miR-217 through SIRT1 regulates the immunotoxicity of cadmium in Cyprinus carpio.

MicroRNAs (miRNAs) play a critical role in regulating the response of animals exposed to heavy metal stress. As a globally dispersed heavy metal in aquatic ecosystems, cadmium (Cd) is highly toxic to many aquatic species. However, little is known about the miRNA response to Cd stress in fish. To investigate the regulatory effect of miRNAs in response to Cd, common carp (Cyprinus carpio) were exposed to Cd2+-containing water (0.005 mg/L, 0.05 mg/L, 0.5 mg/L) for 30 days. After exposure, Cd2+ contents were significantly higher in the kidneys of C. carpio compared to other tissues, when exposed to 0.5 mg/L Cd2+. Hematoxylin and eosin staining images revealed that elevated Cd induced inflammatory damage in the kidneys of C.carpio. Further, miRNA sequencing revealed nine differentially expressed miRNAs (miR-217, miR-205 and seven novel miRNAs) in the kidneys, between 0.5 mg/L Cd2+ exposure and control groups. Potential target mRNAs of miRNAs suggest that miR-217 is involved in immunotoxicity. miR-217 agomir was intraperitoneally administered to C. carpio and RT-PCR revealed that the expression of IL-8 and SIRT1 decreased, while TLR-4, TRAF6, NF-kB, TNF-α, IL-1ß, and TGF-ß increased in the kidneys of C.carpio. Additionally, the expression of SIRT1 decreased, while the expression of other mRNAs increased in kidneys of C. carpio exposed to Cd. According to mRNAs expression in the agomir and Cd treatment, miRNAs inhibit the expressions of target mRNAs. These results demonstrate that miR-217 via SIRT1 plays a regulatory role in the immunotoxicity of Cd to C. carpio.

Proteome of larval metamorphosis induced by epinephrine in the Fujian oyster Crassostrea angulata.

BACKGROUND: The Fujian oyster Crassostrea angulata is an economically important species that has typical settlement and metamorphosis stages. The development of the oyster involves complex morphological and physiological changes, the molecular mechanisms of which are as yet unclear. RESULTS: In this study, changes in proteins were investigated during larval settlement and metamorphosis of Crassostrea angulata using epinephrine induction. Protein abundance and identity were characterized using label-free quantitative proteomics, tandem mass spectrometry (MS/ MS), and Mascot methods. The results showed that more than 50% (764 out of 1471) of the quantified proteins were characterized as differentially expressed. Notably, more than two-thirds of the differentially expressed proteins were down-regulated in epinephrine-induced larvae. The results showed that "metabolic process" was closely related to the development of settlement and metamorphosis; 5 × 10- 4 M epinephrine induced direct metamorphosis of larvae and was non-toxic. Calmodulin and MAPK pathways were involved in the regulation of settlement of the oyster. Expression levels of immune-related proteins increased during metamorphosis. Hepatic lectin-like proteins, cadherins, calmodulin, calreticulin, and cytoskeletal proteins were involved in metamorphosis. The nervous system may be remodeled in larval metamorphosis induced by epinephrine. Expression levels of proteins that were enriched in the epinephrine signaling pathway may reflect the developmental stage of the larvae, that may reflect whether or not larvae were directly involved in metamorphosis when the larvae were treated with epinephrine. CONCLUSION: The study provides insight into proteins that function in energy metabolism, immune responses, settlement and metamorphosis, and shell formation in C. angulata. The results contribute valuable information for further research on larval settlement and metamorphosis.

MicroRNA-155 and microRNA-181a, via HO-1, participate in regulating the immunotoxicity of cadmium in the kidneys of exposed Cyprinus carpio.

