Identification and characterization of TatD DNase in planarian Dugesia japonica and its antibiofilm effect.

TatD DNase, a key enzyme in vertebrates and invertebrates, plays a pivotal role in various physiological processes. Dugesia japonica (D. japonica), a flatworm species, has remarkable regenerative capabilities and possesses a simplified immune system. However, the existence and biological functions of TatD DNase in D. japonica require further investigation. Here, we obtained the open reading frame (ORF) of DjTatD and demonstrated its conservation. The three-dimensional structure of DjTatD revealed its active site and binding mechanism. To investigate its enzymological properties, we overexpressed, purified, and characterized recombinant DjTatD (rDjTatD). We observed that DjTatD was primarily expressed in the pharynx and its expression could be significantly challenged upon stimulation with lipopolysaccharide, peptidoglycan, gram-positive and gram-negative bacteria. RNA interference results indicated that both DjTatD and DjDN2s play a role in pharyngeal regeneration and may serve as functional complements to each other. Additionally, we found that rDjTatD and recombinant T7DjTatD effectively reduce biofilm formation regardless of their bacterial origin. Together, our results demonstrated that DjTatD may be involved in the planarian immune response and pharyngeal regeneration. Furthermore, after further optimization in the future, rDjTatD and T7DjTatD can be considered highly effective antibiofilm agents.

Evolution and Function of the Notch Signaling Pathway: An Invertebrate Perspective.

The highly conserved Notch signaling pathway affects embryonic development, neurogenesis, homeostasis, tissue repair, immunity, and numerous other essential processes. Although previous studies have demonstrated the location and function of the core components of Notch signaling in various animal phyla, a more comprehensive summary of the Notch core components in lower organisms is still required. In this review, we objectively summarize the molecular features of the Notch signaling pathway constituents, their current expression profiles, and their functions in invertebrates, with emphasis on their effects on neurogenesis and regeneration. We also analyze the evolution and other facets of Notch signaling and hope that the contents of this review will be useful to interested researchers.

Extracellular matrix-regulator MMPA is required for the orderly proliferation of neoblasts and differentiation of ectodermal progenitor cells in the planarian Dugesia japonica.

Matrix metalloproteinases (MMPs) are members of a family of zinc-dependent metallopeptidase proteins that are widely found in plants, animals, and microorganisms. As the regulators of the extracellular matrix and basement membrane, MMPs play an important role in embryogenesis, development, innate immunity, and regeneration. However, the function of MMP family in planarian, a model for regeneration research, is still ambiguous. Here, we cloned 5 MMPs genes from Dugesia japonica and found that DjMMPA was associated with the process of regeneration, neoblasts cell maintenance confusion and destruction. Loss of DjMMPA led to homeostasis confusion and eventually death, owing to neoblasts proliferation disorder. Additionally, DjMMPA RNAi-treated animals had impaired regeneration after amputation. Furthermore, knockdown of DjMMPA had noticeable defects in cell differentiation of ectoderm, especially in eyes and neural progenitor cells, possibly by inhibiting Wnt signaling. Our results suggest that extracellular matrix-regulator MMPA is required for the orderly proliferation of neoblasts and differentiation of ectodermal progenitor cells in the planarian, which provide valuable information for further explorations into the molecular mechanism of MMPS, stem cells, and regeneration.

Characterization of the lipidomic profile of clam Meretrix petechialis in response to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a devastating pathogen of clam Meretrix petechialis, which brings about huge economic losses in aquaculture breeding industry. In our previous study, we have found that Vibrio infection is closely associated with lipid metabolism of clams. In this study, an untargeted lipidomics approach was used to explore the lipid profiling changes upon Vibrio infection. The results demonstrated that the hepatopancreas of clams was composed of five lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids and sterol lipids. And the content of lipid classes altered during Vibrio infection, implying that Vibrio infection altered intracellular lipid homeostasis in clams. Meanwhile, a total of 200 lipid species including 82 up-regulated and 118 down-regulated significantly were identified in response to Vibrio infection, of which ceramide (Cer), phosphatidylcholine (PC) and triglyceride (TG) accounted for the largest proportion. Notably, all Cers showed a significantly decreased trend while nearly all TG species were increased significantly during Vibrio infection, which suggested that Cer and TG could be determined as effective biomarkers. Furthermore, these differentially expressed lipid species were enriched in 20 metabolic pathways and sphingolipid metabolism was one of the most enriched pathways. These results evidenced how the lipid metabolism altered in the process of Vibrio infection and opened a new perspective on the response of marine bivalves to pathogen infection.

