Identification of a tyrosinase gene and its functional analysis in melanin synthesis of Pteria penguin.

Tyrosinase is a key rate-limiting enzyme in melanin synthesis. In this study, a new tyrosinase gene (Tyr) was identified from Pteria penguin and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpTyr was 1728 bp long, containing a 5'untranslated region (UTR) of 11 bp, a 3'UTR of 295 bp, and an open reading fragment of 1422 bp encoding 473 amino acids. Amino acid alignment showed PpTyr had the highest (50%) identity to tyrosinase-like protein 1 from Pinctada fucata. Phylogenetic tree analysis classified PpTyr into α-subclass of type-3 copper protein. Tissue expression analysis indicated that PpTyr was highly expressed in mantle, a nacre formation related tissue. After PpTyr RNA interference, PpTyr mRNA was significantly inhibited by 71.0% (P < 0.05). For other melanin-related genes, PpCreb2 and PpPax3 expression showed no significant change, but PpBcl2 was obviously increased. By liquid chromatograph-tandem mass spectrometer (LC-MS/MS) analysis, the total content of PDCA (pyrrole-2, 3-dicarboxylic acid) and PTCA (pyrrole-2,3,5-tricarboxylic acid), as main markers for eumelanin, was sharply decreased by 66.6% after PpTyr RNAi (P < 0.05). The percentage of PDCA was also obviously decreased from 20.1% to 13.9%. This indicated that tyrosinase played a key role in melanin synthesis and color formation of P. penguin.

Catalytic mechanism of the tyrosinase reaction toward the Tyr98 residue in the caddie protein.

Tyrosinase (EC 1.14.18.1), a copper-containing monooxygenase, catalyzes the conversion of phenol to the corresponding ortho-quinone. The Streptomyces tyrosinase is generated as a complex with a "caddie" protein that facilitates the transport of two copper ions into the active center. In our previous study, the Tyr98 residue in the caddie protein, which is accommodated in the pocket of active center of tyrosinase, has been found to be converted to a reactive quinone through the formations of the µ-η2:η2-peroxo-dicopper(II) and Cu(II)-dopasemiquinone intermediates. Until now-despite extensive studies for the tyrosinase reaction based on the crystallographic analysis, low-molecular-weight models, and computer simulations-the catalytic mechanism has been unable to be made clear at an atomic level. To make the catalytic mechanism of tyrosinase clear, in the present study, the cryo-trapped crystal structures were determined at very high resolutions (1.16-1.70 Å). The structures suggest the existence of an important step for the tyrosinase reaction that has not yet been found: that is, the hydroxylation reaction is triggered by the movement of CuA, which induces the syn-to-anti rearrangement of the copper ligands after the formation of µ-η2:η2-peroxo-dicopper(II) core. By the rearrangement, the hydroxyl group of the substrate is placed in an equatorial position, allowing the electrophilic attack to the aromatic ring by the Cu2O2 oxidant.

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis.

Codon usage bias (CUB) is a unique property of genomes and has contributed to the better understanding of the molecular features and the evolution processes of particular gene. In this study, genetic indices associated with CUB, including relative synonymous codon usage and effective numbers of codons, as well as the nucleotide composition, were investigated in the Clonorchis sinensis tyrosinase genes and their platyhelminth orthologs, which play an important role in the eggshell formation. The relative synonymous codon usage patterns substantially differed among tyrosinase genes examined. In a neutrality analysis, the correlation between GC12 and GC3 was statistically significant, and the regression line had a relatively gradual slope (0.218). NC-plot, i.e., GC3 vs effective number of codons (ENC), showed that most of the tyrosinase genes were below the expected curve. The codon adaptation index (CAI) values of the platyhelminth tyrosinases had a narrow distribution between 0.685/0.714 and 0.797/0.837, and were negatively correlated with their ENC. Taken together, these results suggested that CUB in the tyrosinase genes seemed to be basically governed by selection pressures rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and Opisthorchis viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias is much more inclined to selection pressure in highly expressed C. sinensis genes, than in poorly expressed genes.

Temperature increases, hypoxia, and changes in food availability affect immunological biomarkers in the marine mussel Mytilus galloprovincialis.

