Gastropod diversification represents one of the most spectacular evolutionary radiations, underpinned by a fundamentally asymmetric body plan and diverse pigmentation patterns. To understand the genetic underpinnings of these cardinal traits, we constructed a chromosome-level genome assembly for the rainbow abalone, Haliotis iris . Integrating comparative genomics, histoembryology, and molecular assays, we identify a conserved regulatory association between the long non-coding RNA lncRNA1 and pitx that is associated with asymmetric mantle development. In H. iris , this association is supported by chromatin-contact evidence and exploratory data suggesting a possible miRNA-associated post-transcriptional component. Furthermore, we identify a wnt-mitf-tyr framework for melanogenesis and show that the mantle is the primary site of melanin synthesis. Concurrently, we identify mantle-enriched prestin genes that are vibration-responsive, consistent with a possible sensory specialization of the mantle. These findings provide molecular insight into asymmetry, pigmentation, and mantle multifunctionality in gastropods.

In benthic aquaculture, the use of substrates enhances farming efficiency and also induces microbial biofilm formation. The presence of biofilms is closely associated with the aquacultural environment and potentially influences the growth and development of aquatic animals. Strongylocentrotus intermedius is a highly valued species in aquaculture due to its commercially significant gonads. To date, however, few studies have investigated the associations between gut microbial community structures and functions in S. intermedius and substrate surface biofilms. In this study, we examined dynamic changes in the microbial communities on substrate biofilms in S. intermedius culture and assessed their potential effects on sea urchin health. Scanning electron microscopy, contact angle measurement, and quantitative analyses of biofilms revealed that biofilms on the surface of polyvinyl chloride (PVC) substrates gradually increased over time. Fourier-transform infrared spectrometry analysis indicated that PVC substrates gradually age with prolonged cultivation time. Microbial community analysis revealed a succession of dominant bacterial genera in PVC substrate biofilms, particularly those in genera with plastic-degradation ability, including Pseudomonas, Cladosporium, and Alternaria, which may promote PVC aging. Comparative microbial analyses revealed that Vibrio was highly abundant in S. intermedius grown in indoor and offshore culture environments, Cobetia and Debaryomyces were highly abundant in the intestine of offshore-cultured S. intermedius, and Pseudoalteromonas, Candidatus_Hepatoplasma, Cladosporium, and Aspergillus were highly abundant in the guts of indoor-cultured S. intermedius, among which only Candidatus_Hepatoplasma was not detected in PVC substrates. These differences are assumed to reflect the ecological interactions and nutritional requirements of S. intermedius in different habitats, as well as their adaptability to environmental change. Our research demonstrates that the relationship between the biofilms on sea urchin attachment bases and their gut microbiota must be better understood to optimise aquaculture. In particular, the role of beneficial microbes, notably fungi, within these attachment bases requires further investigation. Creating a beneficial bacterial biofilm can optimise the gut environment, improve the growth rate, and promote the overall health of sea urchins.

Asymmetric development, in which functional differences occur between left-right symmetrical organs, is widespread in organisms, including fish and mollusks. However, the asymmetry of symmetrical sensory structures in Haliotis discus hannai, a gastropod with a sensitive sensory system, remains unknown. This study analyzed the transcriptomes of three sensory structures (eyestalks, cephalic tentacles, and epipodial tentacles) to explore potential asymmetries in this species. RNA-seq revealed functional differences in sensory ability and sperm-egg recognition between right and left eyestalks, with cephalic tentacles displaying asymmetry in cytoskeletal organization and cell cycle regulation. Epipodial tentacles showed similar asymmetries, including immune response differences. Moreover, the cAMP-protein kinase A (PKA)-CREB-binding protein (CBP) signaling pathway responded asymmetrically, with PKA responding to activators and inhibitors on both sides and CBP showing a stronger response on the right. These findings provide insights into sensory asymmetry in mollusks and guidance for further investigations of the molecular mechanisms underlying asymmetry in symmetrical organs.

