Histone H3K27 acetylation mediated by KAT8 maintains antiviral trained immunity in shrimp induced by inactivated white spot syndrome virus

Lv, Li-Xia; Zhang, Peng; Ma, Ying; Sun, Yue; Wang, Hao; Zhao, Xiao-Fan; Wang, Jin-Xing

doi:10.1038/s42003-025-08767-5

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Published: 30 August 2025

Histone H3K27 acetylation mediated by KAT8 maintains antiviral trained immunity in shrimp induced by inactivated white spot syndrome virus

Li-Xia Lv¹,
Peng Zhang¹,
Ying Ma¹,
Yue Sun¹,
Hao Wang¹,
Xiao-Fan Zhao ORCID: orcid.org/0000-0003-1809-4730¹ &
…
Jin-Xing Wang ORCID: orcid.org/0000-0003-0283-3930^1,2

Communications Biology volume 8, Article number: 1314 (2025) Cite this article

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Abstract

Shrimp aquaculture is critically important for global food security, but viral diseases like white spot syndrome virus (WSSV) cause devastating economic losses, highlighting the urgent need for effective disease control strategies. While trained immunity has been observed in invertebrates like shrimp after exposure to pathogens, the underlying molecular mechanisms remain elusive. Here we reveal that lysine acetyltransferase KAT8-mediated histone H3K27ac is critical for antiviral defense in shrimp Marsupenaeus japonicus. We demonstrate that ultraviolet-inactivated WSSV (UV-WSSV) induces antiviral trained immunity in the shrimp via KAT8-dependent H3K27ac. UV-WSSV training enhances glycolysis and the tricarboxylic acid (TCA) cycle, increasing acetyl-CoA production. This acetyl-CoA fuels KAT8 activity, depositing H3K27ac marks at the promoter of the NF-κB-like transcription factor Dorsal. This epigenetic modification upregulates Dorsal expression, leading to the enhanced production of the antiviral cytokine Vago5 and antimicrobial peptides (AMPs) upon subsequent WSSV challenge. Furthermore, H3K27ac directly activates key glycolytic genes (Hk2, Pk, Ldh), creating a feedforward loop that sustains metabolic reprogramming. Our work reveals a conserved KAT8-H3K27ac axis driving trained immunity in invertebrates through integrated metabolic-epigenetic crosstalk, analogous to mammalian systems. These findings provide a crucial theoretical foundation for developing antiviral vaccines and sustainable immunostimulants to control disease in shrimp aquaculture.

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Introduction

The vertebrate immune system involves both innate and adaptive immune responses. Invertebrates typically possess only innate immunity against invasive pathogens and lack immunological memory. However, research over the past two decades has demonstrated that innate immunity can exhibit memory, a phenomenon called innate immune memory, or trained innate immunity¹. Trained immunity is characterized by faster and more effective immune responses upon restimulation and provides long-term protection against foreign invaders^2,3. The mechanisms of trained immunity are well known in the innate immune cells of mammals. Trained immunity is driven by the integration of signaling, metabolic, epigenetic and founctional rewiring: Immunological stimuli are initially recognized by different pattern recognition receptors (PRRs). These receptors trigger specific trained immunity-inducing signaling pathways that are crucial to trained phenotypes and subsequently mediate metabolic reprogramming (including glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid metabolism) and epigenetic modifications [including DNA methylation, histone methylation (such as H3K4me3), and acetylation (such as H3K27ac)]. This leads to a rapid and augmented innate immune response upon reinfection^2,4. Trained immunity has also been found in invertebrates, including insects and shrimp^5,6. For example, multiple lines of evidence establish that Marsupenaeus japonicus develops significantly enhanced resistance to WSSV upon secondary challenge when primed through oral delivery or immunization with formalin- or UV-inactivated WSSV^7,8,9. The innate immune memory appeared to persist over a longer time span, or sometimes across generations¹⁰. Several possible mechanisms have been proposed to explain the induction of innate immune memory in invertebrates. These include the upregulation of immune-related pathways (such as Toll, Imd, JNK and JAK/STAT pathways) and phenotypic changes in immune cell populations (such as increased abundance of granulocytes)^11,12,13, however, it is unclear whether epigenetic modifications and metabolic reprogramming are involved in the innate immune memory of invertebrates. On the other hand, although the study of trained immunity has taken many advances over the past decade, there are many key knowledge gaps that hamper our ability to use trained immunity to combat pathogens¹⁴.

Lysine acetylation is an evolutionarily conserved post-translational modification (PTM) that occurs in both prokaryotes and eukaryotes. It is jointly regulated by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs)^15,16. Lysine acetyltransferase 8 (KAT8), also known as MYST1 or MOF (males absent on the first), is important in catalyzing the acetylation of histone H4 at K16 (H4K16) in mammalian cells^17,18,19. It is also responsible for the acetylation of nonhistone substrates such as p53²⁰ and interferon regulatory factor 3 (IRF3)²¹. KAT8 is involved in diverse biological processes, ranging from DNA damage repair, DNA transcription, and embryonic development to apoptosis^22,23. However, the function of KAT8 in trained innate immunity is unclear.

Penaeid shrimp species are valuable for marine aquaculture. However, diseases caused by different pathogens, especially white spot disease (WSD), cause high mortality rates and significant economic losses^24,25. White spot syndrome virus (WSSV) is an infectious pathogen of shrimp and other crustaceans. Approximately 80% of cultured shrimp losses are due to WSSV^26,27. There are currently no effective control strategies for WSSV in shrimp aquaculture. The study of trained immunity in shrimp will facilitate the development of more reliable, cost-effective, and sustainable disease control strategies (such as the application of antiviral vaccines) in shrimp aquaculture. In the present study, we established a trained immunity model using ultraviolet light-inactivated WSSV (UV-WSSV) in the shrimp Marsupenaeus japonicus. We analyzed the profiles of H3K27ac modifications and identified that KAT8 is involved in the antiviral trained immunity of shrimp. The roles and underlying mechanisms of KAT8 in UV-WSSV-induced antiviral trained immunity were analyzed.

Results

H3K27ac and H4K16ac modifications mediated by KAT8 are enhanced in WSSV infected shrimp

Alterations in epigenetic modifications and reprogramming of cellular metabolism are two pillars of trained immunity. In our previous study, we reported that inactivated WSSV induced trained immunity in shrimp⁹. To reveal the mechanisms of trained immunity induced by inactivated WSSV, we first detected changes in histone acetylation, including histone pan-acetylation (pan-Hac), H3K27ac, H4K16ac, and H3K9ac, in the hemocytes of shrimp challenged with WSSV via the use of corresponding antibodies. The results revealed that the abundance of all of the above modifications was increased in the hemocytes of WSSV-infected shrimp, and H3K27ac and H4K16ac abundance were particularly high at 12 h post-WSSV infection (Fig. 1a, a’). Therefore, we focused on H3K27ac and H4K16ac in the following study.

