Glutathione peroxidase (Gpx) is an important member of antioxidant enzymes, which can play a vital role in metabolizing reactive oxygen species (ROS) and in maintaining cell homeostasis. In order to study the evolutionary dynamics of gpx gene family in allotetraploid fish species, we identified a total of 14 gpx genes in common carp Cyprinus carpio, while 9 gpx genes were discovered in the diploid progenitor-like species Poropuntius huangchuchieni. Comparative genomic analysis and phylogenetic analysis revealed that the common carp gpx genes had significant expansion and were divided into five distinct subclades. Exon-intron distribution patterns and conserved motif analysis revealed highly conserved evolutionary patterns. Transcript profiles suggested that different gpx genes had specific patterns of regulation during early embryonic development. In adult tissues, gpx genes had a relatively broad expression distribution, most of which were highly expressed in the gills, intestines, and gonads. RT-qPCR studies showed that most gpx genes were downregulated during the initial cd2+ treatment stage. Dietary supplementation of Bacillus coagulans at different concentrations (Group 2 of 1.0 × 107 cfu/g, Group 3 of 1.0 × 108 cfu/g, and Group 4 of 1.0 × 109 cfu/g) induced different regulatory responses of gpx subclades. This result suggested that the appropriate concentration of B. coagulans can improve gpx gene expression when exposed to heavy metal cadmium treatment, which may play a vital role in the resistance to oxidative stress and immune responses. This study has expanded our understanding of the functional evolution of the gpx gene family in common carp.

Bone morphogenetic proteins (Bmps) are a group of signaling molecules known to play important roles during formation and maintenance of various organs, not only bone, but also muscle, blood and so on. Common carp (Cyprinus carpio) is one of the most intensively studied fish due to its economic and environmental importance. Besides, common carp has encountered an additional round of whole genome duplication (WGD) compared with many closely related diploid teleost, which make it one of the most important models for genome evolutionary studies in teleost. Comprehensive genome resources of common carp have been developed recently, which facilitate the thorough characterization of bmp gene family in the tetraploidized common carp genome. We identified a total of 44 bmps from the common carp genome, which are twice as many as that of zebrafish. Phylogenetic analysis revealed that most of bmps are highly conserved. Comparative analysis was performed across six typical vertebrate genomes. It appeared that all the bmp genes in common carp were duplicated. Obviously, the expansion of the bmp gene family in common carp was due to the latest additional round of whole genome duplication and made it more abundant than other diploid teleosts. Expression signatures were assessed in major tissues, including gill, intestine, liver, spleen, skin, heart, gonad, muscle, kidney, head kidney, brain and blood, which demonstrated the comprehensive expression profiles of bmp genes in the tetraploidized genome. Significant gene expression divergences were observed which revealed substantial functional divergences of those duplicated bmp genes post the latest WGD event. The conserved synteny blocks of bmp5s revealed the genome rearrangement of common carp post the 4R WGD. The whole set of bmp gene family in common carp provides insight into gene fate of tetraploidized common carp genome post recent WGD.

Interleukin-17 (IL17) family cytokines are well known for having pro-inflammatory actions as important mediators of mucosal immune responses and are tightly regulated by various kinds of signals. However, most studies of IL17 genes have focused on mammals, and much less is known about IL17 genes in fish species. To better understand the scope and actions of the IL17 gene family in common carp, we characterized seven IL17 gene homologs from genomic and transcriptomic databases that could be classified into three subclasses according to different comparative genomic analyses. Phylogenetic analysis revealed that most IL17s are highly conserved, though recent gene duplication and gene loss events do exist. Through observation, we found that IL17D has undergone gene duplication in common carp and that all the IL17E genes were lost in vertebrates except mammals. The expression patterns of IL17 genes in common carp were examined during early developmental stages and in various healthy tissues, and the results indicated that most IL17 genes are ubiquitously expressed during early development and show particular tissue-specific expression in various healthy tissues, with relatively high levels in the spleen, liver, and kidney. To gain insights into the mucosal actions of inflammatory processes, the expression profiles of IL17 genes in gills from common carp were investigated after experimental challenge with Aeromonas hydrophila. After A. hydrophila infection, most IL17 genes were upregulated at 4 h postinfection in the gill and then gradually declined, while IL17A/F2 and IL17N were generally upregulated at 12 h postinfection, and IL17D2 maintained an increasing tendency. In contrast, IL17D showed the third phenomenon, rising expression, suggesting that immunogenes have different response strategies to bacterial invasion. Overall, the expression of IL17 in unstimulated tissues and toxicity attack test results demonstrated that these genes play critical roles under normal conditions and during bacterial infection. Moreover, this common carp IL17 gene family research provides a genomic resource for future studies on IL17 gene evolution, fish disease management and immune regulation.

