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. 2026 Aug 29;11:200312. doi: 10.1016/j.cirep.2026.200312

miR-182-5p and miR-750 regulate the immune response by modulating apoptosis, ROS level and esterase activity in white shrimp Litopenaeus vannamei after infection with Vibrio parahaemolyticus

Cairui Jiang a,b, Yongfeng Zou a,b, Junru Hu c, Jia Cai a,b, Xiaowen Zhu a,c, Hui Guo a,b,⁎
PMCID: PMC13572012

Highlights

  • •

    Lva-miR-182-5p and Lva-miR-750 were cloned and characterized from L. vannamei.

  • •

    Lva-miR-182-5pand Lva-miR-750 act as positive regulators of innate immunity, effectively mitigating hepatopancreatic histopathological damage.

  • •

    These miRNAs each enhance immune defense by increasing esterase activity, suppressing ROS levels, and reducing apoptosis in hemocytes.

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    Target prediction identifies C-type lectin 1 and TRAF6 as candidate targets of Lva-miR-750.

Keywords: MicroRNA, Litopenaeus vannamei, Lva-miR-182-5p, Lva-miR-750

Abstract

MicroRNAs (miRNAs) have been reported to play essential roles in inflammation and immune responses during pathogen infection. miR-182-5p and miR-750 have been shown to mitigate inflammation by regulating cytokine release in mammalian immune pathways. However, the regulatory mechanisms of these two miRNAs in the immune response of Litopenaeus vannamei remain unclear. In the present study, Lva-miR-182-5p and Lva-miR-750 from L. vannamei were cloned, and their tissue-specific expression patterns were quantitatively analyzed. After injection of Vibrio parahaemolyticus (1.0 × 10⁹ CFU/mL), the temporal expression dynamics of these two miRNAs in the hepatopancreas were examined. Furthermore, the alterations of histological structure in hepatopancreas, apoptosis rate, reactive oxygen species (ROS) level, and esterase activity in hemocytes were also detected after injection with miRNAs mimics and inhibitors in shrimp that infected with V. parahaemolyticus to elucidate their functional roles in immune response. Multiple sequence alignment revealed high conservation of both Lva-miR-182-5p and Lva-miR-750 across species. Their expression profiles, however, differed markedly: Lva-miR-182-5p was predominantly expressed in gills and hemocytes, whereas Lva-miR-750 showed the highest abundance in the stomach and muscle. Following infection with V. parahaemolyticus, both miRNAs were significantly up-regulated in the hepatopancreas. Histological analysis revealed that mimics-treated groups maintained an intact hepatopancreatic structure with normal nuclei. In contrast, the inhibitors-treated group exhibited tissue edema along with nuclear enlargement and fragmentation. At the cellular level, under V.parahaemolyticus infection, ROS level and the apoptosis rate were significantly increased, while esterase activity were decreased in hemocytes from the Lva-miR-182-5p inhibitors-treated group compared to the Vibrio-infected PBS control. In contrast, the Lva-miR-182-5p mimics-treated group showed reduced ROS level and apoptosis rate, along with enhanced esteraes activity. Similarly, Lva-miR-750 inhibitors-treated group exhibited a significant increase in apoptosis rate and decrease in esterase activity, whereas the Lva-miR-750 mimics-treated group showed enhanced esterase activity and reduced ROS level. Collectively, these findings indicated that Lva-miR-182-5p and Lva-miR-750 function as positive regulators of innate immunity in L. vannamei against V. parahaemolyticus. They appear to mitigate hepatopancreatic damage and enhance immune defenses by reducing the apoptosis rate, modulating ROS levels, and enhancing esterase activity, thereby elucidating their key regulatory roles in the crustacean immune response.