Cadmium (Cd) is a nonessential metal that is a contaminant in aquatic ecosystems. Cd can accumulate in aquatic animals, leading to detrimental effects in tissues, and Cd exposure can induce immunotoxicity in fish. MicroRNAs (miRNAs) play critical roles in immune responses, yet the participation of miRNAs in Cd-induced immunotoxicity remains poorly understood. The present study evaluated the effects of Cd exposure on the immune responses and the mRNAs and miRNAs expressions of immune-related genes in Cyprinus carpio (C. carpio). Then, microRNA-155 (miR-155) was overexpressed and microRNA-181a (miR-181a) was knocked down to determine which miRNA plays a key role in the immune response to Cd. The results showed that 0.5 mg/L Cd2+ significantly decreased the activity of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the kidneys of C. carpio. Cd exposure upregulated the mRNA expressions of interleukin (IL)-1ß, IL-8, nuclear factor-kappa B (NF-κB), tumour necrosis factor-α (TNF-α), and Toll-like receptor 4(TLR-4) and downregulated those of IL-10 and heme oxygenase-1 (HO-1) in C. carpio kidneys. Cd exposure also led to upregulation of miR-155 and miR-181a expressions. Furthermore, AKP and ACP activity in the kidneys was markedly changed after intraperitoneal injection of C. carpio with miR-155 agomir and miR-181a antagomir. All detected mRNA expressions were significantly decreased after injection of miR-155 agomir, and IL-10, NF-κB, TNF-α, and HO-1 mRNA expressions were markedly increased after injection of miR-181a antagomir. The results of this study demonstrate that Cd exposure can immunocompromise C. carpio by targeting HO-1 through miR-155 and miR-181a. This is the first study to reveal that Cd exposure induces immunotoxicity through miR-155 and miR-181a in the kidneys of C. carpio.

Molecular characteristics, pathogenicity and medication regimen of Aeromonas hydrophila isolated from common carp (Cyprinus carpio L.).

Aeromonas hydrophila causes disease in fish known as Motile Aeromonas Septicemia (MAS), also named as bacterial hemorrhagic septicemia. In this study, a pathogenic A. hydrophila strain was isolated from common carp Cyprinus carpio L., which were suffering from severe hemorrhagic septicemia. According to the phylogenetic analysis derived from 16S rDNA sequence, the isolate formed a single branch in the A. hydrophila group, named AhHN1. Artificial infection results indicated that AhHN1 showed strong pathogenicity in C. carpio and the LD50 was 1.38 × 106 CFU/fish, the clinical symptoms and pathological features of infected fish were similar to those observed in natural infections. The antimicrobial susceptibility testing revealed that AhHN1 resistance to more than 13 kinds of antimicrobial agents. However, the AhHN1 strain exhibited an extremely sensitivity to enrofloxacin, the in vitro activities of enrofloxacin were subsequently investigated and drug selection window (MSW) was 0.0016-0.0125 µg/ml. Pharmacokinetics data showed that plasma concentration of enrofloxacin was 0.0016, 0.0148 and 0.0282 µg/ml at 24 hr after orally administered with 2.5, 5 and 10 mg/kg enrofloxacin. Moreover, dosing once a day of 2.5, 5 and 10 mg/kg enrofloxacin, which the relative protection ratio (RPS) was amounted to 33.3, 66.7, and 83.3%, respectively. Therefore, 5 mg/kg enrofloxacin was considered to be the rational regimen for controlling AhHN1 infection in C. carpio in the countries where the use of enrofloxacin is permitted in aquaculture. The aim of this study was to establish a scientific medication regimen for the prevention and therapy of the mutidrug-resistant A. hydrophila infection.

Effects of acidification on the proteome during early development of Babylonia areolata.