Taste receptors (TRs) are seven trans-membrane G protein-coupled receptors as well as the interface between internal and external milieus, which playing pivotal roles in nutrient identification and acquisition. To better understand the scope and function of tr gene family in common carp, one of the most economic and important breeding fish species, which has undergone an additional round of whole genome duplication (WGD), we characterized 13 tr gene homologues including eight type I and five type II taste receptor genes from common carp genome, which were more than any other teleosts. Phylogenetic and syntenic analysis revealed the evolution dynamics of tr gene family, which was highly conserved, though gene duplication and gene loss do exist recently. Furthermore, the motif and dN/dS analyses indicated that these receptors were under different negative selection pressure. Additionally, the gene expression divergences were observed in 12 health tissues of common carp, with a relatively high level in barbel and head kidney, demonstrating tissue-specific expression of tr genes in the tetraploidized genome. The overarching goals of this study were to identify the abundance of tr genes in common carp, compare the gene divergence among species with varied feeding habits and provide genomic resources for future studies on teleost taste sensation.

Hexokinase family includes hexokinases I, II, III and IV, that catalyze the phosphorylation of glucose to produce glucose 6-phosphate. Hexokinase IV, also known as glucokinase, is only half size of the other types of hexokinases that contain two hexokinase domains. Despite the enormous progress in the study of hexokinases, the evolutionary relationship between glucokinase and other hexokinases is still uncertain, and the molecular processes leading to the emergence of hexokinases in vertebrates remain controversial. Here we clearly demonstrated the presence of a single hexokinase-like gene in the amphioxus Branchiostoma japonicum, Bjhk, which shows a tissue-specific expression pattern, with the most abundant expression in the hepatic caecum, testis and ovary. The phylogenetic and synteny analyses both reveal that BjHK is the archetype of vertebrate hexokinases IV, i.e. glucokinases. We also found for the first time that recombinant BjHK showed functional enzyme activity resembling vertebrate hexokinases I, II, III and IV. In addition, a native glucokinase activity was detected in the hepatic caecum. Finally, glucokinase activity in the hepatic caecum was markedly reduced by fasting, whereas it was considerably increased by feeding. Altogether, these suggest that Bjhk represents the archetype of glucokinases, from which vertebrate hexokinase gene family was evolved by gene duplication, and that the hepatic caecum plays a role in the control of glucose homeostasis in amphioxus, in favor of the notion that the hepatic caecum is a tissue homologous to liver.

The solute carrier family 9 (slc9) genes, especially slc9a isoform coding proteins contribute to electroneutral countertransport of H+ for Na+ across the plasmalemmal and organellar membranes, intracellular pH and cellular volume regulation as well as the electrolyte, acid-base, and fluid volume homeostasis at the systemic level. These functional properties determine a potential basis for organisms to challenge stressful conditions. However, these well-done researches have been reported more in mammals. Thus, in this study, a total of eleven slc9 genes were identified from the latest version genome of L. waleckii, a cyprinid fish that could tolerate extremely alkaline environments (pH 9.6). The evolutionary footprint of slc9 genes was uncovered via the analysis of copy numbers, gene structure, motif composition, chromosome location and phylogenetic relationship. More importantly, there were two SNPs located on 5' UTR and three non-synonymous mutations in the coding region of the slc9a3.2 gene by comparing freshwater with alkaline water populations attached to resequencing technology. Slc9a3.2 gene was a statistically significant low expression in gill tissue with extremely alkaline pressure. Generally, slc9 gene family in L. waleckii was highly conserved. Several important SNPs with high Fst values were identified where non-synonymous mutations occurred between freshwater and alkaline water populations, and they may play an important role in specific functional differentiation. Slc9 genes had clear tissue expression preferences and were involved in abiotic stress response, indicating their roles in physiological function and strong self-regulating capacity. Our insight into the genetic variations that take place in the individual genes under extreme conditions could provide a feasible example for studying specific molecular mechanisms based on genomic data with increasing environmental stress.

