Regulation of vtg and VtgR in mud crab Scylla paramamosain by miR-34.

BACKGROUND: Vitellogenin (Vtg) is the precursor of major yolk protein and plays a crucial role in the maturation of oocytes and the production of eggs in oviparous animals. Vitellogenin receptor (VtgR) mediates the transport of Vtg explicitly to oocytes in the membrane. In a previous study, we found that miR-34 can regulate the expression of some eyestalk genes and affect reproduction in mud crab Scylla paramamosain, one of the most important economic crabs on the coasts of southern China. METHODS AND RESULTS: In this study, firstly, we found that miR-34 can target at 3'-UTR of Vtg and VtgR genes by using bioinformatic tools and predicted miR-34 might depress the expression of Vtg and VtgR. Secondly, the relative luciferase activity of HEK293T cells co-transfected with miRNA mimic and pmir-RB-REPORTTM-Vtg/VtgR-3'UTR was significantly lower than those of cells co-transfected with mimic NC and pmir-RB-REPORTTM-Vtg/VtgR-3'UTR. Finally, in vivo experiments showed that agomiR-34 could repress the expression of Vtg and VtgR genes, while Antigomir-34 could promote the expression of these two genes. CONCLUSIONS: These results confirm our hypothesis and previous published results that miR-34 may indirectly regulate ovarian development by binding to the 3'-UTR of Vtg and VtgR genes and inhibiting their expression.

Cloning, expression, and function of the Spdmrt-like gene in Scylla paramamosain.

BACKGROUND: The mud crab Scylla paramamosain is an economically important species for aquaculture in China and has sexually dimorphic between females and males. Understanding sex differentiation in this species is essential for the development of monosex aquaculture. The Dmrt genes play a vital role in sex differentiation in animals. METHODS AND RESULTS: In this study, two dmrt-like transcript variants, Spdmrt-like-tv1 and Spdmrt-like-v2, were cloned. SpDmrt-like-tv1 contained a DM domain, while SpDmrt-like-tv2 contained a DM and a DMA domain. Spdmrt-like-tv1 and Spdmrt-like-tv2 were both specifically expressed in testis. During testicular development, the expression level of Spdmrt-like-tv1 increased from stage I to stage II (P > 0.05) and then decreased from stage II to stage III (P < 0.05). The expression level of Spdmrt-like-tv2 in stages I and II was significantly higher than that in stage III (P < 0.05). During embryonic development, the expression level of Spdmrt-like-tv1 was higher in the mid-embryonic stage compared with the early and late stages, but the differences were not significant. Moreover, the expression level of Spdmrt-like-tv2 was stable and remained high throughout embryonic development. Furthermore, the expression level of Spdmrt-like-tv2 was significantly higher than that of Spdmrt-like-tv1. Knockdown of Spdmrt-like variants indicated that the regulative target gene of Spdmrt-like-tv1 was Spsox21, and the regulative target genes of Spdmrt-like-tv2 were Spfoxl2 and Spsox21. Combined with the results in our previously published peer-reviewed articles that the expression of Spfoxl2 in the testis was significantly higher than that in the ovary, and Spfoxl2 negatively regulated Spvtg expression. Spsox21 played a role in the development and maintenance of testis as well as in the process of neural development and regulation of body segmentation. CONCLUSION: Therefore, we suggest that Spdmrt-like-tv1 and Spdmrt-like-tv2 might be involved in testicular development and embryonic development, and Spdmrt-like-tv2 might play more important roles in these two developmental processes by regulating the expression of Spfoxl2 and Spsox21 due to its high expression.

Characterization of the glucocorticoid receptor of large yellow croaker (Larimichthys crocea) and its expression in response to salinity and immune stressors.