Increases in atmospheric CO2 partial pressure have lowered seawater pH in marine ecosystems, a process called ocean acidification (OA). The effects of OA during the critical stages of larval development may have disastrous consequences for some marine species, including Babylonia areolata (Link 1807), a commercially important sea snail in China and South East Asia. To investigate how OA affects the proteome of Babylonia areolata, here we used label-free proteomics to study protein changes in response to acidified (pH 7.6) or ambient seawater (pH 8.1) during three larvae developmental stages of B. areolata, namely, the veliger larvae before attachment (E1), veliger larvae after attachment (E2), and carnivorous juvenile snail (E3). In total, we identified 720 proteins. This result suggested that acidification seriously affects late veliger stage after attachment (E2). Further examination of the roles of differentially expressed proteins, which include glutaredoxin, heat-shock protein 70, thioredoxin, catalase, cytochrome-c-oxidase, peroxiredoxin 6, troponin T, CaM kinase II alpha, proteasome subunit N3 and cathepsin L, will be important for understanding the molecular mechanisms underlying pH reduction.

Differential proteomic profiles and characterizations between hyalinocytes and granulocytes in ivory shell Babylonia areolata.

The haemocytes of the ivory shell, Babylonia areolata are classified by morphologic observation into the following types: hyalinocytes (H) and granulocytes (G). Haemocytes comprise diverse cell types with morphological and functional heterogene and play indispensable roles in immunological homeostasis of invertebrates. In the present study, two types of haemocytes were morphologically identified and separated as H and G by Percoll density gradient centrifugation. The differentially expressed proteins were investigated between H and G using mass spectrometry. The results showed that total quantitative proteins between H and G samples were 1644, the number of up-regulated proteins in G was 215, and the number of down-regulated proteins in G was 378. Among them, cathepsin, p38 MAPK, toll-interacting protein-like and beta-adrenergic receptor kinase 2-like were up-regulated in G; alpha-2-macroglobulin-like protein, C-type lectin, galectin-2-1, galectin-3, ß-1,3-glucan-binding protein, ferritin, mega-hemocyanin, mucin-17-like, mucin-5AC-like and catalytic subunit of protein kinase A were down-regulated in G. The results showed that the most significantly enriched KEGG pathways were the pathways related to ribosome, phagosome, endocytosis, carbon metabolism, protein processing in endoplasmic reticulum and oxidative phosphorylation. For phagosome and endocytosis pathway, the number of down-regulation proteins in G was more than that of up-regulation proteins. For lysosome pathway, the number of up-regulation proteins in G was more than that of down-regulation proteins. These results suggested that two sub-population haemocytes perform the different immune functions in B. areolata.

Biomechanical response of a submerged, rosette-forming macrophyte to wave action in a eutrophic lake on the Yungui Plateau, China.

Few studies have focused on the biomechanical responses of submerged, rosette-forming macrophytes to wave action, water depth, or their co-occurrence in naturally eutrophic systems. The plant architecture, root anchorage strength-related traits, leaf morphology, and biomechanics of Vallisneria natans inhabiting a range of water depths were examined along three transects (T1, T2, and T3) in a eutrophic lake, Lake Erhai, in Yunnan Province, China. These transects were exposed to weak wave action and hyper-eutrophication (T1), moderate wave action and eutrophication (T2), or strong wave action and eutrophication (T3). The results showed that V. natans was mainly distributed at intermediate depths, with the widest colonization depth in T1. The values of plant architecture, root anchorage strength-related traits, leaf morphology, and biomechanics were generally highest in T3 and smallest in T2. Along the depth gradient, these values were generally highest at 3.5, 2.5, and 2.5 m for the plants growing in T1, T2, and T3, respectively. These findings suggest that V. natans adopts a "tolerance" strategy to cope with the effects of strong wave action in eutrophic habitats and an "avoidance" strategy when exposed to moderate wave action in eutrophic areas. Since the absence of an avoidance strategy increases the resistance to low-light stress at the expense of increased drag forces, there is a limit to the wave action that V. natans can withstand. This study indicates that biomechanics could be important when determining the distribution pattern of V. natans in Lake Erhai.

Author Correction: Proteomics Studies on the three Larval Stages of Development and Metamorphosis of Babylonia areolata.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Proteomics Studies on the three Larval Stages of Development and Metamorphosis of Babylonia areolata.