Hyalomma anatolicum anatolicum (H.a. anatolicum) (Acari: Ixodidae) ticks are globally distributed ectoparasites with veterinary and medical importance. These ticks not only weaken animals by sucking their blood but also transmit different species of parasitic protozoans. Multiple factors influence these parasitic infections including miRNAs, which are non-coding, small regulatory RNA molecules essential for the complex life cycle of parasites. To identify and characterize miRNAs in H.a. anatolicum, we developed an integrative approach combining deep sequencing, bioinformatics and real-time PCR analysis. Here we report the use of this approach to identify miRNA expression, family distribution, and nucleotide characteristics, and discovered novel miRNAs in H.a. anatolicum. The result showed that miR-1-3p, miR-275-3p, and miR-92a were expressed abundantly. There was a strong bias on miRNA, family members, and nucleotide compositions at certain positions in H.a. anatolicum miRNA. Uracil was the dominant nucleotide, particularly at positions 1, 6, 16, and 18, which were located approximately at the beginning, middle, and end of conserved miRNAs. Analysis of the conserved miRNAs indicated that miRNAs in H.a. anatolicum were concentrated along three diverse phylogenetic branches of bilaterians, insects and coelomates. Two possible roles for the use of miRNA in H.a. anatolicum could be presumed based on its parasitic life cycle: to maintain a large category of miRNA families of different animals, and/or to preserve stringent conserved seed regions with active changes in other places of miRNAs mainly in the middle and the end regions. These might help the parasite to undergo its complex life style in different hosts and adapt more readily to the host changes. The present study represents the first large scale characterization of H.a. anatolicum miRNAs, which could further the understanding of the complex biology of this zoonotic parasite, as well as initiate miRNA studies in other related species such as Haemaphysalis longicornis and Rhipicephalus sanguineus of human and animal health significance.

The Japanese ornamental carp (Cyprinus carpio var. Koi) is famous for multifarious colors and patterns, making it commonly culture and trade across the world. Although functional genes and inheritance of color traits have been commonly studied, seldom attentions were focused on the genetic regulation during the developmental process of pigmentation. To better understand the mechanism of skin color development, we observed the morphogenesis of pigment cells during the post-embryonic stages and analysed the temporal expression pattern of mRNAs/miRNAs profiles in four distinct developmental stages. 59 and 103 differentially expressed genes/miRNAs (DEGs/DEMs) associated with pigmentation and skin were identified, including pax7, mitf, tyr, tyrp1, etc., and the highest DEGs were detected at 11 days post hatching (dph). In addition, the functional characteristics of mRNAs/miRNAs associated with pteridine and carotenoid pathway were also examined. Furthermore, 65 miRNA-mRNA interaction pairs related to pigmentation, pteridines and carotenoids metabolism were detected between different stages. Interestingly, the largest pairs appeared in the transition from 11 dph to 48 dph, which had the similar trend with DEGs further manifesting the importance of 11 dph. This study produced a comprehensive programme of DEGs/DEMs during color development, which will provide resources to understand the regulation mechanism in color formation. The understanding of genetic basis in color formation might promote the production and breeding of the Koi carp.