Temperature increases, hypoxia, and changes in food availability are predicted to occur in the future. There is growing concern for the health status of wild and farmed organisms, since environmental stressors alter organism functions, and elicit coordinated physiological responses for homeostasis. Mussels are good bioindicators of environmental conditions. Their ability to maintain unaltered immunosurveillance under adverse environmental conditions may enhance their survival capability. Few studies are currently concerned with the relationships and feedback among multiple stressors. Here, food concentration, temperature, and oxygenation treatments were evaluated for their effects on immune enzymatic parameters of Mytilus galloprovincialis detected in the digestive gland and the lysosomal viability by neutral red uptake. Mussels were exposed to three temperatures (12, 20, and 28 °C) under normoxic (8 mg O2l-1) and anoxic conditions and specimens were fed with six food concentrations, ranging 0.2-5 g chlorophyll l-1. Temperature increases affected esterase and alkaline phophatase enzyme functionality, and addition of food buffered detrimental effects generated by harsh conditions, such as those provided by low oxygen concentrations. Kinetics of the phenoloxidase was negatively correlated with increasing temperature. In this case, food had a buffering effect that counteracted the limiting temperature only under normoxic conditions. In addition, the stability of the lysosomal membrane was altered under conditions of thermal stress and food change, under normoxic and anoxic conditions. Overall, environmental stress factors affected immune biomarkers of Mediterranean mussels, and the level of food acted as a buffer, increasing the thermal resistance of the specimens.

Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1.

Developmental eye defects in X-linked ocular albinism type 1 are caused by G-protein coupled receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most G protein-coupled receptors, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for ocular albinism type I. Many G protein-coupled receptors require association with other proteins to function. These G protein-coupled receptor-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 coimmunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when coexpressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using fluorescence resonance energy transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis.

Tyrosinase, could it be a missing link in ochronosis in alkaptonuria?

The hypothesis that is proposed is that tyrosinase, an enzyme widely found within the human body is implicated in the ochronosis that occurs in alkaptonuria; an autosomal recessive condition first used by Archibald Garrod to describe the theory of "Inborn Errors of Metabolism." The disease results from the absence of a single enzyme in the liver that breaks down homogentisic acid; this molecule becomes systemically elevated in sufferers. The condition is characterised by a clinical triad of symptoms; homogentisic aciduria from birth, ochronosis (darkening) of collagenous tissues (from ∼30years of age) and ochronotic osteoarthropathy in weight bearing joints due to long term ochronosis in them (from ∼40years of age). Tyrosinase, a polyphenol oxidase has been shown in many species to contribute to the darkening of tissues in many organisms; including humans in the production of melanin. Tyrosinase under the right conditions shows alterations in its substrate specificity and may contribute to the darkening seen in AKU where it moves away from polymerising tyrosine but also homogentisic acid, the causative molecule in alkaptonuria, that is present in excess.

Activity of Redox Enzymes in the Thallus of Anthoceros natalensis.

Anthocerotophyta (hornworts) belong to a group of ancient nonvascular plants and originate from a common ancestor with contemporary vascular plants. Hornworts represent a unique model for investigating mechanisms of formation of stress resistance in higher plants due to their high tolerance to the action of adverse environmental factors. In this work, we demonstrate that the thallus of Anthoceros natalensis exhibits high redox activity changing under stress. Dehydration of the thallus is accompanied by the decrease in activities of intracellular peroxidases, DOPA-peroxidases, and tyrosinases, while catalase activity increases. Subsequent rehydration results in the increase in peroxidase and catalase activities. Kinetic features of peroxidases and tyrosinases were characterized as well as the peroxidase isoenzyme composition of different fractions of the hornwort cell wall proteins. It was shown that the hornwort peroxidases are functionally similar to peroxidases of higher vascular plants including their ability to form superoxide anion-radical. The biochemical mechanism was elucidated, supporting the possible participation of peroxidases in the formation of reactive oxygen species (ROS) via substrate-substrate interactions in the hornwort thallus. It has been suggested that the ROS formation by peroxidases is an evolutionarily ancient process that emerged as a protective mechanism for enhancing adaptive responses of higher land plants and their adaptation to changing environmental conditions and successful colonization of various ecological niches.

Identification of a novel microRNA important for melanogenesis in alpaca (Vicugna pacos).