From marine to freshwater environment: A review of the ecotoxicological effects of microplastics.

Microplastics (MPs) have been widely detected in the world's water, which may pose a significant threat to the ecosystem as a whole and have been a subject of much attention because their presence impacts seas, lakes, rivers, and even the Polar Regions. There have been numerous studies that report direct adverse effects on marine organisms, but only a few have explored their ecological effects on freshwater organisms. In this field, there is still a lack of a systematic overview of the toxic effects and mechanisms of MPs on aquatic organisms, as well as a consistent understanding of the potential ecological consequences. This review describes the fate and impact on marine and freshwater aquatic organisms. Further, we examine the toxicology of MPs in order to uncover the relationship between aquatic organism responses to MPs and ecological disorders. In addition, an overview of the factors that may affect the toxicity effects of MPs on aquatic organisms was presented along with a brief examination of their identification and characterization. MPs were discussed in terms of their physicochemical properties in relation to their toxicological concerns regarding their bioavailability and environmental impact. This paper focuses on the progress of the toxicological studies of MPs on aquatic organisms (bacteria, algae, Daphnia, and fish, etc.) of different trophic levels, and explores its toxic mechanism, such as behavioral alternations, metabolism disorders, immune response, and poses a threat to the composition and stability of the ecosystem. We also review the main factors affecting the toxicity of MPs to aquatic organisms, including direct factors (polymer types, sizes, shapes, surface chemistry, etc.) and indirect factors (persistent organic pollutants, heavy metal ions, additives, and monomer, etc.), and the future research trends of MPs ecotoxicology are also pointed out. The findings of this study will be helpful in guiding future marine and freshwater rubbish studies and management strategies.

Graphene oxide disruption of homeostasis and regeneration processes in freshwater planarian Dugesia japonica via intracellular redox deviation and apoptosis.

The aquatic system is a major sink for engineered nanomaterials released into the environment. Here, we assessed the toxicity of graphene oxide (GO) using the freshwater planarian Dugesia japonica, an invertebrate model that has been widely used for studying the effects of toxins on tissue regeneration and neuronal development. GO not only impaired the growth of normal (homeostatic) worms, but also inhibited the regeneration processes of regenerating (amputated) worms, with LC10 values of 9.86 mg/L and 9.32 mg/L for the 48-h acute toxicity test, respectively. High concentration (200 mg/L) of GO killed all the worms after 3 (regenerating) or 4 (homeostasis) days of exposure. Whole-mount in situ hybridization (WISH) and immunofluorescence analyses suggest GO impaired stem cell proliferation and differentiation, and subsequently caused cell apoptosis and oxidative DNA damage during planarian regeneration. Mechanistic analysis suggests that GO disturbed the antioxidative system (enzymatic and non-enzymatic) and energy metabolism in the planarian at both molecular and genetic levels, thus causing reactive oxygen species (ROS) over accumulation and oxidative damage, including oxidative DNA damage, loss of mitochondrial membrane integrity, lack of energy supply for cell differentiation and proliferation leading to retardance of neuron regeneration. The intrinsic oxidative potential of GO contributes to the GO-induced toxicity in planarians. These data suggest that GO in aquatic systems can cause oxidative stress and neurotoxicity in planarians. Overall, regenerated tissues are more sensitive to GO toxicity than homeostatic ones, suggesting that careful handling and appropriate decisions are needed in the application of GO to achieve healing and tissue regeneration.

The Physiological Function of nNOS-Associated CAPON Proteins and the Roles of CAPON in Diseases.