Microplastics (MPs) aging in natural ecosystems are caused by solar irradiation. Photo-aged MPs in aquatic systems are a major threat to molluscs. In this study, polystyrene (PS) photo-aging was simulated using a sunlight simulator. After exposure of Crassostrea gigas to photo-aged PS, a decreased gonadosomatic index, coupled with histological alterations, suggested an inhibitory effect on the gonadal development of bivalves. As the concentration of aged PS increased, the inhibitory effects on gonadal development became more severe. The sex hormone (testosterone and estradiol) and energy metabolism (glycogen, lipid, and protein content) differences between C. gigas males and females suggested a disruption of sex hormonal homeostasis and a shift in energy allocation strategy, which may have affected reproduction, especially female oysters. In addition, the substantial downregulation of SOX-8, SOX-E, Piwi1, and TGF-β genes may be contributing factors causing the inhibitory effect of aged PS on the gonadal development of C. gigas. This study provides an essential reference for evaluating the reproductive health risks posed by aged MPs and offers novel insights and perspectives for exploring the impact of MPs under natural conditions.

Ambient ultraviolet radiation (UVB) from solar and artificial light presents serious environmental risks to aquatic ecosystems. The Pacific oyster, Crassostrea gigas, perceives changes in the external environment primarily through its mantle tissue, which contains many nerve fibers and tentacles. Changes within the mantles can typically illustrate the injury of ambient UVB. In this study, a comprehensive analysis of phenotypic, behavioral, and physiological changes demonstrated that extreme UVB radiation (10 W/m²) directly suppressed the behavioral activities of C. gigas. Conversely, under ambient UVB radiation (5 W/m²), various physiological processes exhibited significant alterations in C. gigas, despite the behavior remaining relatively unaffected. Using mathematical model analysis, the integrated analysis of the full-length transcriptome, proteome, and metabolome showed that ambient UVB significantly affected the metabolic processes (saccharide, lipid, and protein metabolism) and cellular biology processes (autophagy, apoptosis, oxidative stress) of the C. gigas mantle. Subsequently, using Procrustes analysis and Pearson correlation analysis, the association between multi-omics data and physiological changes, as well as their biomarkers, revealed the effect of UVB on three crucial biological processes: activation of autophagy signaling (key factors: Ca2+, LC3B, BECN1, caspase-7), response to oxidative stress (reactive oxygen species, heat shock 70, cytochrome c oxidase), and recalibration of energy metabolism (saccharide, succinic acid, translation initiation factor IF-2). These findings offer a fresh perspective on the integration of multi-data from non-model animals in ambient UVB risk assessment.

As economically significant species, bivalves maintain intimate contact with external microorganisms during respiration and filter feeding, rendering them prime candidates for investigating microbial communities. Although numerous studies have been conducted on the microbial communities inhabiting the mantles, the composition and functions of the microbial community residing within the coloured mantles remain unclear. The pigments in the coloured areas may attract some microorganisms related to pigments. Thus, in vitro culture can be utilized to rapidly assess the fundamental characteristics of the microbial community present in the coloured mantles. Our results indicated that the microbial community in the coloured mantles of three bivalves (Crassostrea gigas, Mytilus edulis, and Chlamys farreri) responded differently to various media, resulting in pigmentation occurring on tyrosine medium. Further analyses by Metabarcoding revealed a relatively low abundance of melanin-producing strains on the surface of coloured mantles, which may be related to the requirement of melanin-producing strains for a specific nutritional environment. Then, we observed that there were significant differences in the composition of the microbial communities in coloured mantles of the three bivalves, with different bivalves possessing distinct biomarkers. These microorganisms affect the host by regulating the immune system and aiding digestion, nutrient absorption, and nitrification. Correlation analysis showed that multiple metabolites of the three bivalves were strongly correlated with bacterial biomarkers and may play an important role in maintaining the dynamic balance of surface microbial communities. In conclusion, this study discussed the multifaceted effects of microorganisms on the dynamic equilibrium of bivalves microbial communities, exploring the intricate connection between the host and the microbe, as well as the composition of microbial communities. This lays the foundation for comprehending the intricate symbiotic relationship between bivalves and their microbial counterparts and offers a theoretical foundation for the future development of microbial preparations intended for bivalves body surfaces.

