Large yellow croaker (Larimichthys crocea), the native species of East Asia, has the highest aquaculture yield among marine fish in China (257,683 tons in 2022) (MOA, 2023). However, the intensive net-cage inshore cultivation model, the degrading farming environment, outbreaks of various abiotic and biotic stressors like hypoxia and Aeromonas hydrophila infection, and other factors, had caused significant economic losses to the aquaculture industry (Ding et al., 2022; Zhang et al., 2022). Many previous studies had confirmed that Toll-like receptor genes played a vital role in innate immunity (Ni et al., 2022; Yao et al., 2023; Gao et al., 2024), however, research on their role in environmental adaptability was still in its early stages. Therefore, the research of biotic and abiotic environmental factors was on the front-page of L.crocea’s survival. This experiment would lay a foundation for the specific role of Toll-like receptor genes in the host environmental adaptation to resistance to hypoxic response and Aeromonas hydrophila infection.

Dissolved oxygen (DO), or molecular oxygen dissolved in water, is the primary source of oxygen for a variety of aquatic organisms. As L. crocea cultures grow and their densities rise, the culture environment degrades and the water exchange capacity eventually drops, leaving large areas with low DO levels. Marine ecosystems are more vulnerable to hypoxic stress than terrestrial environments are (Lee et al., 2024). Low dissolved oxygen levels have a negative impact on fish physiological and biochemical processes, including apoptosis (Liu et al., 2022b), energy metabolism (Jaworski et al., 2019), and other processes, which can result in a variety of illnesses and injuries (Shun et al., 2024). Reactive oxygen species (ROS) are produced by the organism from part of the oxygen taken up by fish respiration. A tiny percentage of ROS control gene expression and cell activity through their involvement in cell signaling pathways (Shi et al., 2024). According to Abdel-Tawwab et al. (2019), fish that experience oxidative stress due to excessive ROS production during hypoxia eventually undergo apoptosis and sustain tissue damage. Aquatic species’ growth, survival, behavior, immunity, and reproduction are all significantly impacted by such hypoxic environments. According to Ding et al. (2020), hypoxia in L.crocea causes a rise in mortality and is now the primary barrier to the growth of its sustainable mariculture. On the other hand, the breeding of L.crocea was threatened by bacterial diseases (Mu et al., 2018). Previous studies had shown that the diseased L. crocea had suffered a variety of severe illness symptoms after Gram-negative bacteria A. hydrophila infection (Chen et al., 2010; Mu et al., 2010; Zhang et al., 2022), as well as the verification of TLRs differential expression following an A. hydrophila challenge in common carp (Gong et al., 2017). Therefore, in order to allow large yellow croak culture population, exploit a better ecological opportunity under the stress of environmental factors such as hypoxia and A. hydrophila infection, researchers were committed to exploring the molecular mechanism of resistance regulation of TLR genes.

To date, studies have shown that TLRs bear the responsibility of playing important roles in innate immunity in organisms (Verma et al., 2024). Innate immune cells, such as dendritic cells (DCs), are activated when the immune system detects microorganisms through a variety of receptors known as pattern recognition receptors (PRRs), which are encoded by germ-line genes and recognize a wide range of microbial structures (Wang et al., 2024). Up to now, many PRRs have been found, including C-type lectin receptors, scavenger receptors (SR), intracellular receptors, lipopolysaccharides (LPS) and β-1, toll-like receptors (TLRs), and Down syndrome cell adhesion molecules (Dscam) (Tran et al., 2019). Among these PRRs, the role of TLRs in innate immune responses during evolution is thought to be highly conserved, usually acting as transmembrane receptors that recognized different microbial components and directly activated immune cells. Exposure of immune cells to the ligands of receptors can activate intracellular signaling cascades, and then rapidly induce the expression of a variety of overlapping and unique genes involved in inflammation and immune responses. After pathogen infection, pathogen-associated molecular patterns (PAMPs) are recognized, myeloid differentiation factor 88 (MyD88) and adaptors containing TIR domains are recruited by TLRs to induce interferon-beta (IFN-β) level (Dias et al., 2022; Ryan and O’Neill, 2023), thereby leading to the activation of protein 1 (AP-1) transcription factor and nuclear factor kappa-B (NF-κB) (Wang et al., 2017). This induces the production of pro-inflammatory cytokines, such as TNF-α and IL1b, to activate innate and adaptive immune responses (Vidya et al., 2018). Structurally, TLR consists of several domains, including N-terminal signaling domains, multiple extracellular leucine-rich repeat (LRRs) domains, transmembrane domains, and intracellular Toll/interleukin-1 receptor (TIR). The TIR domain functions as a protein scaffold, attracting downstream molecules and initiating signaling cascades that activate the expression of target genes, such as antimicrobial peptides, while the extracellular LRR domain recognizes the PAMP ligands of pathogenic microorganisms.