**Fig. 1: KAT8-mediated H3K27ac and H4K16ac are upregulated in WSSV-infected shrimp.**

To identify the lysine acetyltransferases (KATs) critical for histone H3K27ac epigenetic modification in shrimp, RNA interference (RNAi) screening was performed. Nine KATs were identified in our shrimp transcriptome sequencing data and online sources of the genome sequences of M. japonicus (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Supplementary Fig. 1). We chose three KATs (KAT8, Tip60, and KAT2a) that were potentially involved in this modification for further study. After the expression of Kat8, Tip60, and Kat2a was knocked down in shrimp following WSSV infection (Fig. 1b–d), the H3K27ac level in shrimp hemocytes was significantly lower in dsKat8 injection group compared with that in the dsGfp group, no obvious change in H3K27ac was detected after RNAi of the other KATs (Fig. 1e).

Next, we detected H4K16ac in addition to H3K27ac after the knockdown of Kat8 (Fig. 1f), and the results revealed that H4K16ac and H3K27ac modifications were significantly decreased in shrimp hemocytes and the intestinal tissues at 24 h after WSSV infection (Fig. 1g, g’). Hence, KAT8 is also involved in H4K16ac modification in shrimp. The 6-alkylsalicylate MG149, an inhibitor of KAT8^28,29 was used to confirm these results after optimizing the injection dosage of the inhibitor (Supplementary Fig. 2a). Treatment with MG149 significantly inhibited H3K27ac and H4K16ac modifications in a dose-dependent manner in shrimp hemocytes (Supplementary Fig. 2b, b’) and the intestinal tissues (Supplementary Fig. 2c, c’). These results suggest that histone H3K27 and H4K16 acetylation mediated by KAT8 were increased in WSSV infected shrimp.

KAT8 is significantly upregulated and inhibits WSSV replication in shrimp

Shrimp KAT8 is a relatively conserved protein with 65% similarity to the amino acid sequence of Homo sapiens, and contains the CHROMO (Chromatin organization modifier) domain and histone acetyltransferase activity domain (Supplementary Fig. 3a, b). Phylogenetic analysis revealed that M. japonicus KAT8 clustered with human KAT8 and was closely related to Litopenaeus vannamei KAT8 (Supplementary Fig. 3c).

To characterize KAT8 tissue distribution, expression dynamics, and subcellular localization, we first generated specific polyclonal antibodies against the recombinantly expressed the protein (Supplementary Fig. 4a). Subsequent analyses revealed widespread distribution of both Kat8 mRNA and KAT8 protein across hemocytes and multiple tissues, including the heart, gills, stomach, and intestine (Supplementary Fig. 4b). Following WSSV infection, Kat8 transcript levels increased from 6 hpi, peaked at 24 hpi, and subsequently declined (Supplementary Fig. 4c). Consistent with transcriptional upregulation, KAT8 protein levels also increased from 6 hpi, and reaching maximal expression at 24 hpi (Supplementary Fig. 4d–f). The subcellular distribution of KAT8 was analyzed by immunocytochemical and western blot assays. As shown in Supplementary Fig. 4g, g’, KAT8 localized to both the cytoplasm and nucleus of normal shrimp hemocytes. However, 24 h post WSSV infection, its localization shifted predominantly to the nucleus. KAT8 was also detected in the cytosol and nucleus of intestinal cells by western blotting, and similar results were obtained (Supplementary Fig. 4h, h’). Our previous study revealed that KAT8 expression is regulated by the transcription factor Forkhead box O (FOXO) in insects³⁰. The FOXO-binding element (FOXOBE) was also identified in the promoter of the shrimp Kat8 gene via the JASPAR website (http://jaspar.genereg.net/) (Supplementary Fig. 4i). To determine whether WSSV promotes KAT8 transcription through FOXO, we knocked down FoxO using RNAi and examined Kat8 expression. The results showed that FoxO knockdown markedly reduced Kat8 expression in shrimp hemocytes and the intestinal tissues at 24 h post-WSSV infection (Supplementary Fig. 4j). These results demonstrate that KAT8 expression is regulated by FOXO in response to WSSV infection and function in the nucleus for H3K27ac modification.

To explore the functions of KAT8 in shrimp infected with WSSV, RNAi of Kat8 was performed, followed by WSSV replication analysis via the expression of Vp28 (envelope protein-encoding gene of WSSV) and Ie1 (immediate early gene 1 of WSSV) as indicators. The results revealed that the expression of Vp28 (Fig. 2a) and Ie1 (Fig. 2b) increased significantly in shrimp hemocytes and the intestinal tissues at 24 hpi. Similar results were obtained for the expression of the VP28 protein (Fig. 2c). The number of WSSV copies was significantly increased in the intestinal tissues of Kat8-knockdown shrimp challenged with WSSV (Fig. 2d). Compared with that of the control shrimp, the survival of the Kat8-knockdown shrimp significantly decreased (Fig. 2e). These results suggest that KAT8 has antiviral functions mediated through H3K27ac modification.

**Fig. 2: *Kat8* knockdown or MG149 treatment promotes WSSV proliferation in shrimp.**

To verify the role of KAT8 in shrimp infected with WSSV, we treated shrimp with the KAT8 inhibitor MG149 followed by analysis of WSSV replication. The results showed that WSSV replication increased significantly in the hemocytes and intestinal tissues of MG149-treated shrimp infected with WSSV (Fig. 2f, g). The VP28 protein in hemocytes and intestinal tissues was also increased in the MG149 injection group, followed by WSSV infection (Fig. 2h). The survival rate of MG149-treated shrimp infected with WSSV was significantly lower than that of control shrimp (Fig. 2i). These results suggested that KAT8 upregulated in WSSV-infected shrimp, and significantly reduced WSSV infection and increased the shrimp survival rate.

Resistance to WSSV infection is increased in UV-WSSV-trained shrimp through H3K27ac mediated by KAT8

To decipher the underlying mechanisms of trained immunity in the shrimp M. japonicus, we established a model of trained immunity using WSSV inactivated by ultraviolet light (UV-WSSV) as an inducer⁹ (Fig. 3a, b). Compared with that of the control shrimp, the survival rate of UV-WSSV-trained shrimp significantly increased after infection with WSSV (Fig. 3c). These results suggest that UV-WSSV induced trained immunity in shrimp.

**Fig. 3: H3K27ac modification mediated by KAT8 is critical for antiviral trained immunity.**

To explore whether epigenetic modification was involved in UV-WSSV-induced trained immunity in shrimp, we detected variations in H3K27ac, H4K16ac, and H3K9ac marks in the hemocytes of UV-WSSV-trained shrimp using the training model and observed that only the H3K27ac level in hemocytes increased significantly 12 h after UV-WSSV training and it was restored to the basal level on the 5^th d (120 h after UV-WSSV injection); after infection with WSSV, the H3K27ac level in the UV-WSSV group was significantly greater than that in the PBS control group at 3 h postinfection. This finding suggested a faster and stronger response of H3K27ac to the second stimulus with live WSSV (Fig. 3d, d’). However, there was no significant change in H4K16ac and H3K9ac in the UV-WSSV-induced training model. These results suggest that H3K27ac modification, but not H4K16ac or H3K9ac, is involved in UV-WSSV-induced trained immunity, which enhances shrimp resistance to WSSV infection.