The environment contains a large extent of chemical information, which could be detected as olfactory sense. Olfactory in vertebrates plays important roles on many aspects during life time, including localizing prey or food, avoiding predators, mating behavior and social communication. Considering the essential role of olfactory receptors in the specific recognition of diverse stimuli, understanding the evolutionary dynamics of olfactory receptors in teleost means a lot, especially in the allotetraploid common carp, who has undergone the fourth whole-genome duplication event. Here, we identified the whole set of olfactory receptor genes in representative teleosts and found a significant contraction in common carp when compared with other teleosts. Odorant receptor genes (OR) occupy the most among four groups of olfactory receptors, including 33 functional genes and 16 pseudogenes. Furthermore, 6 trace amine-associated receptor (TAAR) genes (including 1 pseudogene), 7 odorant-related-A receptor genes, and 10 olfactory C family receptor genes (including 3 pseudogenes) were identified in common carp. Phylogenetic and motif analysis were performed to illustrate the phylogenetic relationship and structural conservation of teleost olfactory receptors. Selection pressure analysis suggested that olfactory receptor groups in common carp were all under relaxed purifying-selection. Additionally, gene expression divergences for olfactory receptor genes were investigated during embryonic development stages of common carp. We aim to determine the abundance of common carp olfactory receptor genes, explore the evolutionary fate and expression dynamics, and provide some genomic clues for the evolution of polyploid olfactory after whole-genome duplication and for future studies of teleost olfactory.

Metacaspases (MCAs) are cysteine proteases that share sequence homology with caspases, and may play roles in programmed cell death (PCD). In the present study, we identified a novel MCA gene (CpMCA) from the red tide dinoflagellate Cochlodinium polykrikoides, and examined its molecular characteristics and gene expression in response to algicide-induced cell death. CpMCA cDNA is 1164 bp in length, containing a dinoflagellate spliced leader sequence (dinoSL), an 879-bp open reading frame (ORF), which codes for a 293-aa protein, and a poly (A) tail. Multi-sequence comparison indicated that CpMCA belongs to type I MCA, but it has a different structure at the N-terminal. Phylogenetic analysis showed that C. polykrikoides may have acquired the MCA gene from bacteria by means of horizontal gene transfer (HGT). In addition, expressions of CpMCA significantly increased following exposure to the common algicides copper sulfate and oxidizing chlorine, which trigger cell death in dinoflagellates, suggesting that CpMCA may be involved in cell death.

Superoxide dismutase (SOD) acts as the first line of defence against reactive oxygen species (ROS) within cells. In this study, we characterized a novel SOD gene (PmCuZnSOD) from the dinoflagellate Prorocentrum minimum, and examined its structural features, putative origin and gene expression. The SOD cDNA is 895 bp in length, containing dinoflagellate splice-leader (dinoSL) sequence, 714-bp ORF (237 aa), and poly (A) tail. In addition, PmCuZnSOD is coded on the dinoflagellate nuclear genome without introns and in a non-tandem repeat manner; however, the encoded protein is probably localized in chloroplasts. Phylogenetic analysis indicated that it might be acquired from cyanobacteria via horizontal gene transfer (HGT) and then the gene possibly relocated from the chloroplast to the nuclear genome. Excess copper dramatically increased the PmCuZnSOD transcripts and SOD activity in cells, caused by ROS generation and decrease of photosynthetic efficiency in the treated cells. These suggest that CuZnSOD may function to defend against oxidative stress for the survival of the dinoflagellate.

SOX9 plays a crucial, extensive and conservative role in the process of somatic tissue development and adult regeneration through the positive self-regulation mediated by SOM across all vertebrates. In this study, we have cloned SOX9 from the kidney of hatchling Alligator sinensis. The full-length of SOX9 cDNA is 3878 bp with an open reading frame encoding 494 amino acids. Amino acid alignment analyses indicated that the SOX9 exhibit highly conserved functional domains. Using the droplet digital PCR, the mRNA abundances of SOX9 during nephrogenesis in A. sinensis showed prominent changes in the embryonic development, suggesting that SOX9 might combines a vital role in the regulation of complex renal development. Interestingly, we detected the nucleocytoplasmic shuttling of SOX9 protein using immunofluorescence, implying that nucleocytoplasmic shuttling is critical to the regulation of SOX9 in the renal embryonic development. Collectively, these data provide an important foundation for further studies on renal developmental biology and molecular biology of non-mammalian SOX9. Furthermore, it provides new insights into the phenomenon of SOX9 nucleocytoplasmic shuttling in Alligator sinensis, which is probably of great significance to the development of kidney metanephros embryo.