Femtosecond pulsed laser photodynamic therapy activates melanin and eradicates malignant melanoma.

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

Adaptive cooling strategy via human hair: High optothermal conversion efficiency of solar radiation into thermal dissipation.

Natural species have developed complex nanostructures in a hierarchical pattern to control the absorption, reflection, or transmission of desired solar and infrared wavelengths. This bio-inspired structure is a promising method to manipulating solar energy and thermal management. In particular, human hair is used in this article to highlight the optothermal properties of bio-inspired structures. This study investigated how melanin, an effective solar absorber, and the structural morphology of aligned domains of keratin polymer chains, leading to a significant increase in solar path length, which effectively scatter and absorb solar radiation across the hair structure, as well as enhance thermal ramifications from solar absorption by fitting its radiative wavelength to atmospheric transmittance for high-yield radiative cooling with realistic human body thermal emission.

Emergent properties of melanin-inspired peptide/RNA condensates.

Most biocatalytic processes in eukaryotic cells are regulated by subcellular microenvironments such as membrane-bound or membraneless organelles. These natural compartmentalization systems have inspired the design of synthetic compartments composed of a variety of building blocks. Recently, the emerging field of liquid-liquid phase separation has facilitated the design of biomolecular condensates composed of proteins and nucleic acids, with controllable properties including polarity, diffusivity, surface tension, and encapsulation efficiency. However, utilizing phase-separated condensates as optical sensors has not yet been attempted. Here, we were inspired by the biosynthesis of melanin pigments, a key biocatalytic process that is regulated by compartmentalization in organelles, to design minimalistic biomolecular condensates with emergent optical properties. Melanins are ubiquitous pigment materials with a range of functionalities including photoprotection, coloration, and free radical scavenging activity. Their biosynthesis in the confined melanosomes involves oxidation-polymerization of tyrosine (Tyr), catalyzed by the enzyme tyrosinase. We have now developed condensates that are formed by an interaction between a Tyr-containing peptide and RNA and can serve as both microreactors and substrates for tyrosinase. Importantly, partitioning of Tyr into the condensates and subsequent oxidation-polymerization gives rise to unique optical properties including far-red fluorescence. We now demonstrate that individual condensates can serve as sensors to detect tyrosinase activity, with a limit of detection similar to that of synthetic fluorescent probes. This approach opens opportunities to utilize designer biomolecular condensates as diagnostic tools for various disorders involving abnormal enzymatic activity.

Exploratory Rearing Is Governed by Hypothalamic Melanin-Concentrating Hormone Neurons According to Locus Ceruleus.

Information seeking, such as standing on tiptoes to look around in humans, is observed across animals and helps survival. Its rodent analog-unsupported rearing on hind legs-was a classic model in deciphering neural signals of cognition and is of intense renewed interest in preclinical modeling of neuropsychiatric states. Neural signals and circuits controlling this dedicated decision to seek information remain largely unknown. While studying subsecond timing of spontaneous behavioral acts and activity of melanin-concentrating hormone (MCH) neurons (MNs) in behaving male and female mice, we observed large MN activity spikes that aligned to unsupported rears. Complementary causal, loss and gain of function, analyses revealed specific control of rear frequency and duration by MNs and MCHR1 receptors. Activity in a key stress center of the brain-the locus ceruleus noradrenaline cells-rapidly inhibited MNs and required functional MCH receptors for its endogenous modulation of rearing. By defining a neural module that both tracks and controls rearing, these findings may facilitate further insights into biology of information seeking.

Genetically Encoded Lizard Color Divergence for Camouflage and Thermoregulation.