The expression patterns of KAT8 were subsequently analyzed in UV-WSSV-trained shrimp, and the analysis revealed that Kat8 was upregulated at 12 hpi and then declined to basal levels at 120 h after training. Kat8 was then upregulated from 3 to 24 h after WSSV infection (Fig. 3e), suggesting a faster and stronger response after infection with WSSV, which is similar to the increase in H3K27ac in the training model. These results indicate that KAT8 might be involved in trained immunity.

To determine whether KAT8 contributes to antiviral trained immunity, we knocked down Kat8 expression via RNAi, and assessed H3K27ac modification and WSSV replication. RNAi-mediated Kat8 knockdown abrogated UV-WSSV-induced increase in H3K27ac levels in shrimp hemocytes (Fig. 3f). Compared to UV-WSSV-trained controls, Kat8-knockdown shrimp exhibited significantly elevated Vp28 and Ie1 mRNA expression (Fig. 3g), increased VP28 protein levels (Fig. 3h), and markedly reduced survival rates (Fig. 3i). These findings were corroborated using the KAT8 inhibitor MG149 as an independent loss-of-function approach (Supplementary Fig. 5a–c). Collectively, these results indicate that KAT8 mediated H3K27ac modification is required for maintaining antiviral trained immunity in UV-WSSV-primed shrimp.

The glycolysis is promoted in shrimp trained with UV-WSSV

Metabolic reprogramming and epigenetic modifications are the two pillars of trained immunity⁴. To determine whether metabolic rewiring (such as glycolysis) is involved in UV-WSSV-induced trained immunity, we first detected the consumption of glucose and the production of lactic acid (LA) in the hemocytes of UV-WSSV-trained shrimp. The results showed that the contents of glucose decreased and LA increased significantly at 12 h and 24 h post UV-WSSV training (Fig. 4a, b). Next, we analyzed the expression patterns of key genes involved in glycolysis [hexokinase2 (HK2), pyruvate kinase (PK), L-lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH)], and the results showed that Hk2, Pk, Ldh and Pdh were upregulated in UV-WSSV-trained shrimp (Fig. 4c). We then detected the contents of glycolytic metabolite pyruvate (PA) in the hemocytes of UV-WSSV-trained shrimp. The results indicated that the contents of PA increased at 12 h post UV-WSSV training (the first stage of the training model) and increased significantly at 3 h or 6 h post WSSV infection (the 2nd stage of the training model) (Fig. 4d). To further confirm the involvement of glycolysis in the trained immunity, the UV-WSSV-trained shrimp were treated with 2-deoxy-D-glucose (2-DG), a glucose analog that competitively inhibits hexokinase in the glycolysis pathway³¹, the expression of Pk, Ldh and Pdh (Fig. 4e), and the contents of PA (Fig. 4f), LA (Fig. 4g) and acetyl-CoA (Fig. 4h), and H3K27ac modification were significantly decreased (Fig. 4i) in the shrimp. The results indicated that glycolysis is involved in H3K27ac modification in UV-WSSV-induced trained immunity.

**Fig. 4: The glycolysis is enhanced in UV-WSSV-trained shrimp.**

In our previous study, we reported that Hypoxia-inducible factor 1 alpha (HIF-1α) interacts with the binding site of the HK promoter and initiates the transcription of HK³². To investigate how glycolysis is upregulated in shrimp trained with UV-WSSV, we detected the expression of Hk after Hif-1α was knocked down and found a significant decrease in the Hk mRNA level in hemocytes (Fig. 4j). H3K27ac modification was reduced after Hif-1α was knocked down (Fig. 4k), indicating that HK expression was regulated by Hif-1α, a key transcription factor of the mTOR signaling pathway, thereby activating glycolysis in UV-WSSV-trained shrimp. These data demonstrated that glycolysis is involved in the trained immunity induced by UV-WSSV training in shrimp.

The TCA cycle is involved in trained immunity in shrimp induced with UV-WSSV

To determine whether the TCA cycle is involved in UV-WSSV-induced trained immunity, we first analyzed the expression patterns of key genes involved in the TCA cycle [citrate synthase (CS), isocitrate dehydrogenase (IDH), 2-oxoglutarate dehydrogenase (OGDH) and ATP-citrate synthase (ACLY)], and the results showed that Cs, Idh, Ogdh and Acly were upregulated in UV-WSSV-trained shrimp (Fig. 5a). We also detected the contents of key metabolites of TCA cycle, citric acid (CA), and acetyl-coenzyme A (Ac-CoA) in the hemocytes of UV-WSSV-trained shrimp. The results revealed that the contents of CA (Fig. 5b) and Ac-CoA (Fig. 5c) increased significantly at 6 h or 12 h post UV-WSSV training and at 3 h or 6 h post WSSV infection. To further confirm the involvement of the TCA cycle in the H3K27ac modification induced by UV-WSSV training, shrimp were pretreated with SB204990 (SB), a specific inhibitor of ACLY enzyme³³. We found that the contents of Ac-CoA (Fig. 5d) and the level of H3K27ac (Fig. 5e, e’) were declined in UV-WSSV trained shrimp. Next, we knocked down the TCA cycle key enzyme Cs through RNAi and found that the expression of Ogdh and Acly (Fig. 5f), the contents of CA (Fig. 5g) and Ac-CoA (Fig. 5h) were decreased and the H3K27ac modification (Fig. 5i, i’) were significantly inhibited after Cs was successfully knocked down in UV-WSSV trained shrimp. Overall, these data indicate that TCA cycle plays important roles in the UV-WSSV-induced trained immunity by promoting H3K27ac modification via supply of the cofactor (Ac-CoA) of KAT8.

**Fig. 5: The TCA cycle is enhanced in UV-WSSV-trained shrimp.**

Identification of target genes regulated by H3K27ac modification

To reveal the underlying mechanisms mediated by the site-specific histone acetylation, the target genes directly regulated by H3K27ac modification were characterized through a chromatin immunoprecipitation sequencing (ChIP-seq) assay. The hemocytes from UV-WSSV- and PBS-trained shrimp were subjected to genome-wide ChIP-seq analysis. The results revealed that the H3K27ac signal intensity was greater in the transcription start site (TSS)-related region than in the control region (Fig. 6a). H3K27ac modification in the promoter region of genes increased after UV-WSSV training; most of the DNAs bound to H3K27ac were in intergenic regions, and 36.93% of the DNAs bound to H3K27ac were located within the promoter sequences in UV-WSSV-trained shrimp, while 29.96% was in the control (Fig. 6b). The promoter of 2361 genes bound to H3K27ac (Fig. 6c), indicating that the expression of these target genes was regulated by site-specific histone acetylation (H3K27ac). Gene Ontology (GO) functional analysis revealed that the enriched target genes were related to cellular components (nucleus, cytosol, cytoplasm and plasma membrane), cell migration and RNA transcription (Fig. 6d). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the target genes regulated by H3K27ac were enriched in cellular signaling pathways and metabolic pathways (Supplementary Fig. 6a), suggesting the regulatory roles of histone acetylation in trained immunity. A comparison of the gene expression of UV-WSSV trained- and control shrimp revealed that 2305 genes were upregulated in UV-WSSV-trained shrimp. To confirm the genes regulated by H3K27ac modification in UV-WSSV trained shrimp, integrative Genomics Viewer (IGV) tracks and ChIP-qPCR analysis were performed. The results revealed significant enrichment of H3K27ac signal peaks around the transcription start sites (TSSs) of casein kinase I (Ckl), eukaryotic translation initiation factor 4E (Eif4E), Ras-related protein 21 (Rab21), NF-κB signaling pathway related genes [ubiquitin-conjugating enzyme E2 A (UbE2A), inhibitor of nuclear factor kappa-B kinase (IKKβ) and transcription factor p65 (Dorsal)] in UV-WSSV-trained shrimp, and a significant difference in the H3K27ac peak was detected between the UV-WSSV-trained and PBS groups in IGV track analysis (Supplementary Fig. 6b–g). Same results were obtained in ChIP-qPCR assay (Fig. 6e–j). The results suggest that the above genes are the target genes of H3K27ac modification.