The molecular mechanisms underlying the formation of coat colors in animals are poorly understood. Recent studies have demonstrated that microRNA play important roles in the control of melanogenesis and coat color in mammals. In a previous study, we characterized the miRNA expression profiles in alpaca skin with brown and white coat color and identified a novel miRNA (named lpa-miR-nov-66) that is expressed significantly higher in white skin compared to brown skin. The present study was conducted to determine the functional roles of this novel miRNA in the regulation of melanogenesis in alpaca melanocytes. lpa-miR-nov-66 is predicted to target the soluble guanylate cyclase (sGC) gene based on presence of a binding site in the sGC coding sequence (CDS). Overexpression of lpa-miR-nov-66 in alpaca melanocyes upregulated the expression of sGC both at the mRNA and protein level. Overexpression of lpa-miR-nov-66 in melanocyes also resulted in decreased expression of key melanogenic genes including tyrosinase (TYR), tyrosinase related protein 1 (TYRP1), and microphthalmia transcription factor (MITF). Our ELISA assays showed increased cyclic guanosine monophosphate (cGMP) but decreased cyclic adenosine monophosphate (cAMP) production in melanocytes overexpressing lpa-miR-nov-66. In addition, overexpression of lpa-miR-nov-66 also reduced melanin production in cultured melanocytes. Results support a role of lpa-miR-nov-66 in melanocytes by directly or indirectly targeting , which regulates melanogenesis via the cAMP pathway.

[Leukoderma caused by chemicals: mechanisms underlying 4-alkyl/aryl-substituted phenols- and rhododendrol-induced melanocyte loss].

Chemical leukoderma is a skin depigmentation disorder known to occur in manufactural workplace through contact with chemicals, such as monobenzyl ether of hydroquinone (MBEH) and 4-tert- butylphenol (4-TBP). In the skin depigmented -legions induced by these chemicals, the number of melanocyte was severely decreased. Anti-melanoma agent 4-cysteaminylphenol (4-SCAP) and its derivatives are also known to cause leukoderma. Evidence has accumulated supporting that typical class of chemicals causing leukoderma is "4-alkyl/aryl-substituted phenols/catechols", which are structurally similar to melanin precursor tyrosine. Tyrosinase-mediated oxidation of these chemicals yields toxic ortho-quinones which bind to cellular proteins and produce reactive oxygen species. Accordingly, this tyrosinase-dependent metabolic activation is thought to cause melanocyte-specific damage and subsequent immune reactions toward melanocytes. Recently, rhododendrol, an inhibitor of tyrosinase developed for so-called lightening/whitening cosmetics, was shown to cause leukoderma in the users. In this review, I document the causes of known chemical leukoderma and rhododendrol- induced leukoderma, focusing on their common mechanisms underlying melanocyte loss.

Diverse immune functions of hemocyanins.

Substantial evidence gathered recently has revealed the multiple functionalities of hemocyanin. Contrary to previous claims that this ancient protein is involved solely in oxygen transport within the hemolymph of invertebrates, hemocyanin and hemocyanin-derived peptides have been linked to key aspects of innate immunity, in particular, antiviral and phenoloxidase-like activities. Both phenoloxidase and hemocyanin belong to the family of type-3 copper proteins and share a high degree of sequence homology. While the importance of phenoloxidase in immunity and development is well characterised, the contribution of hemocyanin to biological defence systems within invertebrates is not recognised widely. This review focusses on the conversion of hemocyanin into a phenoloxidase-like enzyme and the array of hemocyanin-derived immune responses documented to date.

L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions.

There is evidence that L-tyrosine and L-dihydroxyphenylalanine (L-DOPA), besides serving as substrates and intermediates of melanogenesis, are also bioregulatory agents acting not only as inducers and positive regulators of melanogenesis but also as regulators of other cellular functions. These can be mediated through action on specific receptors or through non-receptor-mediated mechanisms. The substrate induced (L-tyrosine and/or L-DOPA) melanogenic pathway would autoregulate itself as well as regulate the melanocyte functions through the activity of its structural or regulatory proteins and through intermediates of melanogenesis and melanin itself. Dissection of regulatory and autoregulatory elements of this process may elucidate how substrate-induced autoregulatory pathways have evolved from prokaryotic or simple eukaryotic organisms to complex systems in vertebrates. This could substantiate an older theory proposing that receptors for amino acid-derived hormones arose from the receptors for those amino acids, and that nuclear receptors evolved from primitive intracellular receptors binding nutritional factors or metabolic intermediates.

Tyrosinase is the modifier of retinoschisis in mice.

