Molecular cloning and functional characterization of the hypoxia-inducible factor-1α in bighead carp (Aristichthys nobilis).

HIF-l is the earliest documented and most widely studied hypoxia-inducible factor (HIF) and plays a key role in the cell hypoxia signal transduction pathway. Particularly, the HIF-1α protein is sensitive to oxygen and plays a critical role in hypoxia regulation. This study is the first to report on the molecular cloning and characterization of HIF-1α in bighead carp (Aristichthys nobilis; anHIF-1α). The full-length cDNA of anHIF-1α was 2361 bp, and encodes an estimated 674 amino acids with a predicted molecular mass of 76.10 kDa and a theoretical isoelectric point of 7.72. Moreover, the conserved basic Helix-Loop-Helix domain along with two Per-ARNT-Sim domains (A/B), and C-TAD were identified in this protein. Interestingly, the tertiary structure of the anHIF-1α protein was found to be extremely similar to that of mice. Multiple comparison and phylogenetic tree results demonstrated that anHIF-1α was highly conserved. Under normoxic conditions, anHIF-1α mRNA transcripts could be detected in all tissues examined with the highest expression level in the heart. With gradually decreasing oxygen concentrations, anHIF-1α mRNA level was upregulated significantly in the gill, liver, kidney, spleen, intestine, brain, and muscle tissues (P < 0.05). Similarly, anHIF-1α was expressed in all examined bighead carp tissues, and the results suggested that the upregulation of anHIF-1α at the transcriptional level may be an important stress response adaptation to hypoxia in bighead carp. Finally, based on the tertiary structure comparative analyses between anHIF-1α with mouse HIF-1α, we think the physiological function, and protein structure of HIF-1α could be compared between fish and mammal in the future.

In Vivo Analysis of miR-34a Regulated Glucose Metabolism Related Genes in Megalobrama amblycephala.

The Megalobrama amblycephala (M. amblycephala) is one of the most important economic freshwater fish in China. The molecular mechanism under the glucose intolerance responses which affects the growth performance and feed utilization is still confused. miR-34a was reported as a key regulator in the glucose metabolism, but how did the miR-34a exert its function in the metabolism of glucose/insulin in M. amblycephala was still unclear. In this study, we intraperitoneally injected the miR-34a inhibitor (80 nmol/100 g body weight) into M. amblycephala (fed with high starch diet, 45% starch) for 12 h, and then analyzed the gene expression profiling in livers by RNA-seq. The results showed that miR-34a expression in M. amblycephala livers was inhibited by injection of miR-34a inhibitor, and a total of 2212 differentially expressed genes (DEGs) were dysregulated (including 1183 up- and 1029 downregulated DEGs). Function enrichment analysis of DEGs showed that most of them were enriched in the peroxisome proliferator-activated receptor (PPAR), insulin, AMP-activated protein kinase (AMPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways, which were all associated with the glucose/lipid metabolic and biosynthetic processes. In addition, we examined and verified the differential expression levels of some genes involved in AMPK signaling pathway by qRT-PCR. These results demonstrated that the inhibition of miR-34a might regulate glucose metabolism in M. amblycephala through downstream target genes.

Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis.

Nitrite (NO2-) can cause oxidative stress in aquatic animal when it accumulates in the organism, resulting in different toxic effects on fish. In the present study, we investigated the effects of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of Bighead carp (Aristichthys nobilis). Fish [Initial average weight: (180.05 ±â€¯0.092) g] were exposed to 48.634 mg/L nitrite for 96 h, and a subsequent 96 h for the recovery test. Fish livers were collected to assay antioxidant enzymes activity, hepatic structure and expression of genes after 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h of exposure and12 h, 24 h, 48 h, 72 h, 96 h of recovery. The results showed that the activity of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) increased significantly in the early stages of nitrite exposure. The study also showed that nitrite significantly up-regulated the mRNA levels of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) after 6, 48, and 72 h of exposure respectively. Nitrite also increased the formation of malondialdehyde (MDA), oxidized glutathione (GSSG), and the activity of catalase (CAT). Nitrite was observed to reduce the activity of superoxide dismutase (SOD) and the level of glutathione (GSH). In the recovery test, GSH and the GSSG recovered but did not return to pre-stress levels. The results suggested that the glutathione system played important roles in nitrite-induced oxidative stress in fish. The bighead carp responds to oxidative stress by enhancing the activity of GSH-Px, GST, GR and up-regulating the expression level of GSH-Px, GST, GR, a whilst simultaneously maintaining the dynamic balance of GSH/GSSG. CAT was also indispensable. They could reduce the degree of lipid peroxidation, and ultimately protect the body from oxidative damage.