Introduction

As the most widely farmed shrimp species globally, the stable development of Litopenaeus vannamei aquaculture is essential for food security and economic sustainability [1,2]. However, intensive farming practices are vulnerable to disease outbreaks, with acute hepatopancreatic necrosis disease (AHPND), caused by V. parahaemolyticus, emerging as one of the most devastating threats [3]. V. parahaemolyticus carries the pVA1 plasmid, encoding the virulence proteins PirA and PirB, which specifically target the shrimp hepatopancreas and induce extensive apoptosis and necrosis of tubular epithelial cells. This pathogenesis results in high shrimp mortality rates and severe economic losses [4,5]. Although antibiotics have long been used to control AHPND, their prolonged and indiscriminate use has resulted in the emergence and dissemination of multidrug-resistant (MDR) V. parahaemolyticus [6,7]. In response to this challenge, current research is pivoting toward sustainable strategies that target the regulation of shrimp innate immunity as novel therapeutic avenues.

MicroRNAs (miRNAs) are single-stranded RNA molecules consisting of 19–25 nucleotides that play critical regulatory roles in cell differentiation, proliferation, apoptosis, and immune homeostasis [8]. They function primarily by negatively regulating immune-related target genes, thereby modulating key immune signaling pathways, such as apoptosis and inflammation [9]. Such regulatory activity influences the intensity of host immune responses and associated tissue pathology [10,11]. For example, the expression of specific miRNAs in Strongylocentrotus intermedius wassignificantly changed after infection with Vibrio harveyi [12]. Similarly, 55 differentially expressed miRNAs associated with shrimp innate immunity were identified in Marsupenaeus japonicus following Vibrio alginolyticus infection [13]. Furthermore, functional analyses revealed that miR-205-5p and miR-2188-3p regulate apoptosis and anti-inflammatory responses in Ctenopharyngodon Idella infected with Aeromonas hydrophila-susceptible SG [14]. miRNAs are widely recognized as key regulators of immune cell development and immune responses [15,16]. Notably, miR-182 and miR-750 exhibit significant regulatory activity during pathogen infection. For instance, after stimulation by flagellin from V. parahaemolyticus, miR-182-3p was significantly up-regulated in grouper spleen (GS) cells of Epinephelus coioides, where it post-transcriptionally regulating specific EST sequences and thereby inhibiting pro-inflammatory cytokines [17]. Similarly, miR-182-5p has been shown to suppress inflammation and attenuate acute kidney injury (AKI) by regulating dual-specificity phosphatase 1 (DUSP1) [18]. Conversely, inhibition of miR-182-5p in mice was associated with increased asthma severity and airway inflammation [19]. Additionally, administration of miR-182-5p mimics has been shown to inhibit the growth of clear cell renal cell carcinoma (ccRCC) tumors [20]. The immunoregulatory role of miR-750 was further demonstrated in Laodelphax striatellus, where miR-750-3p was significantly down-regulated following infection with rice black-streaked dwarf virus (RBSDV), whereas injection of its mimics significantly reduced viral accumulation [21].

To fundamentally improve shrimp immunity and mitigate disease threats, Lva-miR-182-5p and Lva-miR-750 were selected to study the immune function in shrimp based on the miRNA library we constructed previously. In the current study, we cloned these two miRNAs, analyzed their sequence characteristics and expression patterns. Additionally, we assessed the effects of these miRNAs mimics and inhibitors on histological features, apoptosis rate, ROS level, and esterase activity in hemocytes. This study elucidated the role of Lva-miR-182-5p and Lva-miR-750 in the shrimp immune response against to V. parahaemolyticus, provided a theoretical foundation for understanding miRNA-mediated immune regulation in crustaceans.

Materials and methods

Animals

The experimental shrimp L. vannamei were obtained from an aquaculture facility in Zhanjiang City, Guangdong Province, China. The shrimp were acclimated in cycling-filtered plastic tanks for 2 weeks before the experiment. During the acclimation period, the salinity was maintained as 24, water temperature 26–29 °C, pH 7.9-8.0 and dissolved oxygen (DO) 5.50-6.18 mg/L. Shrimp were fed twice daily with a commercial diet (40% protein, 5.0% fat, 5.0% fibre and 16% ash, supplied by a commercial diet, China) until 24 h before the experimental treatments began. Approximately 50% of the tank water was replaced daily to maintain optimal water quality.