Glucocorticoids are steroidal hormones critical to stress responses in vertebrates. To gain further insight into the role of the glucocorticoid receptor (GR) in acute stress responses in teleost fish, the relevant cDNA of large yellow croaker (Larimichthys crocea; LcGR) was cloned using the rapid amplification of cDNA ends (RACE) technique. Multiple alignment of the amino acids (aa) of LcGR and the GR of other teleosts indicated LcGR contained four commonly conserved domains and lacked the 9-aa insert seen in GR1. Phylogenetic analysis of the amino acid sequence revealed that LcGR grouped most closely with the GR2 of other teleosts and can therefore be considered a GR2 subtype. In healthy L. crocea, Lcgr mRNA was found to be expressed at high levels in the gill, brain, and muscle tissue, expressed at intermediate levels in heart and stomach tissue, and expressed at low levels in the kidney, intestine, head kidney, liver, and spleen tissue. The response of L. crocea to acute low-salinity stress was tested, with a significant increase in plasma cortisol concentration after 3 h, peaking after 6 h, and gradually returning to base levels. Regarding changes of Lcgr expression in different body tissues under the stress, there was up-regulation of the Lcgr transcript in the brain, liver, and gill tissues, but not in muscle tissue. Responses to pathogen mimics were also tested. Injection with lipopolysaccharide resulted in Lcgr expression, with an increase-decrease-increase trend in the head kidney. In contrast, a down-regulation of Lcgr expression in the head kidney was observed throughout the experimental period upon injection of polyinosinic:polycytidylic acid, revealing different roles of Lcgr for different types of pathogens. The results offer novel insights about the effects of different stressors on GR gene expression in L. crocea, and can facilitate further investigations into stress responses in other mariculture fish species.

dLp/HDL-BGBP and MTP Cloning and Expression Profiles During Embryonic Development in the Mud Crab Scylla paramamosain.

Lipids are the main energy source for embryonic development in oviparous animals. Prior to the utilization and catabolism, lipids are primarily transported from the yolk sac to embryonic tissues. In the present study, cDNA encoding a circulatory large lipid transfer protein (LLTP) superfamily member, the precursor of large discoidal lipoprotein (dLp) and high-density lipoprotein/ß-1,3-glucan-binding protein (HDL-BGBP), named dLp/HDL-BGBP of 14,787 bp in length, was cloned from the mud crab Scylla paramamosain. dLp/HDL-BGBP was predicted to encode a 4,831 amino acids (aa) protein that was the precursor of dLp and HDL-BGBP, which were both detected in hemolymph by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. For the intracellular LLTP, three microsomal triglyceride transfer protein (MTP) cDNAs of 2,905, 2,897, and 3,088 bp in length were cloned from the mud crab and were predicted to encode MTP-A of 881 aa, MTP-B of 889 aa, and MTP-C of 919 aa, respectively, which were different merely in the N-terminal region and shared an identical sequence of 866 aa. During embryonic development, the expression level of dLp/HDL-BGBP consecutively increased from the early appendage formation stage to the eye pigment-formation stage, which indicated that HDL-BGBP is probably the scaffolding protein for yolk lipid. For the MTP gene, MTP-C accounted for ~70% of MTP mRNA from the blastocyst stage to the nauplius stage, as well as the pre-hatching stage; MTP-C and MTP-A expression levels were comparable from the early appendage formation stage to the late eye pigment-formation stage; MTP-A was extremely low in blastocyst and gastrula stages; MTP-B was expressed at a relatively low-level throughout embryo development. The variations in the expression profiles among MTP transcripts suggested that MTP might play roles in the lipid droplet maturation and lipoprotein assembly during embryonic development.

Discovery of the Dmrt gene family members based on transcriptome analysis in mud crab Scylla paramamosain.

Doublesex and mab-3 related transcription factors (Dmrts) play crucial roles in sex determination/differentiation and gonad development. The information on Dmrts and their functions are still scarce in mud crab Scylla paramamosain. In this study, 12 published transcriptome data of S. paramamosain were retrieved, pooled, and assembled. From the assembly, 7 Dmrt gene family members were identified and consisted of Spdmrt-like, Spdmrt-1a, Spdmrt-3, Spdmrt-11E, Spidmrt-1, Spdoublesex (Spdsx), and Spidmrt-2. These dmrt genes were predicted to encode 224 aa, 465 aa, 435 aa, 276 aa, 520 aa, 552 aa, and 266 aa protein precursors, respectively. The expression patterns of the dmrt genes were characterized by semi-quantitative PCR. The Spdmrt-like and Spdmrt-1a were exclusively detected in gonads, of which both expression levels in the testis were higher than that in the ovary. The Spdmrt-3, Spdmrt-11E, Spidmrt-1, Spdsx, and Spidmrt-2 were observed in various tissues; all these genes were sexually dimorphic except for dmrt-11E. Specifically, the expression level of Spdmrt-3 and Spidmrt-2 were higher in the testis than that in the ovary. On the contrary, the Spdsx and Spidmrt-1 expression level were higher in ovary than that in testis. The present study's findings provided a fundamental understanding of Dmrt gene family members involving sex determination/differentiation and gonad development in the S. paramamosain.

Large lipid transfer proteins in hepatopancreas of the mud crab Scylla paramamosain.