Local adaptation is critical in speciation and evolution, yet comprehensive studies on proximate and ultimate causes of local adaptation are generally scarce. Here, we integrated field ecological experiments, genome sequencing, and genetic verification to demonstrate both driving forces and molecular mechanisms governing local adaptation of body coloration in a lizard from the Qinghai-Tibet Plateau. We found dark lizards from the cold meadow population had lower spectrum reflectance but higher melanin contents than light counterparts from the warm dune population. Additionally, the colorations of both dark and light lizards facilitated the camouflage and thermoregulation in their respective microhabitat simultaneously. More importantly, by genome resequencing analysis, we detected a novel mutation in Tyrp1 that underpinned this color adaptation. The allele frequencies at the site of SNP 459# in the gene of Tyrp1 are 22.22% G/C and 77.78% C/C in dark lizards and 100% G/G in light lizards. Model-predicted structure and catalytic activity showed that this mutation increased structure flexibility and catalytic activity in enzyme TYRP1, and thereby facilitated the generation of eumelanin in dark lizards. The function of the mutation in Tyrp1 was further verified by more melanin contents and darker coloration detected in the zebrafish injected with the genotype of Tyrp1 from dark lizards. Therefore, our study demonstrates that a novel mutation of a major melanin-generating gene underpins skin color variation co-selected by camouflage and thermoregulation in a lizard. The resulting strong selection may reinforce adaptive genetic divergence and enable the persistence of adjacent populations with distinct body coloration.

The highest-elevation frog provides insights into mechanisms and evolution of defenses against high UV radiation.

Defense against ultraviolet (UV) radiation exposure is essential for survival, especially in high-elevation species. Although some specific genes involved in UV response have been reported, the full view of UV defense mechanisms remains largely unexplored. Herein, we used integrated approaches to analyze UV responses in the highest-elevation frog, Nanorana parkeri. We show less damage and more efficient antioxidant activity in skin of this frog than those of its lower-elevation relatives after UV exposure. We also reveal genes related to UV defense and a corresponding temporal expression pattern in N. parkeri. Genomic and metabolomic analysis along with large-scale transcriptomic profiling revealed a time-dependent coordinated defense mechanism in N. parkeri. We also identified several microRNAs that play important regulatory roles, especially in decreasing the expression levels of cell cycle genes. Moreover, multiple defense genes (i.e., TYR for melanogenesis) exhibit positive selection with function-enhancing substitutions. Thus, both expression shifts and gene mutations contribute to UV adaptation in N. parkeri. Our work demonstrates a genetic framework for evolution of UV defense in a natural environment.

Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis.

Melanins are highly conjugated biopolymer pigments that provide photoprotection in a wide array of organisms, from bacteria to humans. The rate-limiting step in melanin biosynthesis, which is the ortho-hydroxylation of the amino acid L-tyrosine to L-DOPA, is catalyzed by the ubiquitous enzyme tyrosinase (Ty). Ty contains a coupled binuclear copper active site that binds O2 to form a µ:η2:η2-peroxide dicopper(II) intermediate (oxy-Ty), capable of performing the regioselective monooxygenation of para-substituted monophenols to catechols. The mechanism of this critical monooxygenation reaction remains poorly understood despite extensive efforts. In this study, we have employed a combination of spectroscopic, kinetic, and computational methods to trap and characterize the elusive catalytic ternary intermediate (Ty/O2/monophenol) under single-turnover conditions and obtain molecular-level mechanistic insights into its monooxygenation reactivity. Our experimental results, coupled with quantum-mechanics/molecular-mechanics calculations, reveal that the monophenol substrate docks in the active-site pocket of oxy-Ty fully protonated, without coordination to a copper or cleavage of the µ:η2:η2-peroxide O-O bond. Formation of this ternary intermediate involves the displacement of active-site water molecules by the substrate and replacement of their H bonds to the µ:η2:η2-peroxide by a single H bond from the substrate hydroxyl group. This H-bonding interaction in the ternary intermediate enables the unprecedented monooxygenation mechanism, where the µ-η2:η2-peroxide O-O bond is cleaved to accept the phenolic proton, followed by substrate phenolate coordination to a copper site concomitant with its aromatic ortho-hydroxylation by the nonprotonated µ-oxo. This study provides insights into O2 activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.