Clone of miRNAs

Sample collection

Hemolymph was collected using a syringe previously rinsed with ACD anticoagulant solution (containing 0.48 g citric acid, 1.32 g sodium citrate, and 1.47 g glucose, diluted to 100 mL with double-distilled water and sterilized by filtration). The hemolymph sample was centrifuged at 6000 r/min for 5 min at 4 °C, using a refrigerated high-speed centrifuge. After centrifugation, the supernatant was carefully removed, and 500 μL of TRIzol reagent was added to the pellet. The mixture was gently pipetted to ensure homogeneity and immediately stored at −80 °C. Subsequently, eight shrimp tissues (eye-stalks, gills, heart, hepatopancreas, stomach, intestine, muscle and hemocytes) were rapidly excised. To minimize individual variation, three biological replicates were set up for each tissue type, with each replicate consisting of a pooled sample derived from three individual shrimp. The pooled tissues were immediately placed in 2 mL cryotubes, flash-frozn in liquid nitrogen, and stored at −80 °C for long-term preservation.

RNA isolation, cDNA synthesis and PCR amplification

Total RNA was extracted using the TRLzol method. The isolated RNA was then reverse transcribed into cDNA with the TAKARA Reverse Transcription Kit (RR047A) according to the manufacturer's protocol. Based on the sequences of miR-750 and miR-182-5p mature sequences identified through high-throughput sequencing, the miRNAs stem-loop primers (RT primers) were designed for PCR amplification using DNAMAN 7.0 software. PCR amplification was performed by 10-fold dilution of hepatopancreas cDNA stock solution. The PCR amplification was set up according to the following programme: an initial denaturation at 98 °C for 2 min; followed by 40 cycles of denaturation at 98 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, with a final extension at 72 °C for 1 min.

Lva-miR-182-5p and Lva-miR-750 sequence analysis

The PCR product was aspirated, and its length was confirmed by electrophoresis on a 3% agarose gel.

The remaining product was sequenced, and multiple sequence alignment was carried out using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) to compare Lva-miR-182-5p and Lva-miR-750 similarity to others sequence. The 5’-seed (nucleotides 2-8) and anchor (nucleotides 13-16) sequences of the mature miR-182-5p and miR-750 were identified and compared.

Tissue expression of Lva-miR-182-5p and Lva-miR-750

Nine healthy adult L. vannamei were taken and temporarily reared in seawater for one week. Sample collection, total RNA extraction and cDNA synthesis were performed according to 2.2.1 and 2.2.2, respectively. The SYBR Green Realtime PCR Master Mix was thawed at 4 °C before the start of the experiment. The qPCR data were collected by the Light Cycler 96 system and calculated by 2−ΔΔct method.

Differential expression of Lva-miR-182-5p and Lva-miR-750 in response to V. parahaemolyticus

Vibrio virulence assays revealed that shrimp injected with V. parahaemolyticus at a concentration of 1.0 × 109CFU/mL exhibited a cumulative mortality rate of 44 % at 24 h post-injection (hpi). Therefore, 126 healthy L. vannamei were randomly allocated into two groups: the Vibrio-infected experimental group and the PBS-control group. Shrimp in the Vibrio-infected group were injected with 100 μL of V. parahaemolyticus at 1.0 × 109 CFU/mL, while those in the control group were injected with the same volume of sterile phosphate-buffered (PBS, pH 7.4). Hepatopancreas samples were collected from both groups at 0, 1.5, 3, 6, 12, 24, and 48 h post-infection (hpi). Total RNA extraction and cDNA synthesis were performed as described in Section 2.2.2. Hepatopancreatic cDNA from each sampling point was subjected to quantitative real-time PCR (qPCR). RNA isolation and cDNA synthesis followed procedures described in Section 2.2.2. The PCR amplification program was as follows: initial denaturation; followed by 40 cycles of, denaturation at 98 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min, cooling at 37 °C. A blank control without a reverse primer was also prepared.