Large lipid transfer proteins (LLTPs) are extensively involved in various physiological processes. In the present study, five LLTP sequences encoding apolipocrustacein 1 (apoCr 1), apoCr 2, precursor of the large discoidal lipoprotein (dLp) and high density lipoprotein/ß-glucan binding protein (HDL-BGBP) (dLp-BGBP), microsomal triglyceride transfer protein (MTP) and clotting protein (CP) were identified in the hepatopancreas of Scylla paramamosain. Of these, apoCr 2, dLp-BGBP, and MTP were newly identified in this species, and the former two proteins were classified into the APO family while the later into the MTP family in phylogenetic trees. The apoCr 1 expression level was dramatically increased in the hepatopancreas towards ovarian maturation, which was extremely greater than that in the ovaries concurrently, likely to meet the considerable requirements of yolk protein and lipids for embryo development. The dLp-BGBP expression level in male crabs was comparable to that in female crabs, supporting HDL-BGBP acts as a major circulatory lipid carrier. The close phylogenetic relationship between dLp-BGBP and the scaffolding protein of lipid transfer particle implied dLp might facilitate lipid transfer between the hepatopancreas and HDL-BGBP-containing lipoproteins. The MTP expression level was positively related to ovarian development in both the hepatopancreas and ovaries, indicating MTP may be involved in lipoprotein assembly in the hepatopancreas and lipid droplet maturation in the ovaries. CP may play a crucial role in embryo development based on high expression level observed in the testes of mature crabs. Our findings provide novel insights into LLTP superfamily members and their functions in decapods.

The expression regulation of Cyclins and CDKs in ovary via miR-9c and miR-263a of Scylla paramamosain.

Scylla paramamosain is an economically important cultured crab species in China. Cyclins and cyclin-dependent kinases (CDKs) play important roles in regulations of cell cycle and ovarian development. MiRNAs can negatively regulate gene expression at the post-transcriptional level through base-complementary pairing with the 3'-untranslated region (3-UTR) of the target gene. In this study, bioinformatics prediction showed that miR-9c and miR-263a identified from our group's gonad miRNAome of S. paramamosain may bind to the 3' UTR region of cyclin A, cyclin B, cyclin E, cyclin H, CDK1, and CDK2. Furthermore, the results of double luciferase reporter gene assay showed that the luciferase activities of HEK293T cells co-transfected with miR-9c mimics/miR-9c inhibitor and the 3'-UTR plasmid vectors of the five genes (cyclin A, cyclin B, cyclin H, CDK1, and CDK2) were significantly decreased/increased compared with those in the NC (negative control) and BC (blank control) groups. The results in miR-263a were similar to miR-9c, but all of the six genes could be regulated by miR-263a. In in vivo experiments, agomiR-9c (miR-9c enhancer) injection resulted in decreases of cyclin A and CDK1 expression level, and reverse effects were observed by injecting antagomiR-9c. AgomiR-263a decreased the expression of cyclin A, cyclin B, cyclin H, CDK1, and CDK2, but antagomiR-263a increased their expression. Both the in vitro and in vivo experiments confirmed functions of miR-9c and miR-263a in cell cycle progress of ovarian development by expression regulation of cyclin A, cyclin B, cyclin E, cyclin H, CDK1, and CDK2. The findings provide new insights into the reproductive regulation mechanism in mud crab and further enrich the knowledge of cell cycle and ovarian development regulation in invertebrates.

Molecular cloning, characterization, and expression analysis of a sex-biased transcriptional factor sox9 gene of mud crab Scylla paramamosain.

Sox9 gene, a crucial member of the Sox gene family, is present in various organisms and involved in many physiological processes, especially in sex determination and gonad development. In this study, we reported a sox9 gene (designated as Spsox9) from Scylla paramamosain through analyzing published gonad transcriptome data. Meanwhile, the accuracy was validated by PCR technology, and the 3' sequences were cloned with 3' RACE technology. The full-length cDNA of Spsox9 is 2843 bp, consisting of a 243 bp 5' UTR, an 1124 bp 3' UTR, and a 1476 bp ORF encoding 491 amino acids. Furthermore, to better understand its conservation among crustacean species, the sox9 gene ortholog was identified in several other crustaceans species with their published transcriptome data, respectively. All of the Sox9 proteins identified in the current study had the common feature of Sox proteins (HMG domain) and were highly conserved among analyzed crustacean species. In all examined tissues, the Spsox9 was mainly expressed in the gonad (testis and ovary), eyestalk, and cerebral ganglion. During embryo development, Spsox9 was highly expressed in 5 pairs of appendages, 7 pairs of appendages, and eye-pigment formation stage. During ovary development, the expression level of Spsox9 remained stable in the first 4 stages (O1-O4) and decreased in the tertiary vitellogenesis (O5) stage. During testis development, the expression level of Spsox9 was highest in the spermatid stage (T2) and was significantly different from that in the spermatocyte stage (T1) and mature sperm stage (T3) (p < 0.05). In addition, Spsox9 exhibited a sex-biased expression pattern in T1 and O1. These present results indicated that the Spsox9 gene might play crucial roles in the gonad and embryo development of mud crab.