Metal-Loaded Synthetic Melanin via Oxidative Polymerization of Neurotransmitter Norepinephrine Exhibiting High Photothermal Conversion.

Polydopamine (PDA)-derived melanin-like materials exhibit significant photothermal conversion owing to their broad-spectrum light absorption. However, their low near-infrared (NIR) absorption and inadequate hydrophilicity compromise their utilization of solar energy. Herein, we developed metal-loaded poly(norepinephrine) nanoparticles (PNE NPs) by predoping metal ions (Fe3+, Mn3+, Co2+, Ca2+, Ga3+, and Mg2+) with norepinephrine, a neuron-derived biomimetic molecule, to address the limitations of PDA. The chelation between catechol and metal ions induces a ligand-to-metal charge transfer (LMCT) through the formation of donor-acceptor pairs, modulating the light absorption behavior and reducing the band gap. Under 1 sun illumination, the Fe-loaded PNE coated wood evaporator achieved a high seawater evaporation rate and efficiency of 1.75 kg m-2 h-1 and 92.4%, respectively, owing to the superior hydrophilicity and photothermal performance of PNE. Therefore, this study offers a comprehensive exploration of the role of metal ions in enhancing the photothermal properties of synthetic melanins.

Fungal Melanin Rewires Macrophage Metabolism.

Aspergillus fumigatus is a deadly fungal pathogen in immunocompromised patients. A report by Gonçalves et al. reveals that melanin, a secondary metabolite present at the surface of infecting fungal spores, induces glycolysis in macrophages to promote inflammatory responses. This opens a window for the development of innovative host-directed antifungal therapies.

Human Brainstem and Cerebellum Atlas: Chemoarchitecture and Cytoarchitecture Paired to MRI.

Lesion localization is the basis for understanding neurologic disease, which is predicated on neuroanatomical knowledge carefully cataloged from histology and imaging atlases. However, it is often difficult to correlate clinical images of brainstem injury obtained by MRI scans with the details of human brainstem neuroanatomy represented in atlases, which are mostly based on cytoarchitecture using Nissl stain or a single histochemical stain, and usually do not include the cerebellum. Here, we report a high-resolution (200 µm) 7T MRI of a cadaveric male human brainstem and cerebellum paired with detailed, coregistered histology (at 2 µm single-cell resolution) of the immunohistochemically stained cholinergic, serotonergic, and catecholaminergic (dopaminergic, noradrenergic, and adrenergic) neurons, in relationship to each other and to the cerebellum. These immunohistochemical findings provide novel insights into the spatial relationships of brainstem cell types and nuclei, including subpopulations of melanin and TH+ neurons, and allows for more informed structural annotation of cell groups. Moreover, the coregistered MRI-paired histology helps validate imaging findings. This is useful for interpreting both scans and histology, and to understand the cell types affected by lesions. Our detailed chemoarchitecture and cytoarchitecture with corresponding high-resolution MRI builds on previous atlases of the human brainstem and cerebellum, and makes precise identification of brainstem and cerebellar cell groups involved in clinical lesions accessible for both laboratory scientists and clinicians alike.SIGNIFICANCE STATEMENT Clinicians and neuroscientists frequently use cross-sectional anatomy of the human brainstem from MRI scans for both clinical and laboratory investigations, but they must rely on brain atlases to neuroanatomical structures. Such atlases generally lack both detail of brainstem chemical cell types, and the cerebellum, which provides an important spatial reference. Our current atlas maps the distribution of key brainstem cell types (cholinergic, serotonergic, and catecholaminergic neurons) in relationship to each other and the cerebellum, and pairs this histology with 7T MR images from the identical brain. This atlas allows correlation of the chemoarchitecture with corresponding MRI, and makes the identification of cell groups that are often discussed, but rarely identifiable on MRI scan, accessible to clinicians and clinical researchers.

Melanin granules morphology and distribution in human black hair investigated by focused ion beam scanning electron microscopy: Differences between Asian and Caucasian hair.