Histological observations after injection of miRNA mimics and inhibitors

The mimics and inhibitors of Lva-miR-182-5p and Lva-miR-750 were synthesized by Sangon Biotech (Shanghai, China) and diluted to 1 ng/μL using 1 × RNase-free PBS. The solution was then stored at -80 °C for future use.

Under the stimulation of V. parahaemolyticus at 1.0 × 109 CFU/mL concentration, shrimp were injected with 30 ng mimics and inhibitors for immune stimulation. Specifically, Lva-miR-182-5p mimics, Lva-miR-182-5p inhibitors, Lva-miR-750 mimics, and Lva-miR-750 inhibitors were separately injected into distinct experimental groups. Collected gill samples and hepatopancreas samples were fixed in 4 % paraformaldehyde (PFA), followed by ethanol dehydration, xylene transparency, and paraffin embedding. Subsequently, the samples were sliced and stained with 4’,6- diamidino-2-phenylindole (DAPI). DAPI staining was chosen because it specifically binds to DNA, allowing clear visualization of nuclear morphological changes, such as condensation fragmentation, and enlargement, which are hallmark features of apoptosis [22,23]. Histopathological examination was performed under an optical microscope.

Determination of apoptosis rate, ROS level and esterase activity

The sample treatment was the same for the three indices. The shrimp injected with 100 μL of V. parahaemolyticus at a concentration of 1.0 × 109 CFU/mL were divided into five groups (n = 15 per group): PBS-control+Vibrio, Lva-miR-182-5p mimics, Lva-miR-182-5p inhibitors, Lva-miR-750 mimics, Lva-miR-750 inhibitors. The PBS-control group was injected with 30 μL of PBS, while the remaining four groups were each injected with 30 ng of their respective mimics or inhibitors. Hemolymph was collected 6 hpi using a syring rinsed with ACD anticoagulant and placed into a pre-chilled, sterile 1.5-mL microcentrifuge tube, which was then stored on ice until processing.

The Annexin V fluorescein isothiocyanate (FITC) / propidium iodide (PI) apoptosis detection kit (Invitrogen) was used to measure the apoptotic cell ratio of the hemocytes in L. vannamei. The apoptosis rate was calculated using the following formula: Apoptosis rate = (FITC channel fluorescence value + PI channel fluorescence value)/10000 × 100%. The 2’,7’- dichlorofluorescein diacetate (DCFH-DA) was diluted 10-fold, after which 2 μL of the resultant solution was added to the hemocytes and incubated for 20 min on ice protected from light. A flow cytometer was used to record the fluorescence from the hemocytes. The mean fluorescence of DCFH-DA was used to calculate ROS level. The FDA powder was dissolved in dimethyl sulfoxide to a concentration of 5 mg/mL, then diluted 10-fold, after which 2 μL of the resultant solution was added to the hemocytes and incubated for 20 min on ice protected from light. A flow cytometer was used to record the fluorescence from the hemocytes. The mean fluorescence of FDA was used to calculate esterase activity.

Statistical analysis

Flow cytometry data were collected using the BD FACSVerse™ system. qPCR data were collected using LightCycler® system (480 Ⅱ or 96), and relative gene expression was calculated via the 2-ΔΔct method. All data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test using SPSS 18.0 software. The results are presented as the mean±standard deviation (SD). Differences were considered statistically at p < 0.05. All graphs were generated using GraphPad Prism 10 software.