X-linked retinoschisis (XLRS) is a form of macular degeneration with a juvenile onset. This disease is caused by mutations in the retinoschisin (RS1) gene. The major clinical pathologies of this disease include splitting of the retina (schisis) and a loss in synaptic transmission. Human XLRS patients display a broad range in phenotypic severity, even among family members with the same mutation. This variation suggests the existence of genetic modifiers that may contribute to disease severity. Previously, we reported the identification of a modifier locus, named Mor1, which affects severity of schisis in a mouse model of XLRS (the Rs1tmgc1 mouse). Homozygosity for the protective AKR allele of Mor1 restores cell adhesion in Rs1tmgc1 mice. Here, we report our study to identify the Mor1 gene. Through collecting recombinant mice followed by progeny testing, we have localized Mor1 to a 4.4-Mb region on chromosome 7. In this genetic region, the AKR strain is known to carry a mutation in the tyrosinase (Tyr) gene. We observed that the schisis phenotype caused by the Rs1 mutation is rescued by a Tyr mutation in the C57BL/6J genetic background, strongly suggesting that Tyr is the Mor1 gene.

The Drosophila PRR GNBP3 assembles effector complexes involved in antifungal defenses independently of its Toll-pathway activation function.

The Drosophila Toll-signaling pathway controls the systemic antifungal host response. Gram-negative binding protein 3 (GNBP3), a member of the beta-glucan recognition protein family senses fungal infections and activates this pathway. A second detection system perceives the activity of proteolytic fungal virulence factors and redundantly activates Toll. GNBP3(hades) mutant flies succumb more rapidly to Candida albicans and to entomopathogenic fungal infections than WT flies, despite normal triggering of the Toll pathway via the virulence detection system. These observations suggest that GNBP3 triggers antifungal defenses that are not dependent on activation of the Toll pathway. Here, we show that GNBP3 agglutinates fungal cells. Furthermore, it can activate melanization in a Toll-independent manner. Melanization is likely to be an essential defense against some fungal infections given that the entomopathogenic fungus Beauveria bassiana inhibits the activity of the main melanization enzymes, the phenol oxidases. Finally, we show that GNBP3 assembles "attack complexes", which comprise phenoloxidase and the necrotic serpin. We propose that Drosophila GNBP3 targets fungi immediately at the inception of the infection by bringing effector molecules in direct contact with the invading microorganisms.

Innate immunity of the white shrimp Litopenaeus vannamei weakened by the combination of a Vibrio alginolyticus injection and low-salinity stress.

White shrimp Litopenaeus vannamei were reared at a salinity of 35 per thousand without a Vibrio alginolyticus injection (unchallenged group), and other shrimp were reared at 35 per thousand, injected with tryptic-soy broth (TSB)-grown V. alginolyticus at 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) (challenged group), and then examined for the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after transfer to 35 per thousand (control), 25 per thousand, 20 per thousand, and 15 per thousand for 1, 6, 12, 24, 72, and 120 h. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of unchallenged shrimp and challenged shrimp that were transferred to low-salinity levels all began to significantly decrease at 6, 6, 6, and 1 h, respectively, and reached the lowest levels at 12 h. HC, GC, the THC, PO activity, RB, and SOD activity of unchallenged shrimp that were transferred to 15 per thousand decreased by 53%, 41%, 49%, 68%, 39%, and 62%, whereas those parameters of challenged shrimp that were transferred to 15 per thousand decreased by 79%, 78%, 79%, 82%, 54%, and 72%, respectively after 12 h compared to control shrimp. These immune parameters began to recover after 24-72 h for both unchallenged shrimp and challenged shrimp. We concluded that the innate immunity was weakened in white shrimp L. vannamei that received combined stresses of a V. alginolyticus injection, and low-salinity transfer. It was also concluded that shrimp with respectively 21%, 18%, 46%, and 28% lower THC, PO activity, RB, and SOD activity of the original values would be killed due to decreases in their immunity, and resistance to V. alginolyticus infection. Shrimp farming should be maintained at a constant high salinity level to prevent exacerbated decreases in innate immune parameters of shrimp when infected by a pathogen coupled with low-salinity stress leading to mortality.

AAV-mediated tyrosinase gene transfer restores melanogenesis and retinal function in a model of oculo-cutaneous albinism type I (OCA1).