The ivory shell, Babylonia areolata, is a commercially important aquaculture species in the southeast coast of mainland China. The middle veliger stage, later veliger stage, and juvenile stage are distinct larval stages in B. areolata development. In this study, we used label-free quantification proteomics analysis of the three developmental stages of B. areolata. We identified a total of 5,583 proteins, of which 1,419 proteins expression level showed significant differential expression. The results of gene ontology enrichment analysis showed that the number of proteins involved in metabolic and cellular processes were the most abundant. Those proteins mostly had functions such as binding, catalytic activity and transporter activity. The results of Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the number of proteins involved in the ribosome, carbon metabolism, and lysosome pathways were the most abundant, indicating that protein synthesis and the immune response were active during the three stages of development. This is the first study to use proteomics and real-time PCR to study the early developmental stages of B. areolata, which could provide relevant data on gastropod development. Our results provide insights into the novel aspects of protein function in shell formation, body torsion, changes in feeding habits, attachment and metamorphosis, immune-related activities in B. areolata larvae.

Morphological and biomechanical response to eutrophication and hydrodynamic stresses.

Eutrophication and hydrodynamics determine the final distribution patterns of aquatic macrophytes; however, there is limited available knowledge regarding their interactive effects. Morphological and biomechanical responses to eutrophication and hydrodynamic stresses were assessed by sampling five abundant and dominant species, Potamogeton maackianus, P. pectinatus, P. lucens, Ceratophyllum demersum and Myriophyllum spicatum, in three macrophyte beds in Lake Erhai, Yunnan Province, China: one exposed to eutrophication and moderate southeast (SE) wind; one with mesotrophication, but sheltered by the lakeshore, with weak wind disturbance; and one with meso-eutrophication and strong SE wind. The results showed significant interactive effects of eutrophication and hydrodynamics on most biomechanical traits and some morphological traits, suggesting that aquatic macrophytes preferentially undergo biomechanical adjustments to resist the coexisting eutrophication and hydrodynamic stresses. In particular, hydrodynamics increased both the tensile force and tensile strain of P. maackianus under meso-eutrophication and dramatically decreased them in eutrophic areas, suggesting that eutrophication triggers mechanical failure in this species. Additionally, P. pectinatus, C. demersum and M. spicatum showed the lowest and highest values for the biomechanical variables (greater values for M. spicatum) in the most eutrophic and hydrodynamic areas, respectively, implying that increases in hydrodynamics primarily induce mechanical damage in eutrophic species. The plants generally exhibited greater tensile strain in both shallow and deep waters and the greatest tensile force at moderate depths. The stem cross-sectional area, plant height, stem length, internode length, and branch traits were all responsible for determining the biomechanical variables. This study reveals that hydrodynamic changes primarily induce mechanical damage in eutrophic species, whereas eutrophication triggers mechanical damage in sensitive species.

Proteomic analysis of trochophore and veliger larvae development in the small abalone Haliotis diversicolor.

BACKGROUND: Haliotis diversicolor is commercially important species. The trochophore and veliger are distinct larval stages in gastropod development. Their development involves complex morphological and physiological changes. We studied protein changes during the embryonic development of H. diversicolor using two dimensional electrophoresis (2-DE) and label-free methods, tandem mass spectrometry (MS/ MS), and Mascot for protein identification. RESULTS: A total of 150 2-DE gel spots were identified. Protein spots showed upregulation of 15 proteins and downregulation of 28 proteins as H. diversicolor developed from trochophore to veliger larvae. Trochophore and veliger larvae were compared using a label-free quantitative proteomic approach. A total of 526 proteins were identified from both samples, and 104 proteins were differentially expressed (> 1.5 fold). Compared with trochophore larvae, veliger larvae had 55 proteins upregulated and 49 proteins downregulated. These differentially expressed proteins were involved in shell formation, energy metabolism, cellular and stress response processes, protein synthesis and folding, cell cycle, and cell fate determination. Compared with the 5 protein (fructose-bisphosphate aldolase, 14-3-3ε, profilin, actin-depolymerizing factor (ADF)/cofilin) and calreticulin) expression patterns, the mRNA expression exhibited similar patterns except gene of fructose-bisphosphate aldolase. CONCLUSION: Our results provide insight into novel aspects of protein function in shell formation, torsion, and nervous system development, and muscle system differentiation in H. diversicolor larvae. "Quality control" proteins were identified to be involved in abalone larval development.