**Fig. 6: Identification of target genes directly regulated by H3K27ac modification.**

KAT8-mediated H3K27ac suppresses WSSV replication by enhancing expression of NF-κB-like transcription factor Dorsal in UV-WSSV trained shrimp

Humoral immunity is important for the resistance of invertebrates to pathogen infection and is achieved by a range of antiviral genes and antimicrobial peptides (AMPs) which mainly regulated by NF-κB signaling^34,35,36,37. To investigate whether genes related to the NF-κB signaling pathway are regulated by the H3K27ac modification mediated by KAT8, we performed ChIP-qPCR assays using H3K27ac antibody after knocked down Kat8 via RNAi, and found that the enrichments of H3K27ac in the promoter region of UbE2A, Ikkβ and Dorsal were significantly reduced in hemocytes of UV-WSSV trained shrimp (Fig. 7a–c).

**Fig. 7: UV-WSSV-trained shrimp exhibit enhanced Dorsal–Vago5/Amps axes against WSSV infection.**

To further validate the above findings, we examined the expression of NF-κB-like signaling components (UbE2A and Ikkβ), transcription factors (Dorsal and Relish), and Dorsal/Relish-regulated effector genes (Vago5, Ficolin, Amps) in Kat8-silenced shrimp. We found that the expression levels of UbE2A, Ikkβ, Dorsal, Vago5, Ficolin, and Amps, including Anti-lipopolysaccharide factor B1 (Alf-B1), Alf-C2, Alf-D2, Alf-E2, CrusI-1, CrusI-3, and CrusI-5, decreased significantly in the Kat8-silenced shrimp compared with those in the control group. However, there was no difference in the expression of Relish or Alf-C1 (Fig. 7d). Thus, Dorsal, the NF-κB-like transcription factor, was further studied. After knockdown of Dorsal by RNAi in shrimp, the expression of Vago5 and the effectors Ficolin, Amps including Alf-C1, Alf-D2, Alf-E2, CrusI-1, CrusI-3, and CrusI-5, was significantly reduced (Supplementary Fig. 7a, b). To verify whether Dorsal as well as Vago5, Ficolin and AMPs are involved in shrimp trained immunity, we detected the expression patterns of Dorsal, Vago5, Ficolin and Amps in the UV-WSSV trained model and found that Dorsal, Vago5, Ficolin, CrusI-5 (Fig. 7e–h), and Alf-D2, Alf-E2, CrusI-1, and CrusI-3 (Supplementary Fig. 7c–f) exhibited faster and stronger immune responses in the trained immunity of shrimp. After pretreatment with the MG149 inhibitor, the increased expression of Dorsal, Vago5, Ficolin and Amps was inhibited in the training model (Fig. 7i). These results suggest that KAT8-mediated H3K27ac participates antiviral trained immunity by regulating the Dorsal–Vago5–Ficolin and Dorsal–AMPs axes against WSSV in UV-WSSV trained shrimp.

KAT8-mediated H3K27ac promotes the expression of key enzymes Hk2, Pk and Ldh in glycolysis

In the ChIP-seq analysis, significant enrichment of H3K27ac signal peaks was detected around the TSSs of Hk2, Pk, Ldh, Pdh, Cs, Idh and Ogdh, and the peaks of Hk2, Pk and Ldh were significantly increased in UV-WSSV-trained shrimp (Fig. 8a–c). To confirm ChIP-seq results, a ChIP-qPCR assay was performed using H3K27ac antibody after Kat8-RNAi. The results indicated that the expression of Hk2, Pk and Ldh was upregulated in UV-WSSV-trained shrimp (Fig. 8d–f), but decreased following KAT8 knockdown (Fig. 8g–i). Thus, glycolysis key genes, Hk2, Pk and Ldh are also target genes of H3K27ac, suggesting a positive regulatory loop for the expression of the key enzyme-encoding genes in metabolic pathways in trained immunity. The expression of key enzymes involved in glycolysis was positively regulated in a feedforward manner in shrimp trained immunity.

**Fig. 8: KAT8-mediated H3K27ac modification upregulates *Hk2*, Pk, and *Ldh* in UV-WSSV-trained shrimp.**

Taken together, in UV-WSSV-induced trained immunity, glycolysis and the TCA cycle are activated and provide Ac-CoA to increase KAT8 mediated H3K27ac modification, which is deposited at the promoter regions of Dorsal and glycolysis enzymes to enhance gene transcription and metabolic pathways. As a transcription factor, Dorsal promotes the expression of Vago5 and Amps and inhibits the replication of WSSV in shrimp (Fig. 9).

**Fig. 9: Schematic representation of KAT8-mediated H3K27ac involvement in antiviral trained immunity in shrimp.**

Discussion

In the present study, using a model of trained immunity in shrimp induced by UV-WSSV, we demonstrated that UV-WSSV-induced trained immunity in shrimp resulted in a faster and stronger immune response against WSSV infection. UV-WSSV training in shrimp enhanced the glycolysis and TCA cycle metabolic pathways and increased Ac-CoA concentrations. Acting as a cofactor of the enzyme, Ac-CoA promoted the abundance of KAT8 mediated H3K27ac, which was deposited in the promoter region of Dorsal and increased the transcription of Dorsal to further upregulate the expression of the interferon-like cytokine Vago5 and AMP against WSSV infection in shrimp. Moreover, the key enzymes of glycolysis HK2, PK and LDH were also positively regulated by the H3K27ac modification, and further promoted the metabolic pathway in a feedforward manner. This is the first systematic study on the mechanism of invertebrate trained immunity.