Melanin granules (melanosomes) in Asian and Caucasian black hairs were investigated by focused ion beam scanning electron microscopy (FIB-SEM). This technique facilitates a direct evaluation of the three-dimensional distribution and morphology of melanin granules without requiring their isolation from hair. Three-dimensional reconstructed images of melanin granule distribution in hair samples were obtained using serial SEM images observed by FIB-SEM. Melanin granules in black hair tended to be three-dimensionally dense in the outer periphery of the cortex. The morphometric parameters of melanin granules were calculated using the reconstructed three-dimensional images. The results confirmed that melanin granules in Caucasian black hair were much smaller those in Asian black hair. Moreover, it was indicated that the relative frequency distribution of the volume of melanin granules was significantly different between Asians and Caucasians.

Inhibitory actions of melanin-concentrating hormone in the lateral septum.

Melanin-concentrating hormone (MCH) neurons can co-express several neuropeptides or neurotransmitters and send widespread projections throughout the brain. Notably, there is a dense cluster of nerve terminals from MCH neurons in the lateral septum (LS) that innervate LS cells by glutamate release. The LS is also a key region integrating stress- and anxiety-like behaviours, which are also emerging roles of MCH neurons. However, it is not known if or where the MCH peptide acts within the LS. We analysed the projections from MCH neurons in male and female mice anteroposteriorly throughout the LS and found spatial overlap between the distribution pattern of MCH-immunoreactive (MCH-ir) fibres with MCH receptor Mchr1 mRNA hybridization or MCHR1-ir cells. This overlap was most prominent along the ventral and lateral border of the rostral part of the LS (LSr). Most MCHR1-labelled LS neurons lay adjacent to passing MCH-ir fibres, but some MCH-ir varicosities directly contacted the soma or cilium of MCHR1-labelled LS neurons. We thus performed whole-cell patch-clamp recordings from MCHR1-rich LSr regions to determine if and how LS cells respond to MCH. Bath application of MCH to acute brain slices activated a bicuculline-sensitive chloride current that directly hyperpolarized LS cells. This MCH-mediated hyperpolarization was blocked by calphostin C, which suggested that the inhibitory actions of MCH were mediated by protein kinase C-dependent activation of GABAA receptors. Taken together, these findings define potential hotspots within the LS that may elucidate the contributions of MCH to stress- or anxiety-related feeding behaviours. KEY POINTS: Melanin-concentrating hormone (MCH) neurons have dense nerve terminals within the lateral septum (LS), a key region underlying stress- and anxiety-like behaviours that are emerging roles of the MCH system, but the function of MCH in the LS is not known. We found spatial overlap between MCH-immunoreactive fibres, Mchr1 mRNA, and MCHR1 protein expression along the lateral border of the LS. Within MCHR1-rich regions, MCH directly inhibited LS cells by increasing chloride conductance via GABAA receptor activation in a protein kinase C-dependent manner. Electrophysiological MCH effects in brain slices have been elusive, and few studies have described the mechanisms of MCH action. Our findings demonstrated, to our knowledge, the first description of MCHR1 Gq-coupling in brain slices, which was previously predicted in cell or primary culture models only. Together, these findings defined hotspots and mechanistic underpinnings for MCH effects such as in feeding and anxiety-related behaviours.

A role for microsomal glutathione transferase 1 in melanin biosynthesis and melanoma progression.

Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 (MGST1) in biosynthetic pathways for melanin and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative, and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties, and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to nontarget control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.

Fast neurotransmitter identity of MCH neurons: Do contents depend on context?