In this review, the structure, isoform, and physiological role of the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) are summarized. There are three isoforms of CAPON in humans, including long CAPON protein (CAPON-L), short CAPON protein (CAPON-S), and CAPON-S' protein. CAPON-L includes three functional regions: a C-terminal PDZ-binding motif, carboxypeptidase (CPE)-binding region, and N-terminal phosphotyrosine (PTB) structural domain. Both CAPON-S and CAPON-S' only contain the C-terminal PDZ-binding motif. The C-terminal PDZ-binding motif of CAPON can bind with neuronal nitric oxide synthase (nNOS) and participates in regulating NO production and neuronal development. An overview is given on the relationship between CAPON and heart diseases, diabetes, psychiatric disorders, and tumors. This review will clarify future research directions on the signal pathways related to CAPON, which will be helpful for studying the regulatory mechanism of CAPON. CAPON may be used as a drug target, which will provide new ideas and solutions for treating human diseases.

DjApi5 is required for homeostasis in planarian Dugesia japonica.

Orchestrated apoptosis in planarian Dugesia japonica is very important for its degrowth and regeneration. Apoptosis Inhibitor-5 (API5) is an anti-apoptotic factor that negatively regulates cell apoptosis. We characterized the conserved structure of DjApi5, however, the biological function of DjApi5 in planarians needs further investigation. In this study, we found that DjApi5 and its interacting molecular DjAcinus are required for planarian homeostasis, which may be correlated with their specific localization in neoblasts in addition to their anti-apoptosis functions. We further demonstrated the increased expression of DjApi5 during planarian regeneration, and DjApi5 deficiency affects normal regeneration processes. These results indicated new functions of DjApi5 in development.

Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis.

SIRT6 deacetylase activity improves stress resistance via gene silencing and genome maintenance. Here, we reveal a deacetylase-independent function of SIRT6, which promotes anti-apoptotic gene expression via the transcription factor GATA4. SIRT6 recruits TIP60 acetyltransferase to acetylate GATA4 at K328/330, thus enhancing its chromatin binding capacity. In turn, GATA4 inhibits the deacetylase activity of SIRT6, thus ensuring the local chromatin accessibility via TIP60-promoted H3K9 acetylation. Significantly, the treatment of doxorubicin (DOX), an anti-cancer chemotherapeutic, impairs the SIRT6-TIP60-GATA4 trimeric complex, blocking GATA4 acetylation and causing cardiomyocyte apoptosis. While GATA4 hyperacetylation-mimic retains the protective effect against DOX, the hypoacetylation-mimic loses such ability. Thus, the data reveal a novel SIRT6-TIP60-GATA4 axis, which promotes the anti-apoptotic pathway to prevent DOX toxicity. Targeting the trimeric complex constitutes a new strategy to improve the safety of DOX chemotherapy in clinical application.

The Wnt/Ca2+ signaling pathway is essential for the regeneration of GABAergic neurons in planarian Dugesia japonica.

The growth and differentiation of neurons are critical events in the establishment of proper neuron connectivity and function. Planarians have a remarkable ability to completely regenerate a functional nervous system from a pluripotent stem cell population. Thus, planarians provide a powerful model to identify genes required for neuronal differentiation in vivo. The Wnt/Ca2+ signaling pathway is crucial for cancer development, arousing inflammatory responses, and neurodegeneration. We analyzed the expression patterns and RNAi phenotypes for members of the Wnt/Ca2+ signaling pathway in the planarian, Dugesia japonica. The expression of DjWnt5a, DjPLC-ß, DjCamKII, and DjCaln during regeneration was surprisingly similar and revealing in the regenerated brain. RNAi knockdown of DjWnt5a, DjPLC-ß, DjCamKII, and DjCaln led to defects in regenerated brains including brain partial deletions, incompact phenotypes at the posterior of the new brain, and lateral branches, which could not regenerate. Furthermore, the expressions of GAD and the number of GABAergic neurons decreased. Together, these results suggest that the Wnt/Ca2+ signaling pathway is required for GABAergic neuron regeneration.

CCDC114, DNAI2 and TOP2A involves in the effects of tibolone treatment on postmenopausal endometrium.