The acetylation of histones (such as H3K27ac, H3K9ac, and H4K16ac) is associated with active transcriptional activity^{38,39,40,41,42}. KATs (also known as histone acetyltransferases, HATs) and histone deacetylases (HDACs) dynamically regulate these modifications¹⁸. H3K27ac modification is associated with enhanced immune responses in β-glucan-trained murine macrophages⁴³, but which KAT responsible for the modification is unclear. We performed RNAi to identify which the KATs and found that H3K27ac and H4K16ac modifications were significantly reduced after the knockdown of Kat8 and MG149 treatment. KAT8 (MOF or MYST1) is a member of the multiprotein complex primarily responsible for H4K16 acetylation, and it also acetylates nonhistone substrates such as p53 and IRF3^20,21. A recombinant human MOF also has histone acetyltransferase activity directed toward histones H2A, H3, and H4⁴⁴, but the specific acetylation sites of histones H2A and H3 are unknown. In the present study, we found that Kat8 expression was regulated by FOXO in shrimp infected with WSSV. KAT8 was distributed in the cytosol and nucleus and significantly increased in the nuclei of hemocytes and intestinal cells of shrimp challenged with WSSV. Knockdown of the KAT8 gene by RNAi reduced H3K27ac and H4K16ac modifications and promoted WSSV replication in the hemocytes and intestinal tissues of shrimp challenged with WSSV. KAT8 inhibitor MG149 injection produced the same results. However, in our trained immunity model, although the transcription level of Kat8 in hemocytes was significantly increased and the survival rate of UV-WSSV-trained shrimp was increased, the levels of H3K9ac and H4K16ac were not increased. These results suggest that UV-WSSV-induced trained immunity enhances H3K27ac modification and results in resistance to WSSV infection in shrimp. These results indicate that H3K27ac, mediated by KAT8, plays an important role in maintaining antiviral trained immunity in shrimp. Here, we also revealed that KAT8 is responsible for specific site modifications and that H3K27 acetylation and a feedforward loop for metabolic pathways exist in trained immunity.

KAT8 is a catalytic subunit of two independent protein complexes: MSL (male-specific lethal) and NSL (non-specific lethal), and plays a critical role in epigenetic regulation through the two complexes. It is generally believed that both MSL and NSL exert their activity via H4K16ac modification²². In our study, we found although KAT8 catalyzes H4K16ac and H3K27ac modifications, only the latter plays an important role in the trained immunity induced by UV-WSSV. Whether the modification is the complex-dependent need further study. To elucidate the antiviral mechanism and target genes of H3K27ac modification in UV-WSSV-trained shrimp, a genome-wide ChIP‑seq method was used. The results revealed that the H3K27ac peaks in the promoter regions of 2305 genes significantly increased, while 3170 genes significantly decreased in shrimp trained with UV-WSSV. Surprisingly, we found that an NF-κB signaling pathway related genes UbE2A, Ikkβ and Dorsal were the target genes of the H3K27ac modification. Kat8-RNAi and ChIP‒qPCR with H3K27ac antibody confirmed these results. Humoral immunity is important in invertebrate resistance to pathogen infection and is achieved by a series of AMPs and antiviral genes^34,45,46. Three main families of AMPs, i.e., penaeidins, crustins, and ALFs, have been identified in shrimp. These AMPs provide resistance to bacteria, fungi, and viruses^37,47. AMP expression is regulated mainly by the NF-κB (Toll/Drosal and IMD/Relish) signaling pathways in Drosophila⁴⁸. In this study, we detected the expression of NF-κB-like transcription factors, including Dorsal and Relish, and related interferon-like Vago5, as well as the downstream antiviral factor Ficolin, and the expression of related Amps in UV-WSSV-trained shrimp. The results revealed that UV-WSSV training promoted the expression of Dorsal and downstream Vago5, Ficolin, and associated Amps, including Alf-D2, Alf-E2, CrusI-1, CrusI-3, and CrusI-5, in the hemocytes of shrimp. Thus, KAT8 directly activates Dorsal expression by modifying the level of H3K27ac in the Dorsal promoter region, which subsequently promotes the expression of downstream Vago5, Ficolin and Amps to exert antiviral effects. Vago5, an interferon-like cytokine identified in shrimp, activates the JAK/STAT signaling pathway and induces the effector Ficolin through integrin to resist WSSV replication³⁴. The faster and stronger expression of Dorsal associated with Vago5 and Amps in the trained immunity of shrimp is responsible for the significantly higher survival rate of UV-WSSV-trained shrimp than of PBS-trained shrimp.

Metabolic reprogramming is also a key step in regulating trained immunity⁴⁹. To confirm the results, the metabolites and related enzymes in metabolic pathways were analyzed. We found that after training with UV-WSSV, the consumption of glucose and the production of LA significantly increased, indicating an enhanced glycolysis in shrimp. Subsequently, we detected the expression patterns of key genes involved in glycolysis, including Hk2, Pk, Ldh and Pdh, and found that the expression of the key genes of glycolysis was upregulated in UV-WSSV-trained shrimp. We subsequently observed that the key transcription factor Hif-1α of the mTOR signaling pathway enhances glycolysis by regulating the expression of Hk2 in UV-WSSV-trained shrimp. We also found that expression of genes encoding key enzymes in the TCA cycle were significantly increased after UV-WSSV training, and knocking down the TCA cycle key enzyme gene Cs significantly reduced the content of key enzymes Ogdh and Acly, and CA and Ac-CoA production as well as H3K27ac modification. Therefore, the TCA cycle is involved in UV-WSSV induced trained immunity. In the ChIP-seq analysis, genes encoding key enzymes involved in glycolysis (HK2, PK, LDH and PDH) and the TCA cycle (CS, IDH and OGDH) were identified as target genes of H3K27ac. In addition, Hk2, Pk and Ldh showed significant enrichment of H3K27ac signal peaks after UV-WSSV training. All in all, the enhancement of the glycolysis and TCA cycle metabolic pathways provides the acetyl group for H3K27ac modification and the energy for antiviral trained immunity.

Trained immunity mechanisms appear to have been conserved throughout the evolutionary process⁵⁰. Upon stimulation, the activation of gene transcription is accompanied by several metabolic pathways and epigenetic changes in histone epigenetic modifications at the promoters and enhancers of proinflammatory genes¹⁴. However, different stimuli can activate different trained immunity programs. Here, we found that UV-WSSV training promoted the glycolysis and TCA pathways and H3K27ac modification, ultimately promoted the expression of antiviral effectors, which is somewhat similar to β-glucan-induced trained immunity in monocytes/macrophages of mammals. It is reported that the PRRs involved in the initiation of trained immunity in mammals include dectin-1 for β-glucan, nucleotide-binding oligomerization domain 2 (NOD2) for Bacille Calmette-Guerin (BCG) and TLR4 for LPS⁵¹. However, the corresponding molecules of dectin-1 and NOD2 were not identified in shrimp. There might be different PRRs in invertebrates that induce trained immunity.

Previous research demonstrated that M. japonicus primed with formalin-inactivated WSSV exhibited significantly enhanced resistance when challenged at 10 days post-vaccination, though this protection was lost by 30 days post-priming⁸. In contrast, shrimp trained with low-dose live WSSV and challenged with a higher viral dose from 1 week to 3 months post-exposure showed significantly elevated survival rates during re-challenges conducted between 3 weeks and 2 months post-training. However, this protective effect dissipated by 3 months⁵². A comprehensive review analyzing immunological memory in cultured decapods following administration of live, inactivated, and subunit vaccines revealed that protective effects persist for 7–30 days in shrimp and crayfish⁵. Collectively, these studies document immunological memory phenotypes, whereas our findings establish the mechanism of trained immunity in invertebrates.

