b (target gene sif) may co-regulate eye development of embryonic C. quadricarinatus. These miRNAs together with their target genes constitute a network for regulating the development of tissues and organs in the embryo of C. quadricarinatus. Our results lay a foundation for further study on the fundamental molecular and developmental mechanism of crustacean embryogenesis.

Dietary Different Replacement Levels of Fishmeal by Fish Silage Could Influence Growth of Litopenaeus vannamei by Regulating mTOR at Transcriptional Level.

Fish silage (FS) has been confirmed as a high-quality feed ingredient because of its balanced nutrition, low cost, and environmental friendliness. In the present study, we evaluated the performance of replacing fishmeal by FS in the diet of white shrimp, Litopenaeus vannamei. Five isonitrogenous (410 g kg-1) and isolipidic (75 g kg-1) diets were formulated with replacement of fishmeal by 0% (FM), 25% (FS25%), 50% (FS50%), 75% (FS75%), and 100% (FS100%) FS. After an 8-week trial, shrimps fed low FS diets (FM and FS25%) had significantly higher final weight (FW), weight gain (WG), and specific growth ratio (SGR) (P < 0.05). No significant differences were found in body composition and most antioxidant enzyme activities of all groups (P > 0.05). Compared to high FS groups (FS75% and FS100%), low FS replacement levels (0 and 25%) had enhanced trypsin activity. And trypsin transcriptional level presented a similar trend with trypsin activity. In terms of intestinal histopathology, no obvious histological damage was observed in the intestine of all groups. tor and s6k of low replacement level groups (FM and FS25%) were significantly upregulated (P < 0.05), which indicated activation of mammalian target of rapamycin (mTOR) signaling pathway in low FS groups at transcriptional level. The enhanced performances of growth and mTOR signaling pathway in low FS groups (FM and FS25%) provided us some insights into the regulation mechanism of nutrient signal on growth. Based on the above, dietary FS could influence the growth of shrimp by regulating mTOR at the transcriptional level, and FS is a potential substitute of fishmeal in shrimp feed.

A comparative transcriptomic analysis in late embryogenesis of the red claw crayfish Cherax quadricarinatus.

The red claw crayfish (Cherax quadricarinatus) is an emerging and important commercial species in several countries, and is also a potential biological model in crustacean biology. However, its molecular embryonic development mechanism remains largely unknown because of a lack of genomic resources and systematic research. A comprehensive and integrated transcriptomic analysis is necessary to reveal the cell biological function, gene expression profiles, and embryo patterning that occur during embryogenesis. In the present study, transcriptomic profiles of C. quadricarinatus embryos during three developmental stages were investigated by high-throughput Illumina sequencing technology, and the genes related to development were further analyzed. In total, 49,436 unigenes were assembled and clustered, in which 13,727 were annotated in the Nonredundant database, 5087 were classified based on Gene Ontology annotations, and 2735 were associated with 189 Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, gene expression differences among the embryos stages were analyzed, and 6658 differentially expressed genes (DEGs) were identified. In total, 3300, 5211, and 1262 DEGs were identified between the eye pigments forming stage (EP) and prepare-hatching stage (PH), EP and larvae (L), as well as PH and L; meanwhile, 1595, 2540 and 680 DEGs were annotated, respectively. The fundamental developmental genes related to apoptosis, neurogenesis, and segmentation, as well as signaling pathways related to Hedgehog, MAPK, Wnt, TGF-ß and Notch, showed higher expression during the EP stage than in other two stages, indicating that the EP stage has more active biological processes than the latter stages. This transcriptome studies gene expression at different stages of embryonic development and the datasets provide a basis for understanding crustacean developmental biology and guiding seedling production.

Exploration of the influence of surface proteins on the probiotic activity of Lactobacillus pentosus HC-2 in the Litopenaeus vannamei midgut via label-free quantitative proteomic analysis.

Our previous work showed that using Lactobacillus pentosus HC-2 as a probiotic could improve the growth performance, immune response, gut bacterial diversity and disease resistance of Litopenaeus vannamei. However, the probiotic mechanism had not been fully characterized. In the present study, histology and proteomic analysis were performed to explore the influence of HC-2 surface protein on its probiotic effects on L. vannamei after feeding either the intact surface proteins, the probiotic treated with lithium chloride (LiCl) to remove noncovalently bound surface proteins or no probiotic for four weeks. Histological observation found that feeding with normal HC-2 obviously improved the intestinal histology and enhanced the protective effect against pathogen damage, but feeding with LiCl-treated HC-2 did not improve the intestinal environment. A total of over 2764 peptides and 1118 uniproteins were identified from the L. vannamei midgut; 211 proteins were significantly differentially expressed in the normal HC-2 group compared with the control group; 510 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the control group, and 458 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the normal HC-2 group. GO/KEGG enrichment analysis of the significantly different proteins demonstrated that feeding normal HC-2 mainly induced immune response, metabolic, cell adhesion and cell-cell signaling-related protein upregulation, which contributed to bacterial adhesion and colonization in the midgut to improve the shrimp immune system and growth, but these proteins were suppressed after the shrimp were fed bacteria deprived of surface proteins. Taken together, these results indicate that the surface proteins were indispensable for HC-2 to execute probiotic effects in the shrimp midgut.

The effect of rearing density on immune responses of hepatopancreas and intestine in Litopenaeus vananmei against Vibrio paraheamolyticus E1 challenge.

Rearing density and disease management are considered as pivotal factors determining shrimp farm productivity and profitability. To systematically investigate the potential mechanisms for density-related differences between disease susceptibility and rearing densities, we conducted comparative transcriptome analysis of the molecular differences between hepatopancreas and intestine of Litopenaeus vannamei under two different rearing densities (800- and 400- shrimp/m3) for 15 d and further analyzed the differences in immune response to Vibrio parahaemolyticus E1 (VPE1) raised under two density conditions. Totally 45 different expression genes (DEGs) were identified in the hepatopancreas under two different rearing densities, the DEGs were grouped into four processes or pathways related to animal immune system. Then, exposure to the VPE1 resulted in 639 DEGs, involved into fourteen immune related processes or pathways. In the intestine, seventeen processes or pathways related to the immune system were identified among the 5470 DEGs under two different rearing densities. 279 DEGs were identified post VPE1 challenge, classified into five processes or pathways associated with the immune system. Meanwhile, the results of growth performance, histopathology and the activities of antioxidant enzymes in the hepatopancreas and intestines of shrimp showed that high density decreased weight gain rate (63.20 ±â€¯1.67% and 18.73 ±â€¯3.35% in the high and low rearing density groups, respectively), severely destroyed the histopathology and inhibited the antioxidant enzymes activities. This study demonstrated that rearing density in L. vannamei significantly impacts susceptibility to the VPE1, via altered transcriptional challenge responses, and thus higher mortality due to disease.