As human activities increase and environmental changes persist, increased ultraviolet B (UVB) radiation in aquatic ecosystems poses significant threats to aquatic life. This study, through transcriptomic analysis of the mantle tissue of Crassostrea gigas following UVB radiation exposure, identified and validated two key transcription factors, CgRunx1 and CgCBFβ. The highest expression levels of CgRunx1 and CgCBFβ in the mantle suggest their pivotal roles in this tissue. Co-immunoprecipitation experiments revealed that CgRunx1 and CgCBFβ could form heterodimers and interact with each other. Furthermore, this study assessed the impact of UVB radiation on the levels of reactive oxygen species of the C. gigas, speculating that CgRunx1, as a potential redox-sensitive transcription factor, might be regulated by intracellular ROS. Through screening and binding site prediction analysis of target genes, coupled with dual-luciferase reporter assays, we verified that CgRunx1 might participate in regulating the biomineralization and autophagy processes in C. gigas by activating the transcriptional expression of target genes Transport and Golgi organization 1 and V-type proton ATPase catalytic subunit A. These findings provide new insights into the molecular response mechanisms of the C. gigas to UVB radiation and lay an important foundation for studying the adaptive evolution of bivalves to environmental stress.

Sexual dimorphism in immune responses is an essential factor in environmental adaptation. However, the mechanisms involved remain obscure owing to the scarcity of data from sex-role-reversed species in stressed conditions. Benzo[a]pyrene (BaP) is one of the most pervasive and carcinogenic organic pollutants in coastal environments. In this study, we evaluated the potential effects on renal immunotoxicity of the sex-role-reversed lined seahorse (Hippocampus erectus) toward environmental concentrations BaP exposure. Our results discovered the presence of different energy-immunity trade-off strategies adopted by female and male seahorses during BaP exposure. BaP induced more severe renal damage in female seahorses in a concentration-dependent manner. BaP biotransformation and detoxification in seahorses resemble those in mammals. Benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BPDE) and 9-hydroxybenzo[a]pyrene (9-OH-BaP) formed DNA adducts and disrupted Ca2+ homeostasis may together attribute the renal immunotoxicity. Sexual dimorphisms in detoxification of both BPDE and 9-OH-BaP, and in regulation of Ca2+, autophagy and inflammation, mainly determined the extent of renal damage. Moreover, the mechanism of sex hormones regulated sexual dimorphism in immune responses needs to be further elucidated. Collectively, these findings contribute to the understanding of sexual dimorphism in the immunotoxicity induced by BaP exposure in seahorses, which may attribute to the dramatic decline in the biodiversity of the genus.

Massive harmful algal blooms (HABs) have increased the risk of marine organisms encountering the dinoflagellate toxin, okadaic acid (OA). Strongylocentrotus intermedius, a globally significant benthic aquaculture species, has a large appetite for benthic algae. During red tide events, there is a high risk of red tide toxin accumulation. This study systematically evaluated the potential impact of short-term OA exposure on the behavior and physiological functions of juvenile S. intermedius. From typical (5 mu g/L) to extreme OA concentrations (20 mu g/L) during HAB outbreaks, OA exposure gradually inhibited a series of tube foot-related behaviors (sheltering, foraging, righting, and tube-foot tenacity). At OA concentrations during HAB outbreaks (5 mu g/L), the tube foot function of S. intermedius was progressively inhibited. Further physiological indicator analyses revealed that the activity of antioxidants increased over a short period to prevent damage from reactive oxygen species induced by OA. However, OA ultimately suppressed the immune response of S. intermedius, leading to apoptosis. Although HAB-associated concentrations of OA (5 mu g/L) did not induce a continuous increase in the integrated biological response index of S. intermedius, this study speculated that HABs pose a future risk to echinoderm species. Notably, principal component analysis results showed that OA exposure eventually induced significant changes in the production of O2_ , malondialdehyde, and total glutathione, as well as in glutathione S-transferase activity and caspase-7, -8, and -9 levels. This study provides preliminary evidence of OA's toxic effects on sea urchins and essential data for urgent risk assessments of algal toxin pollution in aquaculture during HABs.