Results

Sequence analysis of Lva-miR-182-5p and Lva-miR-750

The sequences of Lva-miR-182-5p and Lva-miR-750 were aligned with miR-182-5p and miR-750 sequences from other species using Clustal Omega for multiple sequence alignment (Fig. 1). Although sequence lengths varied (miR-182-5p: 22–25 nucleotides, miR-750: 22–23 nucleotides), the mature forms of both miRNAs were highly conserved, particularly in the seed sequences (nucleotides 2–8) and anchor sequences (nucleotides 13–17 for miR-182-5p and nucleotides 19–21 for miR-750). The alignment results indicated that Lva-miR-182-5p and Lpa-miR-182-5p were identical, and Lva-miR-750 shared complete sequence identity with miR-750 from multiple species, suggesting functional conservation of miR-182-5p and miR-750 in shrimp.

Fig. 1.

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Sequence alignment of miR-182-5p and miR-750 family members across different species. (A) Multiple sequence alignment of miR-182-5p (hsa:Homo sapiens; dre:Danio rerio; sja:Siniperca chuatsi; rno:Rattus norvegicus; lva: L. vannamei; pol:Paralichthys olivaceus; lpa:Lepisosteus platostomus; gga:Gallus gallus; mmu:Mus musculus; cin:Ciona intestinalis; sko:Saccoglossus kowalevskii); (B) Multiple sequence alignment of miR-750 (bma:Brugia malayi; mse:Manduca sexta; tcf:Triops cancriformis; lgi:Lottia gigantea; isc:Ixodes scapularis; mle:Melibe leonina; pca:Polistes canadensis; dqu:Dinoponera quadriceps).

Tissue-specific expression patterns of Lva-miR-182-5p and Lva-miR-750

The tissue-specific expression patterns of Lva-miR-182-5p and Lva-miR-750 in L. vannamei were delineated using the LightCycler 96 Quantification System across a panel of eight tissues: eye-stalk, gill, heart, hepatopancreas, stomach, intestine, muscle, and hemocytes. Lva-miR-182-5p was found to be predominantly expressed in the gills, with its lowest abundance observed in muscle tissue. In contrast, Lva-miR-750 showed the highest expression level in the stomach, while the lowest expression was detected in the gills, followed closely by that in the muscle (Fig. 2).

Fig. 2.

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Tissue-specific distribution of (A)Lva-miR-182-5p and (B)Lva-miR-750 in L. vannamei. (** indicates P<0.01).

Expression changes of Lva-miR-182-5p and Lva-miR-750 in response to V.parahaemolyticus

Following infection with V. parahaemolyticus (1.0 × 109 CFU/mL), the expression levels of miRNAs in the hepatopancreas were significantly up-regulated. Specifically, Lva-miR-182-5p expression began to increase at 3 hpi, peaked at 48 hpi at a level 6.44-fold higher than the PBS-control group. Similarly, Lva-miR-750 was up-regulated as early as 1.5 hpi, reaching its maximum (at 5.38-fold increase relative to the PBS (at 5.38-fold increase relative to the PBS-control group) at 24 hpi (Fig. 3).

Fig. 3.

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Expression profiles of (A) Lva-miR-182-5p and (B) Lva-miR-750 in the hepatopancreas of L. vannamei following V. parahaemolyticus infection (** indicates P < 0.01).

Histological observation of hepatopancreas after injection of miRNA mimics and inhibitors

Histological examination of the hepatopancreas revealed marked pathological changes in the miRNA inhibitors-treated + Vibrio and PBS-control groups, including noticeable ulceration, hydropic swelling, and extensive nuclear fragmentation. These abnormalities were more severe in the miRNA inhibitors-treated + Vibrio group compared to the PBS-control group. In contrast, the hepatopancreas in the miRNA mimics-treated + Vibrio and Vibrio + PBS-control groups exhibited no significant enlargement and their cellular nuclei remained intact (Fig. 4). Collectively, these results demonstrated that the miRNA mimics effectively protect hepatopancreatic tissue integrity in shrimp.

Fig. 4.

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Histological evaluation of hepatopancreas after miRNA modulation. (A) DAPI staining of hepatopancreas sections (a: Vibrio + PBS-control group, b: PBS-control group, c: Vibrio + mimics-treated group, d: Vibrio + inhibitors-treated group); (B) Hepatopancreas morphology after injection of mimics and inhibitors (a: Vibrio + PBS-control group, b: PBS-control group, c: Vibrio + mimics-treated group, d: Vibrio + inhibitors-treated group).