Label-free proteomic analysis of intestinal mucosa proteins in common carp (Cyprinus carpio) infected with Aeromonas hydrophila.

Outbreaks of infectious diseases in common carp Cyprinus carpio, a major cultured fish in northern regions of China, constantly result in significant economic losses. Until now, information proteomic on immune defence remains limited. In the present study, a profile of intestinal mucosa immune response in Cyprinus carpio was investigated after 0, 12, 36 and 84 h after challenging tissues with Aeromonas hydrophila at a concentration of 1.4 × 108 CFU/mL. Proteomic profiles in different samples were compared using label-free quantitative proteomic approach. Based on MASCOT database search, 1149 proteins were identified in samples after normalisation of proteins. Treated groups 1 (T1) and 2 (T2) were first clustered together and then clustered with control (C group). The distance between C and treated group 3 (T3) represented the maxima according to hierarchical cluster analysis. Therefore, comparative analysis between C and T3 was selected in the following analysis. A total of 115 proteins with differential abundance were detected to show conspicuous expressing variances. A total of 52 up-regulated proteins and 63 down-regulated proteins were detected in T3. Gene ontology analysis showed that identified up-regulated differentially expressed proteins in T3 were mainly localised in the hemoglobin complex, and down-regulated proteins in T3 were mainly localised in the major histocompatibility complex II protein complex. Forty-six proteins of differential abundance (40% of 115) were involved in immune response, with 17 up-regulated and 29 down-regulated proteins detected in T3. This study is the first to report proteome response of carp intestinal mucosa against A. hydrophila infection; information obtained contribute to understanding defence mechanisms of carp intestinal mucosa.

Protein changes in abalone foot muscle from three geographical populations of Haliotis diversicolor based on proteomic approach.

Using two-dimensional gel electrophoresis, the foot muscle proteome of three geographical populations of Haliotis diversicolor were examined, with a total of 922 ± 21 protein spots detected in the Japanese population (JJ), 904 ± 25.6 in the Taiwanese population (TT), and 936 ± 16.2 in the Vietnamese population (VV). Of these, 254 spots showed differential expression and 85 protein spots percentage volumes varied more than twofold. Both "genotype" and "spot" analysis of variance approaches significantly showed differences among the three populations. Hierarchical clustering analysis showed that TT and VV clustered together followed by clustering with JJ, which is consistent with their geographical location. Following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 30 differentially expressed proteins involved in major biological processes including energy production and storage and stress response were identified. Of these proteins, proteins pertaining to muscle contraction and muscle protein regulation showed highest expression levels in VV samples. Proteins involved in energy production and storage, including ATP synthase beta subunit, fructose-1,6-bisphosphate aldolase, arginine kinase, enolase, triosephosphate isomerase, and tauropine dehydrogenase, showed diverse expression patterns among the three populations. For stress-responsive proteins, the expression of heat shock protein 70 was JJ > VV > TT. The expression pattern of Cu/Zn-superoxide dismutase was JJ > VV > TT. Overall, these results may aid in the detection of new differentially expressed proteins within three different abalone populations.

Proteomic profiling of eggs from a hybrid abalone and its parental lines: Haliotis discus hannai Ino and Haliotis gigantea.