BACKGROUND: This study aimed to explore the molecular mechanisms of tibolone treatment in postmenopausal women. METHODS: The gene set enrichment profile, GSE12446, which includes 9 human endometrial samples from postmenopausal women treated with tibolone (tibolone group) and 9 control samples (control group), was downloaded from GEO database for analysis. Differentially expressed genes (DEGs) in tibolone vs. control groups were identified and then used for function and pathway enrichment analysis. Protein-protein interaction (PPI) network and module analyses were also performed. Finally, drug-target interaction was predicted for genes in modules, and then were validated in Pubmed. RESULTS: A total of 238 up-regulated DEGs and 72 down-regulated DEGs were identified. These DEGs were mainly enriched in various biological processed and pathways, such as cilium movement (e.g., CCDC114 and DNAI2), calcium ion homeostasis, regulation of hormone levels and complement/coagulation cascades. PPI network contained 368 interactions and 166 genes, of which IGF1, DNALI1, CCDC114, TOP2A, DNAH5 and DNAI2 were the hue genes. A total of 96 drug-gene interactions were obtained, including 94 drugs and eight genes. TOP2A and HTR2B were found to be targets of 28 drugs and 38 drugs, respectively. Among the 94 obtained drugs, only 12 drugs were reported in studies, of which 7 drugs (e.g., epirubicin) were found to target TOP2A. CONCLUSIONS: CCDC114 and DNAI2 might play important roles in tibolone-treated postmenopausal women via cilium movement function. TOP2A might be a crucial target of tibolone in endometrium of postmenopausal women.

Different classes of small RNAs are essential for head regeneration in the planarian Dugesia japonica.

BACKGROUND: Planarians reliably regenerate all body parts after injury, including a fully functional head and central nervous system. But until now, the expression dynamics and functional role of miRNAs and other small RNAs during the process of head regeneration are not well understood. Furthermore, little is known about the evolutionary conservation of the relevant small RNAs pathways, rendering it difficult to assess whether insights from planarians will apply to other taxa. RESULTS: In this study, we applied high throughput sequencing to identify miRNAs, tRNA fragments and piRNAs that are dynamically expressed during head regeneration in Dugesia japonica. We further show that knockdown of selected small RNAs, including three novel Dugesia-specific miRNAs, during head regeneration induces severe defects including abnormally small-sized eyes, cyclopia and complete absence of eyes. CONCLUSIONS: Our findings suggest that a complex pool of small RNAs takes part in the process of head regeneration in Dugesia japonica and provide novel insights into global small RNA expression profiles and expression changes in response to head amputation. Our study reveals the evolutionary conserved role of miR-124 and brings further promising candidate small RNAs into play that might unveil new avenues for inducing restorative programs in non-regenerative organisms via small RNA mimics based therapies.

Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica.

As a typical organism of platyhelminth, Dugesia japonica attracts more and more attention for its strong regenerative ability. Protein arginine methyltransferase (PRMT) family is composed of a class of enzymes with methyltransferase activities, which play fundamental roles in vivo in many important physiological processes. PRMT1 is a predominant type Ⅰ PRMT, which has been reported to be expressed in Schmidtea mediterranea. Nevertheless, the existence and the specific biological functions of PRMT1 in Dugesia japonica need further investigation. In this study, we acquired the full-length sequence of DjPRMT1 and confirmed it was a conserved protein. Thereafter, whole-mount in situ hybridization results showed DjPRMT1 was mainly expressed in neoblasts of adult worms, and obvious aggregation of DjPRMT1 was observed at the wound site in early stages of regeneration. Silencing of the DjPRMT1 gene retarded the movement of planarians with decreased DjPIWI-A expression, and DjPRMT1 knockdown also affected planarian regeneration with slightly attenuated proliferation around the blastema of posterior-facing wounds regeneration. In summary, these preliminary results demonstrated DjPRMT1 was involved in the regeneration of planarian.

The planarian Vinculin is required for the regeneration of GABAergic neurons in Dugesia japonica.

Vinculin is a cytoskeletal protein associated with cell-cell and cell-matrix junctions, playing an important role in linkage of integrin adhesion molecules to the actin cytoskeleton. The planarian nervous system is a fascinating system for studying the organogenesis during regeneration. In this paper, a homolog gene of Vinculin, DjVinculin, was identified and characterized in Dugesia japonica. The DjVinculin sequence analysis revealed that it contains an opening reading frame encoding a putative protein of 975 amino acids with functionally domains that are highly conserved, including eight anti-parallel α-helical bundles organized into five distinct domains. Whole mount in situ hybridization showed that DjVinculin was predominantly expressed in the brain of intact and regenerating planarians. RNA interference of DjVinculin caused distinct defects in brain morphogenesis and influences the regeneration of planarian GABAergic neurons. The expression level of DjGAD protein was decreased in the DjVinculin-knockdown planarians. These findings suggest that DjVinculin is required for GABAergic neurons regeneration.