In conclusion, metabolic rewiring (activation of glycolysis and the TCA cycle) and epigenetic reprogramming (H3K27ac modification) are involved in UV-WSSV-induced antiviral trained immunity in shrimp (Fig. 9). This study provides evidence for the conservation of mechanisms of trained immunity in mammals and invertebrates. The results of this study reveal the mechanism of training immunity in shrimp and provide a basis for the development of antiviral vaccines for disease prevention and control in shrimp aquaculture.

Limitations of the study

In the process of trained immunity, inducers (such as pathogens and pathogen-associated molecular patterns) are generally recognized by PRRs and initiate a series of intracellular cascades that trigger the upregulation of metabolic pathways. The metabolites obtained from these pathways play crucial roles in the modulation of enzymes involved in epigenetic rewiring, which leads to an increase in transcriptional activation-associated marks (such as H3K27ac and H3K4me3), causing the transcription of proinflammatory genes against pathogens. In this study, we revealed that metabolic pathways and epigenetic modifications (H3K27ac) are involved in trained immunity and induce the transcription of NF-κB-like transcription factor to regulate antiviral effectors expression, but the PRRs of inactivated WSSV in trained immunity need to be identified.

Methods

Animals and virus

Healthy shrimp (M. japonicus, approximately 10 g each) obtained from the Aquatic Product Market of Qingdao, Shandong Province, China, were used in this study. The shrimp were raised in seawater. Prior to experimentation, M. japonicus were acclimated for more than 1 d in a temperature-controlled recirculating aquaculture system (RAS) under the following conditions: water temperature 20–22 °C, salinity 35‰ (Natural seawater), dissolved oxygen ≥5 mg/L (maintained via air stones). Shrimp were fed twice daily with commercial feed at 3%–5% of body weight. WSSV suspensions were prepared in sterile phosphate-buffered saline (PBS). Shrimp were artificially infected via intramuscular injection of purified WSSV³⁵.

Immune challenge and sample collection

Healthy shrimp were intramuscularly injected at the third abdominal segment, with approximately 1.0 × 10⁸ copies of WSSV. PBS-injected shrimp were used as controls. Shrimp hemolymph was collected from the ventral sinus into cold anticoagulant (0.45 M NaCl, 10 mM KCl, 10 mM EDTA, and 10 mM HEPES [pH 7.45]) with a syringe, followed by centrifugation at 800 × g for 8 min at 4 °C to obtain the hemocyte precipitate. Other shrimp tissues (heart, hepatopancreas, gills, stomach, and intestine) were collected simultaneously for the following experiments. The collected tissues were either used immediately or snap-frozen in liquid nitrogen and stored in an ultra-low temperature freezer (−80 °C) for further study.

Establishment of the trained immunity model

WSSV was inactivated by exposure to ultraviolet light for 30 min, and the shrimp trained immunity model was established⁹. The shrimp were divided into two groups. Each shrimp in the experimental group was injected with 50 μl of UV-WSSV (approximately 1.0 × 10⁵ copies), and the shrimp in the control group were injected with 50 µL of PBS. Both groups of shrimp were injected with WSSV (1.0 × 10⁷ copies in 50 µL PBS) 5 days after the first injection. Then, shrimp hemocytes and other tissues were collected at different time points for analysis.

RNA extraction and cDNA synthesis

Total RNA was extracted from hemocytes or ∼100 mg of tissue using TRIzol Reagent (Bioteke, Beijing, China), and the first strand of cDNA was synthesized via a cDNA Synthesis Kit (5x All-in-One RT MasterMix; Applied Biological Materials-abm, Vancouver, Canada) with 2 μg of total RNA according to the manufacturer’s instructions. Prior to reverse transcription, RNA purity was assessed by UV Spectrophotometry (A260/A280 ratio) and integrity was verified via agarose gel electrophoresis, and genomic DNA contamination was eliminated using DNase I.

Identification and analysis of lysine acetyltransferases (KATs) in shrimp

Shrimp KAT sequences in M. japonicus were obtained from both our transcriptome data and publicly available genome data. The transcriptome data were generated by MeRIP-seq using the following procedure: Gills were collected from the shrimp at 24 h post-injection with PBS or UV-WSSV. The samples were flash-frozen in liquid nitrogen and sent to Oebiotech (Shanghai, China) for sequencing. Total RNA was extracted using Trizol™ (Invitrogen, USA). Polyadenylated mRNA was then enriched from the total RNA using the Ambion™ Dynabeads™ mRNA Purification Kit. The enriched mRNA was fragmented and used as a template for cDNA synthesis. Sequencing was performed according to the manufacturer’s protocol on the Illumina NovaSeq™ 6000 platform (Lc-Bio Technologies Co., Ltd., Hangzhou, China). The Input control generated through MeRIP-seq for transcriptome dataset; which has been deposited in NCBI under accession number PRJNA1294137). Human KAT sequences (KAT2a, KAT7, KAT8, CBP/P300, MYST3, Tip60, ATAT, ESCO2 and HAT1) obtained from NCBI were used as query sequences for BLAST analyses. These analyses were performed against our local transcriptome dataset using local BLAST, and against the publicly available Penaeus japonicus genome sequence (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using BLAST. All identified M. japonicus KAT sequences are provided in Supplementary Data 3. Sequence alignments and phylogenetic trees of KATs from different species were constructed using MEGA 7.0 software (http://www.megasoftware.net/).

The sequence of open reading frame (ORF) of Kat8 was amplified via a pair of primers (Kat8-EX-F and Kat8-EX-R; Supplementary Table 1) for sequence confirmation. The corresponding cDNA was subsequently translated to obtain the protein sequence using the ExPASy translation tool (https://web.expasy.org/translate/). The molecular mass and isoelectric point of the peptide were determined using the online tool ExPASy (http://web.expasy.org/compute_pi/). The domain architecture prediction was performed with SMART.

Tissue distribution and expression profile analysis

Semiquantitative RT‒PCR was performed to study the tissue distribution of Kat8 mRNAs via the specific primers listed in Supplementary Table 1 (Kat8-RT-F and Kat8-RT-R). β-actin was used as the control with primers (β-actin-RT-F and β-actin-RT-R) (Supplementary Table 1). RT-PCR amplifications were performed in 50 μL reaction volumes using TransStart FastPfu DNA Polymerase (TransGen Biotech, Beijing, China) Each reaction contained: 1 μL cDNA template (synthesized from 200 ng RNA), 10 μL of 5 × TransStart FastPfu Buffer, 4 μL of 2.5 mM dNTPs (final concentration 0.2 mM), 1 μL of each 10 μM primer (final concentration 0.2 μM each), 1 μL of FastPfu DNA Polymerase (2.5 units), and nuclease-free water to a final volume of 50 μL. Thermal cycling conditions were as follows: 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 10 min; followed by a hold at 4 °C. PCR products were analyzed using 1.2% agarose gel electrophoresis.

Quantitative real-time RT‒PCR (qRT‒PCR, qPCR) analysis was performed in triplicate using SYBR Green PCR Master Mix (TaKaRa, Dalian, China) on a fluorescence quantitative PCR instrument (Analytik Jena, Germany) with gene-specific primers listed in Supplementary Table 1. qPCR amplifications were carried out in 10 μL reactions containing 1 μL cDNA template (synthesized from 100 ng RNA), 3.6 μL RNase-Free Water, 0.2 μL of each primer, and 5 μL qPCR SuperMix. Thermal cycling conditions were as follows: 95 °C for 10 min; 40 cycles of 95 °C for 10 s, 60 °C for 50 s, and 75 °C for 2 s, followed by a melting curve analysis from 68 °C to 95 °C with continuous fluorescence monitoring. PCR data were analyzed using the 2^−ΔΔCt method, and Ef-1α mRNA expression levels were used as internal references. Each experiment was performed in triplicate and analyzed using Student’s t test or one-way ANOVA.