Effects of Lva-miR-182-5p and Lva-miR-750 mimics and inhibitors on apoptosis rate, ROS level, and esterase activity

To investigate the functional roles of Lva-miR-182-5p and Lva-miR-750, we used flow cytometry to analyze hemocytes following injection of 30 ng of miRNA mimics or inhibitors. As shown in Fig. 5A and B, the apoptosis rates in both the Lva-miR-182-5p and Lva-miR-750 inhibitors-treated groups were significantly higher than those in the control group, accompanied by prominent PI absorption peaks. Both miRNAs mimics significantly reduced hemocyte apoptosis rate compared to the PBS-control group, whereas their inhibitors markedly increased apoptosis rate (Fig. 5C). Analysis of intracellular ROS revealed that miRNA inhibitors enhanced ROS level. Specifically, inhibitors of both Lva-miR-182-5p and Lva-miR-750 significantly elevated ROS levels relative to the PBS-control group. Conversely, treatment with their corresponding mimics reduced ROS levels, with Lva-miR-750 mimics decreasing ROS to a level 58% lower than that of the Vibrio-infected group (Fig. 6A). Furthermore, we assessed esterase activity, a marker of cellular immune function. Injection of miRNAs mimics led to a notable increase in esterase activity compared to the Vibrio-infected group, while the inhibitors significantly suppressed it (Fig. 6B). These results collectively demonstrated that Lva-miR-182-5p and Lva-miR-750 positively regulate hemocytes immunity by inhibiting apoptosis rate and ROS level while enhancing esterase activity.

Fig. 5.

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Annexin V/PI double-staining flow cytometry analysis of hemocyte apoptosis. (A) Apoptosis of hemocyte in Lva-miR-182-5p mimics and inhibitors injections; (B) Apoptosis of hemocyte in Lva-miR-750 mimic and inhibitor injections; (C) Hemocyte apoptosis rate after mimic and inhibitor injection (* Indicates P < 0.05, *** indicates P < 0.001, ns indicates no significant difference).

Fig. 6.

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Immune related functional changes in hemocytes of L. vannamei following injection of Lva-miR-182-5p and Lva-miR-750 mimics and inhibitors. (A) ROS level. (B) Esterase activity. (* Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, ns indicates no significant difference).