TLRs are a family of germline-encoded PRRs conserved in function. They are essential for recognizing PAMPs like flagellin, lipoprotein, nucleic acid, LPS, and lipoteichoic acid (Tran et al., 2019). For instance, bacterial flagellin stimulates TLR5, while double-stranded RNA (dsRNA) can activate TLR3 (Adams et al., 2024). Similar in location, structure, and function, TLR7 and TLR8 are recognized to detect single-stranded RNA (ssRNA) from viruses (Wallach et al., 2023). Furthermore, it has been discovered that TLR9 and TLR21 can both identify CpG DNA in fish and that they react preferentially to CpG DNA with distinct CpG (Yeh et al., 2013). Following ligand identification, TLR uses the Toll/Interleukin-1 receptor (TIR) domain and TIR-containing cohesive molecules, such Myd88 and other TIR-domain-containing convergent proteins, to initiate downstream signaling cascades and an immunological response (Sahoo et al., 2012). Activation of the TLR signaling pathway triggers the production of co-stimulatory molecules and inflammatory cytokines, which effectively trigger the host’s antiviral or antimicrobial immune responses (Li et al., 2022).

It is now evident that TLRs promote and regulate the quality of the adaptive immune response in addition to playing a critical role in the innate identification of microorganisms (Manicassamy and Pulendran, 2009). The discovery of 21 distinct TLR types in a range of fish species in recent years has spurred interest in investigating TLRs’ potential as targets for increasing fish immunity and disease resistance (Mahapatra et al., 2023). Twelve TLRs in all were found in the L. crocea examined in this investigation. The number was the same as the closely related species T. rubripes and O. latipes (Xie et al., 2023). The majority of genes were carried by L. crocea, which reached a high level of evolutionary conservativeness. Besides, four “fish-specific” family members (Rebl et al., 2010) of TLRs were identified in L. crocea, including TLR21, 22, 23. These findings corroborated those of earlier research, which revealed that fish TLRs had a great deal of diversity and distinctive traits, most likely due to their varied evolutionary histories and the varied settings they live in (Palti, 2011).

Members of the TLR family in teleosts were divided into six clades based on sequence homology comparison (Palti, 2011; Fan et al., 2019). Additionally, TLRs 18–20 and 22–28 are thought to be special to fish (Palti, 2011). While the majority of fish TLRs have not been documented to recognize comparable PAMPs, the fish TLRs have unique characteristics resulting from their varied evolutionary backgrounds and intricate aquatic environments. Notably, even though L. crocea contained most TLR genes, we found that the large yellow croaker’s genome lacked the TLR4 and TLR13 genes, and we also noticed the similar situation in the genome of D. rerio, P. olivaceus and O. niloticus etc., indicating that gene gain and loss events might occur frequently in fish.

The phylogeny of gene families may be further explained by structural analysis. Here, we found that LcTLR14 had a longer intron structure while others had shorter introns. Besides, it was discovered that the majority of the TLR genes in Cyprinus carpio and Patinopecten yessoensis have intron-less architectures (Gong et al., 2017; Xing et al., 2017). The splicing process of post-transcriptional modification will take longer for gene architectures with longer or more introns (Jeffares et al., 2008), which is not good for the efficient regulation of genes. It was discovered that under stress, genes with short or no introns might express themselves quickly (Guo et al., 2015; Liu et al., 2022a). These findings implied that the TLR gene family might have evolved in a significant way toward gene structural simplification as a result of environmental adaption.

Studies have shown that environmental stressors just like hypoxia stress and bacterial infection could affect TLR expression. In the kidneys, spleen, brain, and fish gills, hypoxia typically results in cell necrosis, apoptosis, and inflammation while also encouraging the growth of new blood vessels (Harper and Wolf, 2009). However, little is known about how TLRs function in fish in response to hypoxia. Prior research has demonstrated that in both human and mouse dendritic cells (DCs), hypoxia preferentially upregulated the expressions of TLR2 and TLR6 (Kuhlicke et al., 2007). According to Stridh et al. (2011), hypoxia in neonatal mice led to an increase in TLR1 and TLR2 expression in the brain. Additionally, research on Tibetan schizothoracine fish (Gymnocypris eckloni) revealed that while TLR2 and TLR3 expressions increased in the head kidney, TLR8a, TLR12 and TLR19 in the spleen, and TLR1 expressions increased in the gill, the majority of TLRs’ mRNA expressions either significantly decreased or remained unchanged under acute hypoxia (Qi et al., 2017). In this study, the expression of LcTLR1, LcTLR9, and LcTLR21 in gill, LcTLR5 in heart, LcTLR3, LcTLR5, LcTLR7, LcTLR8 and LcTLR9 in kidney, and LcTLR2a, LcTLR7, LcTLR8 in spleen were significantly up-regulated, whereas the expressions of other TLRs in the gill, heart, kidney and spleen declined dramatically or remained unchanged in a severe hypoxic environment. This result was consistent with Tibetan schizothoracine fish TLRs expression in response to acute hypoxia. The cessation of the energy-saving mechanism of TLR protein synthesis leads to the reduction of TLR mRNA (Roesner et al., 2006), and TLRs are normally involved in innate immune responses regulated by the expression of damage-associated molecular patterns (DAMP), which are secreted by necrotic or damaged cells (Mkaddem et al., 2010). Thus, the unique function of TLRs in mediating the innate immune response to host defense against hypoxia-induced tissue damage might be connected to tissue-specific TLR expression under hypoxic settings. TLRs offer a great model for examining the natural selection imposed by pathogenic microorganisms on the organism genome because of their direct placement at the host–environment interface and potential for coevolutionary dynamics with their pathogenic counterparts (Barreiro et al., 2009).