Vitamin C inhibits lipid deposition through GSK-3ß/mTOR signaling in the liver of zebrafish.

In this study, the mechanism that VC inhibits lipid deposition through GSK-3ß/mTOR signaling was investigated in the liver of Danio rerio. The results indicated that 0.5- and 1.0-g/kg VC treatments activated mTOR signaling by inhibiting GSK-3ß expression. The mRNA expression of FAS, ACC, and ACL, as well as the content of TG, TC, and NEFA, was decreased by 0.5- and 1.0-g/kg VC treatments. Moreover, to confirm GSK-3ß playing a key role in regulating TSC2 and mTOR, GSK-3ß RNA was interfered and the activity of GSK-3ß was inhibited by 25- and 50-mg/L LiCl treatments, respectively. The results indicated that GSK-3ß inactivation played a significant role in inducing mTOR signaling and inhibiting lipid deposition. VC treatments could induce mTOR signaling by inhibiting GSK-3ß, and mTOR further participated in regulating lipid deposition by controlling lipid profile in the liver of zebrafish.

Met-enkephalin inhibits ROS production through Wnt/ß-catenin signaling in the ZF4 cells of zebrafish.

Opioid neuropeptides are developed early in the course of a long evolutionary process. As the endogenous messengers of immune system, opioid neuropeptides participate in regulating immune response. In this study, the mechanism that Met-enkephalin (M-ENK) inhibits ROS production through Wnt/ß-catenin signaling was investigated in the ZF4 cells of zebrafish. ZF4 cells were exposed to 0, 10, 20, 40, 80, and 160 µM Met-enkephalin (M-ENK) for 24 h, and the cell viability was detected with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The cell viability was significantly increased by 10, 20, 40, 80, and 160 µM M-ENK. After ZF4 cells were exposed to 0, 20, 40, and 80 µM M-ENK for 24 h, the mRNA expression of Wnt10b, ß-catenin, and CCAAT/enhancer binding protein α (C/EBPα) was significantly increased by 40 and 80 µM M-ENK. However, the mRNA and protein expression of GSK-3ß was significantly decreased by 40 and 80 µM M-ENK. The protein expression of ß-catenin was significantly induced by 40 and 80 µM M-ENK, while the protein expression of p-ß-catenin was significantly decreased by 20, 40, and 80 µM M-ENK. In addition, the mRNA expression of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 µM M-ENK. The levels of H2O2, ·OH, and O2·- were significantly decreased, but the activity of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 µM M-ENK. The fluorescence intensity of reactive oxygen species (ROS) was decreased, and that of mitochondrial membrane potential (MMP) was increased with the increase of M-ENK concentration in ZF4 cells. The results showed that M-ENK could induce Wnt/ß-catenin signaling, which further inhibited ROS production through the induction of C/EBPα, MMP, and the activities of antioxidant enzymes.

The inhibition of GSK-3ß promotes the production of reactive oxygen species via ß-catenin/C/EBPα signaling in the spleen of zebrafish (Danio rerio).

In this study, the mechanism that the inhibition of glycogen synthase kinase-3ß (GSK-3ß) promotes the production of reactive oxygen species (ROS) via ß-catenin/CCAAT/enhancer binding protein α (C/EBPα) signaling was investigated in the spleen of zebrafish (Danio rerio). The results demonstrated that the inhibition of GSK-3ß induced the mRNA expression of ß-catenin and C/EBPα by lithium (Li) treatments or GSK-3ß RNA interference. The levels of hydrogen peroxide (H2O2), superoxide anion (O2.-), and hydroxy radical (·OH) as well as the activity of superoxide dismutase (SOD) were increased, while the activities of catalase (CAT) and glutathione peroxidase (GSH-PX) were decreased in the spleen and ZF4 cells of zebrafish by Li+ treatments. In addition, GSK-3ß RNA interference increased ROS levels and decreased the activities of CAT and GSH-PX in the spleen. The fluorescence intensity of ROS was increased but the mitochondrial membrane potential (MMP) was decreased by Li+ treatments in ZF4 cells labeled with 2',7'-dichlorofluorescein diacetate (DCFH-DA) and Rhodamine-123, respectively. The results of present study indicated that the inhibition of GSK-3ß promoted the ROS production via ß-catenin/C/EBPα signaling in the spleen of zebrafish, and the balance between ROS and antioxidants could be destroyed by the GSK-3ß/ß-catenin/C/EBPα signaling. The results may be a valuable contribution to understanding the modulatory mechanism of GSK-3ß/ß-catenin/C/EBPα signaling on the antioxidant system in fish species.