Discussion

To date, research on the regulatory roles of miRNAs in the immune signaling pathways and inflammatory responses of L. vannamei following pathogenic or abiotic stimulation remains limited. In this study, Lva-miR-182-5p and Lva-miR-750 were identified from miRNA transcriptome database using stem-loop RT-PCR and Illumina Genome Analyzer sequencing. Multiple sequence alignments revealed that the mature sequences of these two miRNAs differ by only one or two nucleotides from their homologs in other species, with completely conserved seed regions, indicating a high degree of evolutionary conservation. Analysis of their tissue distriution showed that Lva-miR-182-5p and Lva-miR-750 were ubiquitously expressed but exhibited distinct patterns. Lva-miR-182-5p exhibited the highest expression levels in eye-stalks, gills, and hemocytes, whereas Lva-miR-750 was predominantly expressed in the stomach and muscle. Notably, hemocytes, stomach, hepatopancreas, and gills are vital organs involved in shrimp nonspecific immunity [24]. It is well established that crustaceans lack a complex adaptive immune system and rely primarily on innate immunity to defend against pathogens [[25], [26], [27]]. Within this innate immune system, the hepatopancreas function as a central immune organ [[28], [29], [30]], while hemocytes serve as primary immune effector cells [[31], [32], [33]]. Therefore, although Lva-miR-182-5p and Lva-miR-750 exhibited higher basal expression in the gills and stomach respectively, under healthy conditions, both miRNAs were expressed at moderate levels in the hepatopancreas and were significantly upregulated upon V. parahaemolyticus infection (6.44-fold for Lva-miR-182-5p and 5.38-fold for Lva-miR-750). Given that the hepatopancreas is a core immune organ and the primary target of AHPND [3]. We hypothesize that it serves as a central hub for miRNA-mediated immune responses under pathogenic stimulation—a notion supported by previous findings that L. vannamei combats V. parahaemolyticus via downregulation of Lva-miR-4850 in the hepatopancreas, activating target genes such as proPO2 and PPAF2 [34]. Accordingly, the hepatopancreas was selected for subsequent functional studies. Despite significant progress has been made in understanding miRNA function in terrestrial model organisms, research on their role in crustaceans remains relatively limited. In shrimp aquaculture, Vibrio infections-characterized by symptoms such as empty stomachs and pale hepatopancreas-pose a major threat to production [35,36]. In the present study, experimental infection of L. vannamei with V. parahaemolyticus via ventral sinus injection led to significant upregulation of both Lva-miR-182-5p and Lva-miR-750. Their expression increased as early as 3 hpi with Lva-miR-182-5p peaking at 48 hpi and Lva-miR-750 at 24 hpi. A similar pattern of early immune activation has been observed in the crab Eriocheir sinensis, where V. parahaemolyticus infection for just1 h increased phosphorylation levels of EsHpo, a key component of the Hippo signaling pathway, suggesting the existence of conserved immune regulatory mechanisms across crustaceans [37]. In contrast, a previous study reported that miR-750 levels were markedly decreased in hemocytes of shrimp infected with white spot syndrome virus (WSSV), a discrepancy that may be attributed to pathogen-specific responses or tissue-specific expression patterns [38]. Histopathological examination further demonstrated critical roles of Lva-miR-182-5p and Lva-miR-750 in modulating immune responses. Following V. parahaemolyticus challenge, inhibition of either miRNA significantly aggravated pathological alterations in the hepatopancreas, including extensive tissue ulceration and nuclear fragmentation. Conversely, miRNA mimics preserved hepatopancreatic histological architecture and nuclear integrity. These result suggested that Lva-miR-182-5p and Lva-miR-750 maintained tissue structure and mitigate damage during bacterial infection. The severe tissue damage observed upon miRNA inhibition prompted us to investigate the underlying cellular dysfunctions.

Given the established role of miRNAs in regulating oxidative stress and apoptosis-two processes critical for cell survival, Lva-miR-182-5p and Lva-miR-750 may function as stress regulators involved in environmental adaptation in shrimp. Numerous studies have documented the intricate relationship between oxidative stress and apoptosis. ROS are not merely toxic byproducts but also participate in various physiological and immunological processes, including the regulation of autophagy, apoptosis induction, protein damage, and promotion of cell death [39,40]. For example, overexpression of miR-200 indueced beta-cell apoptosis in mice, whereas suppression of the miR-183/96/182 cluster triggered ROS accumulation, activating the ROS-mediated p53/apoptosis signaling pathway, ultimately leading to apoptosis [41]. Consistent with the pro-apoptotic role of miR-183/96/182 cluster suppression, inhibition of Lva-miR-182-5p significantly increased ROS levels and apoptosis rates in hemocytes, accompanied by nuclear fragmentation in hepatopancreatic cells. Furthermore, injection of Lva-miR-182-5p and Lva-miR-750 inhibitors significantly reduced esterase activity. Previous studies indicated that carboxylesterases, a key class of hydrolases within esterases [42], play a crucial role in regulating immunity in aquatic organisms by hydrolyzing pathogen-associated lipids [43,44]. It had been demonstrated that inhibition of carboxylesterase activity leads to elevated ROS and hydroxyl radical (·OH) levels, subsequently inducing oxidative stress and inflammatory damage [45]. Consistent with this, our finding showed that inhibition of Lva-miR-182-5p or Lva-miR-750 impaired esterase activity, elevated ROS levels, and induced apoptosis. Given that previous studies have mechanistically linked these phenotypes, we propose a coherent hypothesis, termed the "Esterase-ROS-Apoptosis" hypothesis. In this hypothesis, Lva-miR-182-5p and Lva-miR-750 acted as upstream regulators that maintained esterase activity, thereby facilitating the elimination of toxic lipids. Thus, the suppression of these miRNAs led to esterase dysfunction, which in turn induced oxidative stress—a primary activator of the intrinsic apoptosis pathway. Consequently, the observed elevation in apoptosis was a direct outcome of this esterase dysfunction-induced oxidative stress.