Characterization and antimicrobial evaluation of a new Spgly-AMP, glycine-rich antimicrobial peptide from the mud crab Scylla paramamosain.

Antimicrobial peptide (AMP) is a crucial component of the innate immune system in crustaceans. In mud crab, Scylla paramamosain, a commercially important species, a glycine-rich antimicrobial peptide (Spgly-AMP) gene was newly identified and putatively encoded a 26aa signal peptide and 37aa mature peptide. To understand the function of Spgly-AMP, the expression profile of Spgly-amp gene was characterized, which showed Spgly-amp was expressed widely in most tissues of adult crabs with the highest expression level in hemocytes. After Vibrio parahaemolyticus, PGN, or Poly I:C stimulations, the expression level of Spgly-amp was significantly up-regulated in the hemocytes. In antimicrobial assays, chemically synthesized Spgly-AMP peptides exhibited strong antibacterial activities against both Gram-positive and Gram-negative bacteria and high thermal stability after high-temperature heating. These findings in the present study verified the importance of the Spgly-AMP in defense of pathogenic bacteria infection in the mud crab and provided a promising candidate of antimicrobial agents in the crab aquaculture.

Histological Variation in Blubber Morphology of the Endangered East Asian Finless Porpoise (Neophocaena asiaeorientalis sunameri) with Ontogeny and Reproductive States.

The highly lipid-rich blubber in the hypodermis is a specialized structure that functions in thermoregulation, energy storage, buoyancy control, locomotion, and streamlining the body shape in marine mammals. The key objective of this study was to investigate blubber development in the East Asian Finless Porpoise (Neophocaena asiaeorientalis sunameri) across the ontogenetic (fetuses, calves, and adults) and reproductive states (adults, pregnant, and lactating). Blubber samples were collected from East Asian Finless Porpoises (EAFP) that were accidentally caught in the fishing nets in the Bohai/Yellow Sea from late April to mid May of 2015. The mean blubber depth was significantly thinner in fetuses across the ontogenetic groups and significantly thicker in pregnant and lactating vs. adult females across the reproductive states. Across the four regions in each group, we did not find significant variations in blubber depth. However, the correlations between body length and weight vs. blubber depth was significant. Histological observation identified three layers of stratified blubber with a significantly smaller adipocyte cell size in fetuses and a significantly higher area ratio of structural fiber in the middle and inner layers across the ontogenetic groups. Across the reproductive states, we did not observe a statistically significant difference in the adipocyte cell size or area ratio of the structural fiber. Our results suggest that prenatal blubber growth is characterized by an increase in the adipocyte cell count, while postnatal growth is the result of an increase in cell size. They also indicate that ontogeny can affect blubber depths and cellular measurements in the EAFP.

The possible effects of anthropogenic acoustic pollution on marine mammals' reproduction: an emerging threat to animal extinction.

For about 119 species of cetaceans and other aquatic animals, sound is the key source of learning about the environment, navigation, communication, foraging, and avoiding predators. However, in the recent era, the introduction of large quantities of anthropogenic noise into the ocean has significantly altered the ocean's acoustic environment. The anthropogenic noises travel very long distances, blanketing enormous areas. This can affect cetaceans, either by direct killing or compromising hearing, navigation, communication, predation, as well as normal behaviors. It has been suggested that acoustic pollution could possibly negatively affect cetacean reproduction, which is harmful for endangered and threatened species. However, it is still unknown how acoustic pollution can suppress cetacean reproduction. This is the first comprehensive review article, which focuses on the possible consequences affecting the reproduction of marine mammals resulting from acoustic pollution.

Hematologic and biochemical differences between two free ranging Yangtze finless porpoise populations: The implications of habitat.