Inhibitor of κB kinase (IKK) family proteins are key signaling molecules in the animal innate immune system and are considered master regulators of inflammation and innate immunity that act by controlling the activation of transcription factors such as NF-κB. However, few functional studies on IKK in invertebrates have been conducted, especially in marine mollusks. In this study, we cloned the IKK gene in the Zhikong scallop Chlamys farreri and named it CfIKK3. CfIKK3 encodes a 773-amino acid-long protein, and phylogenetic analysis showed that CfIKK3 belongs to the invertebrate TBK1/IKKϵ protein family. Quantitative real-time PCR analysis showed that CfIKK3 mRNA is ubiquitously expressed in all tested scallop tissues. The expression of CfIKK3 transcripts was significantly induced after challenge with lipopolysaccharide, peptidoglycan, or poly(I:C). Co-immunoprecipitation (co-IP) assays confirmed the direct interaction of CfIKK3 with MyD88 (the key adaptor in the TLR pathway) and MAVS (the key adaptor in the RLR pathway), suggesting that this IKK protein plays a crucial role in scallop innate immune signal transduction. In addition, the CfIKK3 protein formed homodimers and bound to CfIKK2, which may be a key step in the activation of its own and downstream transcription factors. Finally, in HEK293T cells, dual-luciferase reporter gene experiments showed that overexpression of CfIKK3 protein activated the NF-κB reporter gene in a dose-dependent manner. In conclusion, our experimental results confirmed that CfIKK3 could respond to PAMPs challenge and participate in scallop TLR and RLR pathway signaling, ultimately activating NF-κB. Therefore, as a key signaling molecule and modulator of immune activity, CfIKK3 plays an important role in the innate immune system of scallops.

Gonadotropin-releasing hormone (GnRH) controls synthesis of sex steroid hormones through hypothalamic-pituitary-gonadal (HPG) axis in vertebrates. But in mollusks, research on neuroendocrine control of gonadal function, such as the function of GnRH during gonadal development is limited. In this study, we investigated the morphology and structure of the nerve ganglia of Zhikong scallop Chlamys farreri by physiological and histological observations. We also cloned the ORF and studied the expression patterns of GnRH in the scallop. Tissue expression analysis showed that GnRH was highly expressed in parietovisceral ganglion (PVG). The in situ hybridization result further confirmed that GnRH mRNA only distributed in some good-sized neurons in the posterior lobe (PL) and some pint-sized neurons in the lateral lobe (LL). In addition, by examining the expression of GnRH during gonadal development in ganglia, we found GnRH displayed higher expression in the female scallops, and showed significant high expression at the growing stage of female scallops in PVG. This study would contribute to gaining insight into the mechanism underlying reproduction regulation by GnRH in the scallop and help to provide a better understanding of reproductive neuroendocrine in mollusks.

Molluscs constitute the second largest phylum of animals in the world, and shell colour is one of their most important phenotypic characteristics. In this study, we found among three folds on the mantle edge of oyster, only the outer fold had the same colour as the shell. Transcriptome and mantle cutting experiment indicated that the outer fold may be mainly reflected in chitin framework formation and biomineralisation. There were obvious differences in SEM structure and protein composition between the black and white shell periostraca. The black shell periostraca had more proteins related to melanin biosynthesis and chitin binding. Additionally, we identified an uncharacterized protein gene (named as CgCBP) ultra-highly expressed only in the black outer fold and confirmed its function of chitin-binding and CaCO3 precipitation promoting. RNAi also indicated that CgCBP knockdown could change the structure of shell periostracum and reduce shell pigmentation. All these results suggest that the mantle outer fold plays multiple key roles in shell periostraca bioprocessing, and shell periostracum structure affected by chitin-binding protein is functionally correlated with shell pigmentation. The investigation of oyster shell periostracum structure and shell colour will provide a better understanding in pigmentation during biological mineralisation in molluscs.