Chronic stress effects of high doses of vitamin D3 on Megalobrama amblycephala.

Dietary vitamin D3 plays an important role in the growth of aquatic animals, but long-term excessive feeding has potential hazards. In this study, Megalobrama amblycephala specimens were fed different experimental diets with 2000 IU/kg or 200,000 IU/kg of vitamin D3 for 90 days, in order to evaluate chronic stress effects of high doses of vitamin D3 on growth, immunity, and structural damage to enterohepatic tissues. The results showed that high doses of vitamin D3 did not have a significant influence on the growth performance of M. amblycephala (P > 0.05), but it significantly reduced the survival rate after infection by Aeromonas hydrophila (P < 0.05). Serum albumin, alkaline phosphatase, and insulin levels, as well as hepatic total antioxidant capacity, were also significantly reduced (P < 0.05). Serum cortisol levels and hepatic heat stress protein 70 expression in M. amblycephala showed that high doses of vitamin D3 significantly inhibit the anti-stress ability of M. amblycephala (P < 0.05). Paraffin tissue sections and electron microscopy showed that high doses of vitamin D3 could cause different degrees of structural damage to enterohepatic tissues of M. amblycephala. Our results indicate that, although M. amblycephala can tolerate high doses of dietary vitamin D3 over a long period, its glycolipid metabolism, immune function, anti-stress function, and resistance to pathogenic infections are adversely affected.

Morphological and molecular characterization of actinosporeans infecting oligochaete Branchiura sowerbyi from Chinese carp ponds.

We surveyed the actinosporean stages of fish myxosporeans at fish farms in Jiangsu Province, China, from 2011 to 2014. During the surveys, we identified 7 actinosporean types from 4 collective groups: echinactinomyxon (1 type), triactinomyxon (1 type), aurantiactinomyxon (1 type), and neoactinomyxum (4 types), released by the oligochaete Branchiura sowerbyi. The morphological characteristics and DNA sequences of these types are described here. Based on 18S rDNA sequence analysis, the actinosporean of echinactinomyxon type CZ with 4 branches at the end of the caudal processes was identified as Myxobolus wulii, and the neoactinomyxum type JD was identified as Thelohanellus wangi Yuan, Xi, Wang, Xie, Zhang, 2015 (JX458816), a recently nominated species from the gills of allogynogenetic gibel carp Carassius auratus gibelio. In addition, actinosporeans of aurantiactinomyxon type JD, neoactinomyxum type CZ-1, neoactinomyxum type CZ-2, and neoactinomyxum type CZ-3 showed high genetic similarity to T. wuhanensis (96.3-96.5%), T. nikolskii (98.0-99.1%), T. wuhanensis (97.8-98.9%), and T. hovorkai (98.7-98.9%), respectively. Phylogenetic analyses showed that these actinosporeans were robustly clustered in the Thelohanellus spp. clade.

Three actinosporean types (Myxozoa) from the oligochaete Branchiura sowerbyi in China.

Three actinosporean types--raabeia, aurantiactinomyxon, and guyenotia--from the oligochaete Branchiura sowerbyi Beddard collected from a crucian carp (Carassius auratus gibelio) pond are described in this report. Compared with the actinospores described previously, the raabeia type presents similar spore shape with the actinospore of Myxobolus cultus, except a much longer caudal process, 250.8 µm (217.5-276.3). The aurantiactinomyxon and guyenotia type significantly differ from other actinospores in its same collective group with different caudal process shape (taper and leaf-like) and dimension, 170.8 µm (167.5-176.3) and 18.5 µm (16.5-20.6), respectively. The partial 18S rDNA sequences of raabeia and aurantiactinomyxon types, and myxospores M. cultus, Myxobolus wulii, Myxobolus pyramidis, and Thelohanellus wuhanensis detected from pond-reared crucian carp were determined. Based on DNA sequences analysis, the raabeia type showed high genetic similarity (99.5-100%) with myxospore M. cultus Yokoyama, Ogawa & Wakabayashi, 1995 on the gills of crucian carp sampled from the same pond. The aurantiactinomyxon showed no more than 85.2% sequence similarity with myxospores determined in this report and other myxozoans available from GenBank.