Identification and functional analysis of invasion associated locus B (IalB) in Bartonella species.

Bartonellosis is caused by the genus Bartonella. Bartonella is widely distributed in the ruminants, cats, dogs, rodents and other mammals including humans. At least 13 species or subspecies of Bartonella are zoonotic, and each species appears to be highly adapted to one or a limited number of reservoir animals in which it is asymptomatic, while it can be transmitted to humans in which a variety of clinical manifestations can be caused. It was reported that Bartonella henselae infection rate among domestic cats was high in nature, making it one of the leading, important, and easily neglected zoonotic diseases. The aims of this study were to identify the expression, localization, immunogenicity and functional mechanism of Bartonella virulence factor IalB. We found that recombinant IalB protein could react with the serum from infected reservoir hosts and anti-IalB polyclonal antibodies could react with different Bartonella species by western blot analysis. According to these results, we proposed that IalB protein and anti-IalB antibodies would be good candidates for diagnosis of Bartonella infection by antigen-based anti-IalB antibodies or antibody-based IalB antigen capture immunoassay, respectively. We also found that IalB had a putative 22-amino-acid signal sequence and little IalB was localized to the outer membrane of Bartonella birtlesii by electron microscopy assay. Incubation with anti-IalB polyclonal antibodies resulted in inhibition of the invasion of mouse erythrocytes by B. birtlesii. According to these results, we propose that IalB could be a secreted protein that facilitates Bartonella entry into erythrocytes. In conclusion, these results improve our understanding of IalB as a candidate for immunodiagnosis and how IalB affects Bartonella-erythrocyte entry.

Planarian myosin essential light chain is involved in the formation of brain lateral branches during regeneration.

The myosin essential light chain (ELC) is a structure component of the actomyosin cross-bridge, however, the functions in the central nervous system (CNS) development and regeneration remain poorly understood. Planarian Dugesia japonica has revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. In this study, the cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. It encodes a deduced protein with highly conserved functionally domains EF-Hand and Ca(2+) binding sites that shares significant similarity with other members of ELC. Whole mount in situ hybridization studies show that DjElc expressed in CNS during embryonic development and regeneration of adult planarians. Loss of function of DjElc by RNA interference during planarian regeneration inhibits brain lateral branches regeneration completely. In conclusion, these results demonstrated that DjElc is required for maintenance of neurons and neurite outgrowth, particularly for involving the brain later branch regeneration.

Cloning, characterization and expression of tyrosinase-like gene in amphioxus Branchiostoma japonicum.

Prophenoloxidase (tyrosinase) widely distributed in invertebrates and vertebrates, and plays a crucial role in the innate immune. In the present study, the full-length cDNA of a tyrosinase-like (designated AmphiTYR) was cloned from amphioxus Branchiostoma japonicum by PCR techniques. The full-length cDNA of AmphiTYR is 2314 bp, and its predicted open-reading frame codes for a protein of 544 amino acids with a predicted molecular mass of approximately 60.9 kDa and an isoelectric point of 5.65. It has a conserved putative copper-binding domain with six histidines in tyrosinase proteins. Six potential N-linked glycosylation sites and 14 conserved cysteine residues were also predicted to be present in B. japonicum tyrosinase. Homology analysis revealed that AmphiTYR was higher similar to vertebrates tyrosinases (32.5-40.5%) than to invertebrates phenoloxidase (6.4-25.4%). In the adult, AmphiTYR mRNA was expressed in the muscle, epidermis, notochord, ovary, hepatic caecum, pharynx and gill, but not in the neural tube, intestines and testis. During the different development stages from unfertilized egg to larvae of amphioxus, AmphiTYR expressed during all the amphioxus development. These results indicated that AmphiTYR gene not only play a pivotal role in innate immune but also play an important role during embryonic development of cephalochordate amphioxus.