The increasing and intensifying ultraviolet B (UVB) radiation in sunlight is an environmental threat to aquatic ecosystems, potentially affecting the entire life cycle of wild or aquacultural Pacific oyster Crassostrea gigas with photoreception. Due to its complex composition, plasma is an important biological specimen for investigating the degree of disturbance from its steady state caused by the external environment in the open-pipe-type hemolymph of mollusks. We performed a multi-omic analysis of C. gigas plasma exposed to daylight UVB radiation. Hub differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified using the functional classification of Clusters of Orthologous Groups of proteins (COGs) through the protein-protein interaction (PPI)-based maximal clique centrality (MCC) algorithm. Our results summarize three types of UVB influences (disruption of the cell membrane, promotion of nucleotide metabolism, and inhibition of energy metabolism) on C. gigas based on transcriptomic, proteomic, and metabolomic analyses. The associated hub DEGs, DEPs (e.g., nucleoside diphosphate kinase, malate dehydrogenase, and hydroxyacyl-coenzyme A dehydrogenase), and metabolites (e.g., uridine, adenine, deoxyguanosine, guanosine, and xylitol) in the plasma were identified as biomarkers of mollusk response to UVB radiation, and could be used to evaluate the influence of environmental UVB on mollusks in future studies.

Nonylphenol (NP) is an endocrine disruptor and environmental hormone representing alkylphenol compounds. Marine mollusks are an important source of protein for people worldwide. Many researchers have begun to study the effect of NP on marine mollusks immune system in view of its toxicity; however, the underlying molecular mechanisms require in-depth analysis. In this study, we focused on the transcriptional expression change of immune-related genes and antioxidant enzymes activities variation after NP exposure in a marine bivalve mollusk, Chlamys farreri, to explore the immunomodulatory capacity of NP in marine mollusks. We identified MAVS (Mitochondrial antiviral signaling protein), a key adaptor molecule in the RLR (RIG-I like receptor) pathway, and studied the expression of multiple immune-related genes in response to different concentrations of NP. The key genes involved in RLR/TLR (Toll like receptor) innate immune pathway, apoptosis, and cellular antioxidation mechanism were investigated. Changes in the enzymatic activities of scallop antioxidant enzymes after NP exposure were also examined. The results revealed that the genes expression and the antioxidant enzymes activities show significant changes, thus proving that NP stimulation affects the scallop immune system. Our research results demonstrate the immunomodulatory capacity of NP in marine bivalve mollusks and lay the foundation for further in-depth analysis of the molecular mechanism of NP toxicity.

Retinoic acid (RA) plays important roles in various biological processes in animals. RA signaling is mediated by two types of nuclear receptors, namely retinoic acid receptor (RAR) and retinoid x receptor (RXR), which regulate gene expression by binding to retinoic acid response elements (RAREs) in the promoters of target genes. Here, we explored the effect of all-trans retinoic acid (ATRA) on the Pacific oyster Crassostera gigas at the transcriptome level. A total of 586 differentially expressed genes (DEGs) were identified in C. gigas upon ATRA treatment, with 309 upregulated and 277 downregulated genes. Bioinformatic analysis revealed that ATRA affects the development, metabolism, reproduction, and immunity of C. gigas. Four tyrosinase genes, including Tyr-6 (LOC105331209), Tyr-9 (LOC105346503), Tyr-20 (LOC105330910), and Tyr-12 (LOC105320007), were upregulated by ATRA according to the transcriptome data and these results were verified by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. In addition, increased expression of Tyr (a melanin-related TYR gene in C. gigas) and Tyr-2 were detected after ATRA treatment. The yeast one-hybrid assay revealed the DNA-binding activity of the RA receptors CgRAR and CgRXR, and the interaction of CgRAR with RARE present in the Tyr-2 promoter. These results provide evidence for the further studies on the role of ATRA and the mechanism of RA receptors in mollusks.