Oculo-cutaneous albinism type 1 (OCA1) is characterized by congenital hypopigmentation and is due to mutations in the TYROSINASE gene (TYR). In this study, we have characterized the morpho-functional consequences of the lack of tyrosinase activity in the spontaneous null mouse model of OCA1 (Tyr(c-2j)). Here, we show that adult Tyr(c-2j) mice have several retinal functional anomalies associated with photoreceptor loss. To test whether these anomalies are reversible upon TYR complementation, we performed intraocular administration of an adeno-associated virus (AAV)-based vector, encoding the human TYR gene, in adult Tyr(c-2j) mice. This resulted in melanosome biogenesis and ex novo synthesis of melanin in both neuroectodermally derived retinal pigment epithelium (RPE) and in neural crest-derived choroid and iris melanocytes. Ocular melanin accumulation prevented progressive photoreceptor degeneration and resulted in restoration of retinal function. Our results reveal novel properties of pigment cells and show that the developmental anomalies of albino mice are associated with defects occurring in postnatal life, adding novel insights on OCA1 disease pathogenesis. In addition, we provide proof-of-principle of an effective gene-based strategy relevant for future application in albino patients.

Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family.

N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.

Inhibition of MITF and tyrosinase by paeonol-stimulated JNK/SAPK to reduction of phosphorylated CREB.

Tyrosinase and its transcriptional regulator microphthalmia-associated transcription factor (MITF) play critical roles in regulation of melanogenesis, and are required for environmental cues or agents in modulation of melanin synthesis. Identifying the signals regulating tyrosinase and MITF is crucial to understanding how pigmentation responds to extracellular stimuli. In this report, we discovered that paeonol down-regulated melanin production via decreasing MITF expression and consequent mRNA and protein levels of tyrosinase. We also found that paeonol reduced phosphorylation of a cAMP responsive element binding protein (phospho-CREB), which binds and activates MITF. A selective inhibitor of c-jun N-terminal or stress-activated protein kinases (JNK/SAPK)-SP600125 significantly reversed paeonol-induced down-regulation of melanogenesis. Inhibition of cAMP/PKA pathway intensified the hypopigmentation response to paeonol. These results identify a mechanism in which paeonol induces the down-regulation of melanogenesis through inhibition of CREB phosphorylation, leading to the expression reduction of MITF and subsequently tyrosinase. The key kinase mediating the effects of paeonol on melanogenesis in B16F10 cells is JNK/SAPK. Additionally, the cAMP/PKA pathway may take part in this process.

Parkin protects against tyrosinase-mediated dopamine neurotoxicity by suppressing stress-activated protein kinase pathways.

Parkinson's disease (PD) motor symptoms are caused by degeneration of nigrostriatal dopaminergic (DAergic) neurons. The most common causes of hereditary PD are mutations in the PARKIN gene. The ubiquitin ligase parkin has been shown to mediate neuroprotection in cell culture and in vivo, but the molecular mechanisms are not well understood. We investigated the effects of parkin in a human SH-SY5Y neuroblastoma cell culture model of PD, in which transcriptional induction of the enzyme tyrosinase causes a neurotoxic overproduction of cellular DA and its oxidative metabolites. Tyrosinase induction caused formation of reactive oxygen species in the cytosol and mitochondria, and neurotoxicity via activation of apoptotic stress-activated protein kinases and caspase 3. Stable transfection of wild-type parkin suppressed tyrosinase-induced apoptosis, and PD-associated mutations abolished the neuroprotective effect of parkin. Expression of wild-type parkin did not affect reactive oxygen species production, but attenuated the tyrosinase-induced activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase as well as their cognate mitogen-activated protein kinase kinases. PD-associated mutations differentially affected the anti-apoptotic signaling of parkin. Thus, parkin contributes to DAergic neuroprotection by suppression of apoptotic stress-activated protein kinase pathways.

Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells.

The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.

Phenoloxidase is an important component of the defense against Aeromonas hydrophila Infection in a crustacean, Pacifastacus leniusculus.

The melanization cascade, in which phenoloxidase is the terminal enzyme, appears to play a key role in recognition of and defense against microbial infections in invertebrates. Here, we show that phenoloxidase activity and melanization are important for the immune defense toward a highly pathogenic bacterium, Aeromonas hydrophila, in the freshwater crayfish, Pacifastacus leniusculus. RNA interference-mediated depletion of crayfish prophenoloxidase leads to increased bacterial growth, lower phagocytosis, lower phenoloxidase activity, lower nodule formation, and higher mortality when infected with this bacterium. In contrast, if RNA interference of pacifastin, an inhibitor of the crayfish prophenoloxidase activation cascade, is performed, it results in lower bacterial growth, increased phagocytosis, increased nodule formation, higher phenoloxidase activity, and delayed mortality. Our data therefore suggest that phenoloxidase is required in crayfish defense against an infection by A. hydrophila, a highly virulent and pathogenic bacterium to crayfish.