RNA interference assay

To analyze KAT8 function, we performed RNAi assays. The Kat8 fragments were amplified with primers (Kat8-RI-F and Kat8-RI-R) linked to the T7 promoter (Supplementary Table 1). The double-stranded RNA fragments were synthesized using an in vitro T7 Transcription Kit (EP0111, Thermo Fisher Scientific, Waltham, MA, USA) with the DNA template. The dsRNA was synthesized in a 50 μL reaction mixture containing: 1 μg DNA template, 2.5 μL of each 25 mM NTP, 4 μL T7 RNA polymerase (50 U/μL), 2 μL RNase inhibitor (20 U/μL), 20 μL 5 × Transcription Buffer, and DEPC-treated water to a final volume of 50 μL. The reaction mixture was incubated at 37 °C for 5 h or overnight. The dsRNA products were purified by phenol-chloroform extraction prior to shrimp injection. Shrimp were then administered 50 μg of dsRNA via microsyringe injection into the abdominal hemocoel, followed by an identical second injection 24 h later. The control group was injected with an equal amount of control dsRNA. The RNA interference efficiency was examined via qPCR or western blotting at 24 h after the second injection. To test the function of KAT8 in virus infection, WSSV infection (1.0 × 10⁸ copies per shrimp) was performed at 24 h after the second dsRNA injection. The same method was used for RNAi of Foxo, Dorsal, Hif-1α and Cs. The expression levels of associated genes were then analyzed via qPCR with the primers listed in Supplementary Table 1. The qPCR conditions for Foxo, Dorsal, Hif-1α, and Cs were identical to those detailed in “Tissue distribution and expression profile analysis” section.

Recombinant expression and antibody preparation

For the recombinant expression of KAT8, a pair of primers (KAT8-EX-F and KAT8-EX-R) (Supplementary Table 1) was designed to amplify a fragment from the open reading frame (ORF) of KAT8. The PCR products were purified using a quick DNA purification kit (Sangon Biotech, Shanghai, China). The purified fragments were ligated into the pET-30a vector via a ClonExpress II One Step Cloning Kit (C112-01, Vazyme, Nanjing, China). The recombinant plasmids were subsequently transformed into E. coli Rosetta (DE3) cells. The expression of recombinant KAT8 was induced using 0.5 mM isopropyl-β-D-thiogalactopyranoside at 28 °C for 8 h. The recombinant protein was then purified⁵³ and used for antiserum preparation⁵⁴.

Protein extraction, SDS‒PAGE, and western blotting

Proteins from hemocytes and other organs were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (R0020, Solarbio, Beijing, China) supplemented with 20 nM phenylmethylsul fonylfluoride (PMSF) and a protease inhibitor cocktail (RM02916, ABclonal, Wuhan, China). The histones were extracted from hemocytes and the intestine according to the manufacturer’s protocol (BB31171, BestBio, Shanghai, China)⁵⁵.

The homogenized samples were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatants were obtained. Protein extracts were mixed with protein loading buffer (5X) for sodium dodecyl phosphate polyacrylamide gel electrophoresis (SDS‒PAGE) and heated at 100 °C for 10 min. Then, 30 μg of protein sample was separated by SDS‒PAGE. The proteins in the SDS‒PAGE gels were transferred to a nitrocellulose (NC) membrane using transfer buffer (25 mM Tris–HCl, 20 mM glycine, 0.037% mM SDS, and 20% ethanol) for 1 h. The NC membranes were incubated in 5% skim milk in TBST (150 mM NaCl, 10 mM Tris-HCl, pH 8.0) for 2 h. Then, they were incubated with the indicated primary antiserum: Anti-KAT8 with 1:100 dilution, anti-ACTB with 1:250 dilution, and anti-VP28 with 1:250 dilution, prepared in our laboratory; anti-histone-H3 polyclonal antibody (17168-1-AP, proteintech, Wuhan, China, 1:5000), anti-acetyl lysine antibody (ICP0380, Immunechem, 1:500), anti-acetyl-histone H3-K27 rabbit pAb (A7253, ABclonal, Wuhan, China, 1:5000), anti-acetyl-histone H3-K9 rabbit pAb (A7255, ABclonal, Wuhan, China, 1:5000), and anti-acetyl-histone H4-K16 rabbit pAb (PTM-122, PTM BIO, Hangzhou, China, 1:1000) and shaken gently overnight at 4 °C. After they were washed three times with TBST, the membranes were incubated with the secondary Ab (ZB2308 ZSGB-Bio, Beijing, China, 1:5,000) for 1 h at room temperature. The protein bands were visualized with enhanced chemiluminescence (ECL) (Tanon, Shanghai, China), using a chemiluminescence instrument (Tanon, Shanghai, China).

Inhibitor assay

The KAT8 inhibitor MG149 (HY-15887) was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and was dissolved in PBS. MG149 was injected in vivo at different concentrations in a volume of 50 μl (0, 1, 5, and 10 μM). The same volume of PBS was used as the control. Shrimp were first treated with MG149 for 6 h. Subsequently, the shrimp were stimulated with 1.0 × 10⁸ copies WSSV for 24 h. Hemocytes and intestinal samples were collected and detected via qPCR and western blotting.

To suppress glycolysis, the shrimp were intramuscularly injected with 100 μl of 36 mM 2-deoxy-D-glucose (2-DG) (M5140, AbMole, Houston, USA) for 24 h; to inhibit ACLY and thus Ac-CoA production and H3K27 acetylation, we treated shrimp with SB (TargetMol, Boston, USA) at a concentration of 100 M for 48 h, and the hemocytes of the shrimp were collected 12 h after UV-WSSV training for detection. PBS was used as a control.

Immunocytochemistry assay

To examine the subcellular localization of KAT8 in the hemocytes of shrimp after WSSV infection, we performed immunocytochemical assays³⁴. Shrimp were injected with WSSV (1.0 × 10⁸ copies), with PBS injected as the control. At 24 h after injection, the hemocytes were collected and analyzed.

Chromatin immunoprecipitation (ChIP)-seq

ChIP assays were performed by Shandong Xiuyue Biotechnology Co., Ltd., according to the standard crosslinking ChIP protocol with modifications⁵⁶. Briefly, hemocytes from UV-WSSV-trained shrimp were harvested and crosslinked with 1% formaldehyde for 10 min at room temperature, and PBS-trained shrimp were used as controls. After sonication, immunoprecipitation was performed with an anti-H3K27ac antibody (8173, Cell Signaling Technology, USA). The immunoprecipitated complex was washed, and the DNA was extracted and purified with a MinElute PCR Purification Kit (QIAGEN, #28006). The ChIP-Seq library was prepared using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, #TD501) according to the manufacturer’s instructions. For ChIP-seq, extracted DNA was ligated to specific adaptors, followed by deep sequencing on the Illumina NovaSeq 6000 platform via 150 bp paired ends.