The Pacific oyster, Crassostrea gigas, is widely distributed and is substantially important to shellfish cultivation. Oysters with melanin-colored shells and soft parts are increasingly popular and are of high economic value. However, the distribution and synthesis of melanin in C. gigas remains unclear and there have been only few relevant studies on it. In this study, transmission electron microscopy (TEM) was used to observe melanin in the epidermis of dark mantle tissue. Melanocytes containing melanosomes were observed below the epidermis, suggesting the possibility of a gradual transfer of melanin from the mantle tissue to the epithelium. The frozen section technique and three melanin staining methods were used to confirm the presence of melanin. The results suggest that enzyme identification (DOPA) is a suitable method of melanin staining in the mantle tissues of C. gigas. This study preliminarily identified the existence of melanin in the mantle tissues of C. gigas and is a foundation for the study of melanin synthesis. Furthermore, it provides new insights into the mechanism of shell color formation.

Molluscs constitute the second largest phylum of animals in the world, and shell colour and stripes are one of their most important phenotypic characteristics. Studies on the mechanism of shell pigmentation help understand the evolutionary and ecological significance of shell colour and serve as the basis for shell colour breeding. In this study, a matched-pair design was used in comparing the black- and white-striped mantles of the same oyster. The result showed that the stripes of shell surface are corresponding to the stripes of the mantle edge. Transcriptomic analysis identified an uncharacterized protein gene (we named it as Crassostrea gigas chitin-binding protein, CgCBP) highly expressed in the black mantles. Three folds (inner fold, middle fold and outer fold) were found on the mantle edge of oyster, but only the outer fold has the same colour as the shell. Transcriptomic comparison indicated that the three folds of the mantle edge are functionally differentiated, and the CgCBP gene is ultra-highly expressed only in the outer fold. We obtained new black and white shell periostraca from the shell notching experiment and found their structural differences by scanning electron microscope. Chitin was successfully extracted and identified from the shell periostraca. Proteomic analysis revealed differences in protein composition between the two shell periostraca. Particularly, the black shell periostraca have more proteins related to melanin biosynthesis and chitin binding compared with the white ones, and melanin particles were observed in the black mantle edge using transmission electron microscope. Magnetic bead binding and Western blot experiments showed that the CgCBP protein can specifically bind to chitin and in vivo RNAi screening indicated that CgCBP knockdown can change the structure of the shell periostracum and reduce its pigmentation. All these results suggest that the outer fold of mantle may have more important roles in shell pigmentation, shell periostracum structure is functionally correlated with shell pigmentation, and the CgCBP gene ultra-highly expressed in the outer fold may influence shell pigmentation by affecting its periostracum structure.

The interferon regulatory factor (IRF) family, a class of transcription factors with key functions, are important in host innate immune defense and stress response. However, further research is required to determine the functions of IRFs in invertebrates. In this study, the coding sequence of an IRF gene was obtained from the Zhikong scallop (Chlamys farreri) and named CfIRF8-like. The open reading frame of CfIRF8-like was 1371 bp long and encoded 456 amino acids. Protein domain prediction revealed a typical IRF domain in the N-terminus of the CfIRF8-like protein and a typical IRF3 domain in the C-terminus. Multiple sequence alignment confirmed the conservation of the amino acid sequences of these two functional protein domains. Phylogenetic analysis showed that CfIRF8-like clustered with mollusk IRF8 proteins and then clustered with vertebrate IRF3, IRF4, and IRF5 subfamily proteins. Quantitative real-time PCR detected CfIRF8-like mRNA in all tested scallop tissues, with the highest expression in the gills. Simultaneously, the expression of CfIRF8-like transcripts in gills was significantly induced by polyinosinic-polycytidylic acid challenge. The results of protein interaction experiments showed that CfIRF8-like could directly bind the TBK1/IKKε family protein of scallop (CfIKK2) via its N-terminal IRF domain, revealing the presence of an ancient functional TBK1/IKKε-IRF signaling axis in scallops. Finally, dual-luciferase reporter assay results showed that the overexpression of CfIRF8-like in human embryonic kidney 293T cells could specifically activate the interferon β promoter of mammals and the interferon-stimulated response element promoter in dose-dependent manners. The findings of this preliminary analysis of the signal transduction and immune functions of scallop CfIRF8-like protein lay a foundation for an in-depth understanding of the innate immune function of invertebrate IRFs and the development of comparative immunology. The experimental results also provide theoretical support for the breeding of scallop disease-resistant strains.