To further explore the downstream regulatory network of these two miRNAs, we performed a preliminary bioinformatics prediction of their target genes using miRanda and RNAhybrid [46], retaining only genes predicted by both tools as high-confidence candidates (miRanda score > 150 and RNAhybrid MFE < -20 kcal/mol). For Lva-miR-750, the high-confidence target genes included C-type lectin 1 and tumor necrosis factor receptor-associated factor 6 (TRAF6). These targets are functionally consistent with the phenotypic changes induced by Lva-miR-750 observed in our study. For instance, C-type lectins are well-known pattern recognition receptors (PRRs) that initiate innate immune responses against bacterial pathogens in crustaceans [47]. TRAF6 is a key adaptor in the Toll/NF-κB signaling pathway, and previous studies have shown that miRNAs can inhibit inflammation by targeting TRAF6 [48]. In contrast to the well-characterized targets of Lva-miR-750, target gene prediction for Lva-miR-182-5p yielded mostly uncharacterized transcripts, precluding reliable functional assignment. Nevertheless, based on the inhibition of apoptosis and reduction of ROS levels phenotype observed in this study, and drawing from previous reports in mammalian systems where miR-182-5p directly targets caspase and genes other associated with apoptosis [49], we speculate that Lva-miR-182-5p may similarly regulate conserved apoptotic pathways in L. vannamei. This hypothesis warrants direct experimental validation in future studies.

Collectively, our findings delineated a critical role for Lva-miR-182-5p and Lva-miR-750 in shrimp innate immunity. These miRNAs orchestrated an integrated defense by enhancing esterase activity, which in turn suppressed ROS-mediated apoptosis. This study thereby proposing a novel miRNA-mediated regulatory hypothesis that fortifies hepatopancreatic integrity against V. parahaemolyticus infection.

Conclusion

In conclusion, this study demonstrated that Lva-miR-182-5p and Lva-miR-750 acted as positive immune regulators in L. vannamei during V. parahaemolyticus infection.These miRNAs mitigated Vibrio-induced hepatopancreatic injury by enhancing esterase activity, which in turn reduced excessive ROS accumulation, thereby suppressing hemocyte apoptosis. The findings provided new insights into the regulatory role of miRNAs in crustacean immunity and highlighted their potential as molecular targets for the development of novel strategies to control AHPND.

CRediT authorship contribution statement

Cairui Jiang: Writing – review & editing, Writing – original draft, Visualization, Supervision, Software, Resources, Investigation, Data curation, Conceptualization. Yongfeng Zou: Supervision, Resources, Project administration, Methodology, Funding acquisition. Junru Hu: Writing – original draft, Visualization, Validation, Data curation. Jia Cai: Visualization, Validation, Project administration. Xiaowen Zhu: Supervision, Resources, Methodology, Investigation. Hui Guo: Writing – review & editing, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was funded by the Department of education of Guangdong Province (2026CXTD27), the Guangdong Basic and Applied Basic Research Foundation (2025A1515010949), the Open Fund of Tianjin Key Laboratory of Aquatic Ecology and Aquaculture (No. TJAE201506). We sincerely appreciate the valuable comments from the anonymous reviewers.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cirep.2026.200312.

Appendix. Supplementary materials

mmc1.docx (11.3KB, docx)

Data availability

Data will be made available on request.

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