Proteomic analysis was performed on the eggs of hybrid abalone and their corresponding parental lines. A total of 915 ± 19 stained protein spots were detected from Haliotis discus hannai♀ × H. discus hannai♂ (DD), 935 ± 16 from H. gigantea♀ × H. gigantea♂ (GG) and 923 ± 13 from H. gigantea♀ × H. discus hannai♂ (GD). The spots from DD and GD were clustered together. The distance between DD and GG was maximal by hierarchical cluster analysis. A total of 112 protein gel spots were identified; of these, 59 were abalone proteins. The proteins were involved in major biological processes including energy metabolism, proliferation, apoptosis, signal transduction, immunity, lipid metabolism, electron carrier proteins, protein biosynthesis and decomposition, and cytoskeletal structure. Three of 20 differential expression protein spots involved in energy metabolism exhibited as upregulated in GD, 13 spots exhibited additivity, and four spots exhibited as downregulated in the offspring. Eleven protein spots were expressed at the highest level in DD. The proteins involved in stress responses included superoxide dismutase, peroxiredoxin 6, thioredoxin peroxidase and glutathione-S-transferase. Two of seven differential expression protein spots involved in response to stress exhibited as upregulated in GD, three exhibited additivity, and two exhibited as downregulated. These results might suggest that proteomic approaches are suitable for the analysis of hybrids and the functional prediction of abalone hybridization.

Phagocytosis and respiratory burst activity of haemocytes from the ivory snail, Babylonia areolata.

Haemocytes from the ivory snail, Babylonia areolata phagocytized Saccharomyces cerevisiae and Vibrio parahaemolyticus after 30 min. Haemocytes phagocytized V. parahaemolyticus at a greater rate than they phagocytized S. cerevisiae. The phagocytic rate (PP) of V. parahaemolyticus by granulocytes to was a little higher than that of S. cerevisiae. The phagocytic index (PI) of V. parahaemolyticus by granulocytes was significantly higher than that of S. cerevisiae. The same was true of hyalinocytes. The PP of granulocytes was significantly higher than that of hyalinocytes for each pathogen. No difference in PI was observed in granulocytes and hyalinocytes. Two defense mechanisms of B. areolata were quantified using flow cytometry. Haemocyte phagocytosis was quantified using fluorescent microbeads and respiratory burst activity was measured using H2O2 increases detected by 2', 7'-dichlorofluorescein diacetate. Both phagocytosis and respiratory burst activity of the haemocytes increased over time. After 90 min the phagocytic rate no longer increased. In the case of respiratory burst, the greatest increase in fluorescence occurred between 30 and 120 min, no further increase was seen after 120 min. These results showed unequivocally that a native (unstimulated) haemocyte oxidative burst was active in B. areolata. The aim of this study was to further the knowledge of immunology in gastropods.

Genetic changes in muscle protein following hybridization between Haliotis diversicolor reeve Japan and Taiwan populations revealed using a proteomic approach.

Protein expression patterns were compared in a Japan and Taiwan population of Haliotis diversicolor and in a hybrid between them using 2DE and MALDI-TOF-TOF analyses. Using the software PDQuest, 924 ± 7 protein spots were detected in the Japan population (RR), 861 ± 11 in the Taiwan population (TT), and 882 ± 9 in the F1 hybrid (TR). RR and TR were clustered together, but the distance between RR and TT was the maximum using hierarchical cluster analysis. A total of 46 gel spots were identified and a total of 15 spots matched with abalone proteins (a 33.6% identification rate). Hybrid exhibiting additivity or overdominance accounted for 73.9% of these 46 identified proteins. The 46 differentially expressed proteins were shown to be involved in major biological processes, including muscle contraction and regulation, energy metabolism, and stress response. The proteins involved in energy metabolism included adenosine triphosphate (ATP) synthase ß subunit, fructose 1, 6-bisphosphate aldolase, triosephosphate isomerase, enolase, arginine kinase, and tauropine dehydrogenase. These proteins exhibited additivity in their offspring. The proteins involved in stress responses included HSP Hsp70 (exhibiting overdominance in the offspring) and Cu/Zn-superoxide dismutase (exhibiting additivity). These results suggested that proteomic approach is suitable for analysis of heterosis and functional prediction of abalone hybridization.