IKK proteins are key signaling molecules in the innate immune system of animals, and act downstream of pattern recognition receptors. However, research on IKKs in invertebrates, especially marine mollusks, remains scarce. In this study, we cloned CfIKK1 gene from the Zhikong scallop (Chlamys farreri) and studied its function and the signaling it mediates. The open reading frame of CfIKK1 was 2190 bp and encoded 729 amino acids. Phylogenetic analysis showed that CfIKK1 belonged to the invertebrate IKKα/IKKβ family. Quantitative real-time PCR analysis revealed the ubiquitous expression of CfIKK1 mRNA in all scallop tissues and challenge with lipopolysaccharide, peptidoglycan, or poly(I:C) significantly upregulated the expression of CfIKK1. Co-immunoprecipitation assays confirmed the interaction of CfIKK1 with scallop MyD88 (Myeloid differentiation actor 88, the key adaptor of the TLR signaling pathway) via its N-terminal kinase domain. Additionally, CfIKK1 protein could form homodimers and even oligomers, with N-terminal kinase domain and C-terminal scaffold dimerization domain playing key roles in this process. Finally, the results of RNAi experiments showed that when the scallop IKK1 gene was suppressed, the expression of IRF genes also decreased significantly. In conclusion, CfIKK1 could respond to PAMPs challenge and interact with MyD88 protein of scallop TLR signaling, with the formation of CfIKK1 dimers or oligomers. At the same time, the results of RNAi experiments revealed the close regulatory relationship between IKK1 and IRF genes of scallop. Therefore, as a key signal transduction molecule and immune activity regulator, CfIKK1 plays important roles in the innate immune system of scallops.

The IκB kinase (IKK) complex, which is essential for the activation of the transcription factor NF-κB, is composed of IKK1 (IKKα), IKK2 (IKKβ), and the key regulatory protein NF-κB essential modulator (NEMO). However, there are few studies on the function of NEMO in invertebrates. In this study, we obtained the complete coding sequence of the NEMO gene of the Zhikong scallop Chlamys farreri and studied its response to pathogen-related molecular pattern (PAMP) stimulation and its interaction with the IKK1 protein. Sequence analysis revealed that Chlamys farreri NEMO (CfNEMO) encodes 778 amino acids, and the encoded protein contains a typical NEMO domain, two coiled coils, a putative leucine zipper motif, and a C-terminal zinc finger domain. Phylogenetic relationship analysis showed that CfNEMO belongs to the NEMO family of mollusks in invertebrates. Real-time quantitative PCR showed that CfNEMO transcripts were expressed in all tissues tested in scallops, with the highest expression in the gills. Furthermore, the expression level of CfNEMO in the gills was significantly induced after lipopolysaccharide, peptidoglycan, and polyinosinic–polycytidylic acid challenge. Finally, the results of co-immunoprecipitation showed that CfNEMO can interact with IKK1 protein (IKKα/IKKβ family member). And by constructing truncated mutants, we found that the interaction between the two proteins is mediated by the NEMO domain of CfNEMO and the SDD (scaffold dimerization domain) domain of IKK1. In conclusion, our results lay the foundation for in-depth understanding of the innate immune function of invertebrate NEMO and the formation of the IKK complex. Our findings provide valuable information on disease control and management in scallop farming.