ChIP-seq data analysis

Raw data (raw reads) in fastq format were first processed through in-house Perl scripts. In this step, clean reads were obtained by removing reads containing adapters, reads containing poly-N sequences and low-quality reads from the raw data. Moreover, the Q20, Q30 and GC contents of the clean data were calculated. All of the downstream analyses were based on high-quality, clean data. Clean reads were mapped to the reference genome using Bowtie2 software. Macs2 was used to call peaks with P < 0.05.

We analyzed the differentially accessible peaks in 3 steps. First, the peak files of each sample were merged via bedtools software. Second, the counts of the reads across the bed were determined for each sample using bedtools multicov. Finally, differentially accessible peaks were assessed via DESeq2. Regions were considered differentially accessible at P < 0.05.

The HOMER findMotifsGenome.pl tool was used for motif analysis. The input files were the peak file and the genome fasta file. The DNA sequence was extracted according to the peak file, and the sequence was compared with the Motif database to obtain the motifs. Peaks were annotated via the ChIPseeker package.

GO and KEGG enrichment analysis

Gene Ontology (GO) analysis was performed to elucidate the biological implications of unique genes in the significant or representative profiles of the genes in the experiment⁵⁷. We downloaded the GO annotations from NCBI, UniProt (http://www.uniprot.org/) and Gene Ontology (http://www.geneontology.org/). Fisher’s exact test was applied to identify the significant GO categories, and FDR was used to correct the p-values.

Pathway analysis was used to determine the significant pathways associated with the genes according to the KEGG database. We used Fisher’s exact test to select the significant pathway, and the threshold of significance was defined by the p-value and FDR⁵⁸.

ChIP‒qPCR assay

A ChIP assay was performed using a ChIP Assay Kit (Beyotime Biotechnology, Wuhan, China) according to the manufacturer’s instructions. Shrimp were trained with UV-WSSV (1.0 × 10⁵ copies), and PBS was used as the control. The hemocytes were collected 12 h post-UV-WSSV training and pooled for ChIP analysis. qPCR assays were used to detect enrichment of the DNA fragments in the Dorsal promoter sequence using DNA obtained from ChIP with the anti-H3K27ac antibody via Dorsal promoter region primers (Supplementary Table 1). IgG antibody was used as a control. The ChIP-qPCR assays for Ckl, Eif4E, Rab21, UbE2A, Iκκβ, Hk2, Pk and Ldh were performed with same method.

Survival rate analysis

The survival rates of Kat8-RNAi- and MG149-treated shrimp challenged with WSSV were determined. Shrimp were randomly divided into two groups (30 shrimp/group) with three replicates: the experimental group, which was injected with dsKat8/MG149, and the control group, which was injected with dsGfp/PBS. At 24 h after the injection of dsKat8/dsGfp or at 6 h after the injection of MG149/PBS, the shrimp were injected with WSSV (1.0 × 10⁷) for virus infection. The number of dead shrimp was counted every 12 h for 5 d or 6 d. Survival rates were analyzed by Kaplan‒Meier methodology using GraphPad Prism 8.0 software (San Diego, CA, USA).

Measurement of glucose, PA, LA, CA, and acetyl-CoA contents

To detect the contents of metabolites involved in glycolysis and the TCA cycle in UV-WSSV-trained shrimp, hemocytes were collected at different time points. The intracellular contents of glucose, PA, LA, CA, and acetyl-CoA were quantified via the Glucose Content Assay Kit (BC2500, Solarbio), PA Content Assay Kit (BC2200, Solarbio), LA Content Assay Kit (BC2230, Solarbio), CA Content Assay Kit (BC2155, Solarbio), and A-CoA (Acetyl Coenzyme A) ELISA Kit (E-EL-0125, Elabscience, Wuhan, China), respectively, according to the manufacturer’s instructions, with a microplate spectrophotometer at 505, 520, 575, 545, and 450 nm, respectively.

Statistics and reproducibility

Statistical analyses were performed using GraphPad Prism 8.0 software. Statistical significance was analyzed by Student’s t test for pairwise comparisons or one-way ANOVA for multiple comparisons. The results are presented as the mean ± standard deviation (SD) from three replicates, and a p-value less than 0.05 was considered statistically significant (ns, not significant). The western blotting bands were digitalized on the basis of three independent replicates with ImageJ software (National Institutes of Health, https://imagej.nih.gov/ij/download.html). All in vivo experiments included three independent biological replicates (n = 3 shrimp per replicate group) and technical replicates. For the survival assay, the survival curves are shown as Kaplan‒Meier plots with the log-rank test.

Ethics statement

All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care & Welfare Committee at the School of Life Sciences of Shandong University (SYDWLL-2021-53).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All of the data generated or analyzed during this study are included in this published article and its supplementary information files. The numerical source data for graphs and charts are available in Supplementary Data 1. ChIP-seq data presented in the study were deposited in NCBI GEO under the accession number GSE273689. The transcriptome data were deposited in NCBI SRA with accession number PRJNA1294137. All ChIP-Seq datasets generated for this study have been processed in detail and are provided as Supplementary Data 2. Uncropped and unedited blot/gel images (Supplementary Figs. 8–29) are provided in the Supplementary Information. Nine KAT sequences are provided as Supplementary Data 3. All other data are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Dr. Xiang-Mei Ren of the Core Facilities for Life and Environmental Sciences, State Key Laboratory of Microbial Technology of Shandong University for help with the microplate spectrophotometer technique, and Dr. Yiteng Xu from the Core Facility and Service Platform at School of Life Sciences, Shandong University for help in confocal microscopy imaging. This work was supported by grants from the National Natural Science Foundation of China (31930112) and Provincial Natural Science Foundation of Shandong (ZR2022QC232).

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Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, Shandong, China
Li-Xia Lv, Peng Zhang, Ying Ma, Yue Sun, Hao Wang, Xiao-Fan Zhao & Jin-Xing Wang
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, China
Jin-Xing Wang

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Li-Xia Lv
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Peng Zhang
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Ying Ma
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Yue Sun
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Hao Wang
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L.X.L., J.X.W. and X.F.Z. designed the research. J.X.W., X.F.Z. and P.Z. supervised all aspects of the research. L.X.L. performed most of the experiments. Y.M., Y.S. and H.W. helped to analyze data. P.Z. and Y.M. performed Western blot, and qPCR. L.X.L. and J.X.W. wrote and finalized the manuscript. All persons have made contributions to this work.

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Correspondence to Xiao-Fan Zhao or Jin-Xing Wang.

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Lv, LX., Zhang, P., Ma, Y. et al. Histone H3K27 acetylation mediated by KAT8 maintains antiviral trained immunity in shrimp induced by inactivated white spot syndrome virus. Commun Biol 8, 1314 (2025). https://doi.org/10.1038/s42003-025-08767-5

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Received: 12 February 2025
Accepted: 21 August 2025
Published: 30 August 2025
Version of record: 30 August 2025
DOI: https://doi.org/10.1038/s42003-025-08767-5