Hypothalamic melanin-concentrating hormone (MCH) neurons participate in many fundamental neuroendocrine processes. While some of their effects can be attributed to MCH itself, others appear to depend on co-released neurotransmitters. Historically, the subject of fast neurotransmitter co-release from MCH neurons has been contentious, with data to support MCH neurons releasing GABA, glutamate, both, and neither. Rather than assuming a position in that debate, this review considers the evidence for all sides and presents an alternative explanation: neurochemical identity, including classical neurotransmitter content, is subject to change. With an emphasis on the variability of experimental details, we posit that MCH neurons may release GABA and/or glutamate at different points according to environmental and contextual factors. Through the lens of the MCH system, we offer evidence that the field of neuroendocrinology would benefit from a more nuanced and dynamic interpretation of neurotransmitter identity.

The appressorium at a glance.

Many plant pathogenic fungi have the capacity to infect their plant hosts using specialised cells called appressoria. These structures act as a gateway between the fungus and host, allowing entry to internal tissues. Appressoria apply enormous physical force to rupture the plant surface, or use a battery of enzymes to digest the cuticle and plant cell wall. Appressoria also facilitate focal secretion of effectors at the point of plant infection to suppress plant immunity. These infection cells develop in response to the physical characteristics of the leaf surface, starvation stress and signals from the plant. Appressorium morphogenesis has been linked to septin-mediated reorganisation of F-actin and microtubule networks of the cytoskeleton, and remodelling of the fungal cell wall. In this Cell Science at a Glance and accompanying poster, we highlight recent advances in our understanding of the mechanisms of appressorium-mediated infection, and compare development on the leaf surface to the biology of invasive growth by pathogenic fungi. Finally, we outline key gaps in our current knowledge of appressorium cell biology.

Melanin-concentrating hormone receptor 1 is discarded by exosomes after internalization.

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1), a G protein-coupled receptor, is poised for interaction with its ligands on the plasma membrane. Analyses of MCHR1 knockout mice suggest that this receptor could be a therapeutic target for the treatment of appetite disorders, glucose metabolism, psychiatric disorders, and inflammation. Binding of MCH to MCHR1 initiates calcium signaling, which is subsequently attenuated through receptor internalization. However, the ultimate destiny of the receptor post-internalization remains unexplored. In this study, we report the extracellular secretion of MCHR1 via exosomes. The recruitment of MCHR1 to exosomes occurs subsequent to its internalization, which is induced by stimulation with the ligand MCH. Although a highly glycosylated form of MCHR1, potentially representing a mature form, is selectively recruited to exosomes, the MCHR1 transferred into other cells does not exhibit functionality. The truncation of MCHR1 at the C-terminus not only impairs its response to MCH but also hinders its recruitment to exosomes. These findings imply that functional MCHR1 could be secreted extracellularly via exosomes, a process that may represent a mechanism for the termination of intracellular MCHR1 signaling.

Image-Guided Photothermal and Immune Therapy of Tumors via Melanin-Producing Genetically Engineered Bacteria.

Photothermal therapy (PTT) is a new treatment modality for tumors. However, the efficient delivery of photothermal agents into tumors remains difficult, especially in hypoxic tumor regions. In this study, an approach to deliver melanin, a natural photothermal agent, into tumors using genetically engineered bacteria for image-guided photothermal and immune therapy is developed. An Escherichia coli MG1655 is transformed with a recombinant plasmid harboring a tyrosinase gene to produce melanin nanoparticles. Melanin-producing genetically engineered bacteria (MG1655-M) are systemically administered to 4T1 tumor-bearing mice. The tumor-targeting properties of MG1655-M in the hypoxic environment integrate the properties of hypoxia targeting, photoacoustic imaging, and photothermal therapeutic agents in an "all-in-one" manner. This eliminates the need for post-modification to achieve image-guided hypoxia-targeted cancer photothermal therapy. Tumor growth is significantly suppressed by irradiating the tumor with an 808 nm laser. Furthermore, strong antitumor immunity is triggered by PTT, thereby producing long-term immune memory effects that effectively inhibit tumor metastasis and recurrence. This work proposes a new photothermal and immune therapy guided by an "all-in-one" melanin-producing genetically engineered bacteria, which can offer broad potential applications in cancer treatment.

