Unlike vertebrates, which deploy both adaptive and innate immune responses, mollusks rely exclusively on innate immunity. Their defense toolkit includes enhanced phagocytic activity, pattern recognition receptors, and effector molecules that identify and neutralize invading microorganisms. Among these molecular sentinels is the mannose receptor, a member of the C-type lectin superfamily and a type I transmembrane protein that functions as what researchers describe as a non-standard pattern recognition receptor. Rather than serving as a conventional first-line sensor, the mannose receptor contributes to host defense primarily by recognizing and binding endogenous ligands and pathogen-associated molecules, thereby modulating immune responses and maintaining immune homeostasis under external stress. Its importance has been demonstrated previously in aquatic animals such as the red swamp crayfish Procambarus clarkii and the orange-spotted grouper Epinephelus coioides, where MR-mediated responses showed antiviral and antibacterial activity against Vibrio species.

Structurally, the mannose receptor is an intricate machine. It comprises an extracellular cysteine-rich domain, a fibronectin type II domain, and eight tandem C-type lectin-like domains, along with a transmembrane segment and a short cytoplasmic tail. Functional studies have shown that the fibronectin type II domain and the second C-type lectin-like domain act synergistically to enhance the uptake of glycosylated collagen, while the fourth domain has been implicated in modulating T cell cytotoxicity. The receptor’s C-type lectin-like domains can specifically recognize and bind carbohydrate ligands such as mannan, trehalose, and N-acetylglucosamine on both endogenous and exogenous molecules, initiating immune responses upon binding. In the bream Megalobrama amblycephala, for example, the mannose receptor binds chitosan oligosaccharide and mediates its uptake by macrophages through lectin-dependent endocytosis, modulating the expression of tumor necrosis factor receptor-associated factors, interleukins, and nitric oxide synthase.

To understand how this receptor family operates in the Manila clam, the researchers mined the clam’s genome, which was previously sequenced and made available under NCBI BioProject PRJNA479743. Using Hidden Markov Model searches based on the C-type lectin-like domain model PF00059.23, they identified a remarkably large repertoire of 13 mannose receptor genes, designated RpMR1 through RpMR13. The predicted proteins displayed considerable diversity, with molecular weights ranging from 11.27 to 320.37 kilodaltons and theoretical isoelectric points between 4.42 and 6.82. Exon numbers varied from 2 to 60, and every RpMR protein contained at least one C-type lectin domain, with copy numbers ranging from 1 to 14 per protein, hinting at substantial functional redundancy or diversification within the family. Conserved motif analysis revealed ten distinct motifs across the proteins, and multiple sequence alignment highlighted conserved cysteine residues, calcium-binding sites, acidic amino acids, and aromatic residues such as phenylalanine and tryptophan, all characteristic features of C-type lectin family proteins.

Chromosomal localization added another layer of organization to the story. Ten of the 13 RpMR genes mapped to six annotated chromosomes, with chromosome 10 harboring three genes in what appears to be a gene cluster, while the remaining three genes sat on unplaced scaffolds. Phylogenetic analysis, built from 142 mannose receptor amino acid sequences across nine representative species, revealed that MR genes cluster into two major branches, with molluscan sequences from R. philippinarum, Crassostrea virginica, C. gigas, and Biomphalaria glabrata grouping predominantly within a single large clade. This pattern suggests that mannose receptor genes in mollusks have undergone evolutionary conservation, reflecting shared ancestry and potentially conserved functional roles across the phylum. Interestingly, when the researchers examined expression across developmental stages, all RpMR genes were expressed as the clams grew, with most showing significantly increased expression at the D-larva stage, indicating that this immune machinery is active from early life onward.

The infection challenge experiments brought the gene family’s defensive role into sharp focus. Wild clams collected from Jinshitan in Dalian were immersed in V. anguillarum at a concentration of 1 × 10⁷ CFU/mL, and hepatopancreas tissues were sampled at intervals from 0 to 96 hours post-challenge. The results showed that RpMR expression surged following infection, with RpMR1, RpMR2, RpMR3, RpMR4, and RpMR6 all peaking at 72 hours post-infection, reaching 3.2-, 6.8-, 8.3-, 1.68-, and 1.1-fold increases respectively relative to baseline. Tissue-specific analysis revealed that the genes were expressed throughout the clam body, including the adductor muscle, mantle, foot, gill, siphon, and digestive gland, but expression was most pronounced in the hepatopancreas, where RpMR2 reached twelvefold higher levels than in the adductor muscle. This organ, the researchers conclude, likely serves as a key site for mannose receptor-mediated immune responses.