There had suggested an idea called TLR/TLR cross-talk, which means that, in comparison to DCs stimulated with individual TLR4 ligand and TLR7 ligand, TLR/TLR synergy, such as simultaneous stimulation of human monocyte-derived DCs with LPS (TLR4 agonist) and R484 (TLR7 agonist), leads in a synergistic rise in anti-inflammatory cytokine IL-10 (Napolitani et al., 2005; Manicassamy and Pulendran, 2009). In this study, A. hydrophila infection was performed to evaluate L. crocea TLR gene alterations, and our results were similar to a research of L. crocea in response to Cryptocaryon irritans infection, demonstrating that TLR5 was distinctly up-regulated following infection (Zhang et al., 2020). This might due to the function of TLR5 responding to PAMP, such as lipids and bacterial proteins (Yang et al., 1999). TLR genes that were up-regulated during the bacterial infection were generally thought to be crucial for identifying bacterial ligands (Zhang et al., 2017). Besides, LcTLR3, LcTLR7, LcTLR9 and LcTLR14 were remarkedly down-regulated after 12 h in our study. The role of TLR3 in immune responses was complex and differed between species. TLR3 in the spleen did not significantly alter following injection of Vibrio parahaemolyticus in the prior research of Pseudosciaena crocea, suggesting that bacterial infection of the spleen might not be the initial cause of the PcTLR3 response (Huang et al., 2011). After bath challenge with a Gram-negative bacterium, rainbow trout’s spleen showed a similar outcome (Rodriguez et al., 2005). However, TLR3 was shown to be significantly down-regulated in the kidney of channel catfish following an infection with Edwardsiella tarda. This finding, along with our results, suggested that this protein might be the most susceptible to bacterial infection, as the subpopulation of phagocytes expressing these genes might quickly migrate from the kidney to the infection sites (Zhang et al., 2013). This variation in expression changes of TLR3 might be due to differences in TLR3-mediated inflammatory responses among different immune organs, further hinting that apart from their recognition of dsRNA, TLR3 was also engaged in fish immune responses to bacterial infections. SmTLR7 was markedly down-regulated in the gill and intestine of turbot (Scophthalmus maximus) following infection with both E. tarda and Vibrio anguillarum (Wang et al., 2022). After A. hydrophila infection, the expression level of MaTLR14 was up-regulated in the liver, spleen, foregut, and hindgut of Asian swamp eels (Monopterus albus) (Liu et al., 2022b). All of these findings revealed that different Gram-negative bacterial components regulated TLRs, suggesting that the immune response to bacterial infection might involve numerous TLR-mediated signaling cascades at the same time. Notably, LcTLR2 had no significant change throughout the process, which was inconsistent with the previous study showing that the LcTLR2 transcripts increased significantly after Vibrio parahaemolyticus, Polyinosinic-polycytidylic acid (Poly(I:C)) and LPS immune challenges (p < 0.05) in the spleen, liver and head-kidney (Fan et al., 2015). This might be due to differences in immune response induced by A. hydrophila and V. parahaemolyticus.

Together with A. hydrophila infection and hypoxic stress, this study revealed that there were six and nine members of LcTLRs showing significant differential expression in immune responses, respectively. These results provided insights into TLRs regulation to biological and abiotic stresses, implying that TLR expressions specific to particular tissues mediated innate immune responses for host defense against tissue damage or physiological changes brought on by bacterial infection and hypoxic stress.

The larger yellow croaker (Larimichthys crocea) was found to harbor a total of 12 TLR genes in this study. Analysis of the TLR genes’ phylogenetic relationships, gene structures, and motif compositions revealed that these genes were highly conserved among different species. Expression pattern analysis and qPCR validation of A. hydrophila infection and hypoxic stress revealed that there were six and nine members of LcTLRs showing significant differential expression in immune responses, respectively. Notably, LcTLR5 was significantly expressed under both biological and abiotic stresses, suggesting that it might play a significant role in the effort to combat environmental stressors. In summary, the identification of TLRs and their roles would advance our knowledge of fish disease resistance mechanisms and open up new avenues for therapeutic intervention to control immune responses.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The animal study was approved by the Institutional Animal Care and Use Committee at the Zhejiang Laboratory Animal Research Center and Ningbo University. The study was conducted in accordance with the local legislation and institutional requirements.

T-YY: Writing – original draft, Methodology, Formal analysis. Q-TM: Writing – original draft, Investigation. X-YS: Writing – original draft, Conceptualization. CR: Writing – original draft, Data curation. Q-PX: Writing – original draft, Formal analysis. X-BW: Writing – review & editing, Supervision, Project administration, Funding acquisition.