The goals of this study were to compare the serum chemistry and hematology values of wild and semi-natural free-ranging Yangtze Finless Porpoises (Neophocaena asiaeorientalis ssp. asiaeorientalis) populations and to ascertain how these values change with the different environmental condition. For this study, samples were collected from 81 YFPs, 35 living in the wild and 46 living in a semi-natural reserve. Each population was divided into 8 life history categories; Male Calf, Female Calf, Juvenile Male, Juvenile Female, Adult Male, Pregnant, Lactating and Pregnant plus Lactating. Statistically significant differences in the various parameters were observed in the same life history categories for both populations. Generally, Lipid Profile, Hepatic Enzymes, Creatine Kinase, Red Blood Cells, Hemoglobin, Hematocrit and Neutrophils were significantly higher in the Tian-E-Zhou Oxbow population while, Creatinine, Phosphate, Lactate Dehydrogenase, Bilirubin and Lymphocytes were significantly higher in the Poyang Lake YFPs. Across the groups in the Tian-E-Zhou Oxbow population, a significant decrease in serum Albumin, Alkaline Phosphatase and Calcium, while a significant increase in the Neutrophils and Platelets was observed. Similarly, in the Poyang Lake, Alkaline Phosphatase levels in the Female Calves group, High Density Lipoprotein Cholesterol in Lactating group, basophil counts in Pregnant plus Lactating group, lymphocytes counts in Juvenile Females group and Globulin and Total Protein levels in Pregnant group were significantly higher. This study in health assessments can help us to understand the effect of sex, age, reproductive status and environmental conditions on the well-being of Yangtze Finless Porpoises.

Assessment of Yangtze Finless Porpoises (Neophocaena asiaorientalis) through Biochemical and Hematological Parameters.

Ghulam Nabi, Yujiang Hao, Xianyuan Zeng, and Ding Wang (2017) For the management of endangered species, a periodic health assessment is important, as diet in captivity is restricted due to cost and some nutrients are lost during the processing, storage and thawing of sh. The objective of this study was to compare and assess the nutritional, as well as the physiological health of both the captive and free-ranging Yangtze Finless Porpoises (YFPs) through biochemical markers, selected electrolytes, enzymes and hematological parameters. Our results showed statistically significantly (P < 0.05) higher levels of some biochemical markers (HDL-c/LDL-c, globulin, TP, Urea, Creatinine and BUN), enzymes (GGT), electrolytes (K+, Na+, PO4 3- and Mg2+) and hematological (WBCs, lymphocytes, eosinophil) parameters in wild compared to the captive populations. However, the captive population also showed significantly (P < 0.05) higher levels of some biochemical markers (LDL-c, albumin and albumin/globulin), enzymes (AMS), electrolytes (Cl-) and complete blood count (neutrophil, monocytes and basophil) parameters versus wild populations. Differences in the parameters of captive YFPs could be due to their limited diet of only three fish species as well as their environment (captivity). Whereas, wild YFPs, continuously feed on a large variety of live fish species and shrimp as they travel long distances. Our results suggest that mineral supplements be added to their diet. As well, improved physical fitness training and hygienic conditions are required for the effective management of captive finless porpoises.

Topographical distribution of blubber in finless porpoises (Neophocaena asiaeorientalis sunameri): a result from adapting to living in coastal waters.

BACKGROUND: Blubber has many functions, among which energy storage, thermoregulation, buoyancy, and hydrodynamic streamlining are the most frequently cited. Within and between taxa, variations in its structure and distribution likely reflect different adaptations of a species to its life history requirements, environment, health, and function. Here, we use ultrasound to describe the distribution of blubber in the finless porpoise (Neophocaena asiaeorientalis sunameri) based on examinations of 34 fresh cadavers recovered as accidental fisheries bycatch. RESULTS: Measurements of blubber depth determined by ultrasound positively correlated with conventional measurements using a scalpel and calipers. Whereas conventional surgical incision and visual examination revealed two layers of blubber, ultrasound revealed up to three layers; thus, ultrasound reveals additional structural detail in blubber while crude necropsy techniques do not. Across life history categories, ultrasound revealed the distribution of inner blubber to be topographically consistent with that of full-depth blubber. Blubber in the dorsal region was stratified into three layers and was significantly thicker than that in the lateral and ventral regions, in which a middle layer was normally absent. CONCLUSIONS: Ultrasoundprovides a fast, effective, and accurate means to determine blubber thickness and structure, and thus, assessment of the health of fresh finless porpoise carcasses. Blubber depth is determined largely by the thickness of the inner and middle layers, wherein lipids are concentrated. The thickening of blubber in the dorsal thoracic-abdominal region suggests multiple roles of thermal insulation, lipid storage, and, we speculate, to facilitate vertical stability in the complex shallow and estuarine waters in which this animal absent of a dorsal fin occurs.