The team then zeroed in on RpMR1 as a functional candidate. They cloned the gene’s coding region into a PET-28A(+) vector, expressed it in E. coli Rosetta (DE3) cells, and purified the recombinant protein, which appeared at its theoretical 123 kilodalton position on SDS-PAGE gels and was confirmed by Western blot. In vitro antibacterial assays tested the protein against eight bacterial strains, and the results were strikingly specific. RpMR1 significantly inhibited growth of three Gram-negative Vibrio pathogens: V. splendidus, with significant suppression observed at 4 hours, and V. anguillarum, with sustained inhibition at 6, 8, and 10 hours, along with V. alginolyticus. No inhibitory activity was detected against Bacillus subtilis, Staphylococcus aureus, Vibrio parahaemolyticus, V. harveyi, or Escherichia coli. The researchers attribute this bactericidal specificity to RpMR1’s affinity for lipopolysaccharides, the dominant outer membrane component of Gram-negative bacteria and a conserved pathogen-associated molecular pattern recognized by Toll-like receptor 4.

The most dramatic evidence came from in vivo experiments. Clams were divided into four groups receiving different injections: phosphate-buffered saline plus recombinant protein, V. anguillarum plus recombinant protein, V. anguillarum plus buffer, and buffer alone. By 96 hours, the cumulative mortality rate in the infected group that received only buffer reached 78.6 percent, while the infected group that also received RpMR1 protein saw mortality drop to 52.7 percent, a 26 percent reduction in deaths. Clams receiving protein without infection showed the same survival as untreated controls, confirming the protein’s safety. Complementary molecular measurements showed that injection of RpMR1 rapidly activated the Toll-like receptor signaling pathway, with the genes TLR, MyD88, TRAF, NF-κB, IKK, and AP-1 all peaking at 6 hours post-infection in the infected, protein-treated group, while most pathway genes in the infected, untreated group did not peak until 96 hours. Nitric oxide synthase activity was also significantly elevated at 12 and 72 hours in the protein-treated infected group, consistent with nitric oxide’s established role in promoting phagolysosome maturation and microbicidal activity.

To establish causality rather than mere correlation, the researchers turned to RNA interference. Injecting synthetic double-stranded RNA targeting RpMR1 successfully silenced the gene, reducing its expression significantly. The knockdown had cascading effects: TRAF6 expression dropped significantly, while TLR4 and AP-1 expression levels were also significantly reduced. This demonstrates that RpMR1 positively regulates components of the TLR signaling pathway during the immune response, confirming a functional interaction between the mannose receptor and TLR4. The finding fits with a broader literature on pattern recognition receptor crosstalk: mannose-binding lectin, another C-type lectin family member, has been shown to potentiate TLR4 signaling through direct interaction with its leucine-rich repeat domain, and cooperative interactions between mannose receptors and TLR4 have been documented in orchestrating pro-inflammatory mediator release, including interleukin-1β, tumor necrosis factor-alpha, and interleukin-6. Because the mannose receptor itself lacks intrinsic signaling capacity, such cooperative interactions with other receptors may be essential for transducing immune activation signals while maintaining immune homeostasis.

The implications for aquaculture are considerable. The authors suggest that the immunological functions of recombinant RpMR1 protein could eventually be applied in Manila clam farming, potentially incorporated into feed as an antimicrobial agent, though they caution that further research is needed to determine optimal dosage and whether sustained antibacterial activity can be maintained at scale. More broadly, the study marks the first functional demonstration of mannose receptor-mediated immunity in mollusks, bridging receptor-mediated pathogen recognition with downstream effector mechanisms such as nitric oxide production and TLR signaling in bivalve host defense. The researchers note that the precise molecular interplay by which RpMR1 confers anti-Vibrio immunity through nitric oxide synthase-dependent mechanisms remains to be fully delineated, and targeted investigations into the tripartite relationship between receptor activation, effector enzyme regulation, and pathogen clearance are still needed. Nevertheless, by identifying a concrete molecular target linked to survival during infection, the work opens a promising avenue for breeding or engineering disease-resistant clam strains, offering a potential lifeline for an aquaculture industry under relentless bacterial pressure.

Subject of Research: Mannose receptor-mediated innate immunity in the Manila clam against Vibrio anguillarum infection

Article Title: Mannose receptor RpMR1 of Manila clam (Ruditapes philippinarum) defense against Vibrio anguillarum infection

Article References: Yin, Z., & Nie, H. (2025). Mannose receptor RpMR1 of Manila clam (Ruditapes philippinarum) defense against Vibrio anguillarum infection. Advanced Biotechnology, 3(3), Article 23. https://doi.org/10.1007/s44307-025-00075-7

Image Credits: AI Generated

DOI: 10.1007/s44307-025-00075-7

Keywords: Manila clam, Ruditapes philippinarum, mannose receptor, RpMR1, Vibrio anguillarum, pattern recognition receptor, innate immunity, TLR4, nitric oxide synthase, RNA interference, aquaculture, bivalve defense