Dual-Polymer Functionalized Melanin-AgNPs Nanocomposite with Hydroxyapatite Binding Ability to Penetrate and Retain in Biofilm Sequentially Treating Periodontitis.

Periodontitis is the leading cause of adult tooth missing. Thorny bacterial biofilm and high reactive oxygen species (ROS) levels in tissue are key elements for the periodontitis process. It is meaningful to develop an advanced therapeutic system with sequential antibacterial/ antioxidant ability to meet the overall goals of periodontitis therapy. Herein, a dual-polymer functionalized melanin-AgNPs (P/D-MNP-Ag) with biofilm penetration, hydroxyapatite binding, and sequentially treatment ability are fabricated. Polymer enriched with 2-(Dimethylamino)ethyl methacrylate (D), can be protonated in an acid environment with enhanced positive charge, promoting penetration in biofilm. The other polymer is rich in phosphate group (P) and can chelate Ca2+, promoting the polymer to adhere to the hydroxyapatite surface. Melanin has good ROS scavenging and photothermal abilities, after in situ reduction Ag, melanin-AgNPs composite has sequentially transitioned between antibacterial and antioxidative ability due to heat and acid accelerated Ag+ release. The released Ag+ and heat have synergistic antibacterial effects for bacterial killing. With Ag+ consumption, the antioxidant ability of MNP recovers to scavenge ROS in the inflammatory area. When applied in the periodontitis model, P/D-MNP-Ag has good therapeutical effects to ablate biofilm, relieve inflammation state, and reduce alveolar bone loss. P/D-MNP-Ag with sequential treatment ability provides a reference for developing advanced oral biofilm eradication systems.

The genomic architecture of continuous plumage colour variation in the European barn owl (Tyto alba).

The maintenance of colour variation in wild populations has long fascinated evolutionary biologists, although most studies have focused on discrete traits exhibiting rather simple inheritance patterns and genetic architectures. However, the study of continuous colour traits and their potentially oligo- or polygenic genetic bases remains rare in wild populations. We studied the genetics of the continuously varying white-to-rufous plumage coloration of the European barn owl (Tyto alba) using a genome-wide association approach on the whole-genome data of 75 individuals. We confirmed a mutation at the melanocortin-1-receptor gene (MC1R) is involved in the coloration and identified two new regions, located in super-scaffolds 9 and 42. The combination of the three regions explains most of the colour variation (80.37%, 95% credible interval 58.45-100%). One discovered region, located in the sex chromosome, differs between the most extreme colorations in owls sharing a specific MC1R genotype. This region may play a role in the colour sex dimorphism of this species, possibly in interaction with the autosomal MC1R. We thus provide insights into the genetic architecture of continuous colour variation, pointing to an oligogenic basis with potential epistatic effects among loci that should aid future studies understanding how continuous colour variation is maintained in nature.

Role of Pmk1, Mpk1, or Hog1 in the mitogen-activated protein kinase pathway of Aspergillus cristatus.

Aspergillus cristatus is a probiotic fungus known for its safety and abundant secondary metabolites, making it a promising candidate for various applications. However, limited progress has been made in researching A. cristatus due to challenges in genetic manipulation. The mitogen-activated protein kinase (MAPK) signaling pathway is involved in numerous physiological processes, but its specific role in A. cristatus remains unclear. In this study, we successfully developed an efficient polyethylene glycol (PEG)-mediated protoplast transformation method for A. cristatus, enabling us to investigate the function of Pmk1, Mpk1, and Hog1 in the MAPK signaling pathway. Our findings revealed that Pmk1, Mpk1, and Hog1 are crucial for sexual reproduction, melanin synthesis, and response to external stress in A. cristatus. Notably, the deletion of Pmk1, Mpk1, or Hog1 resulted in the loss of sexual reproduction capability in A. cristatus. Overall, this research on MAPK will contribute to the continued understanding of the reproductive strategy and melanin synthesis mechanism of A. cristatus.