Gonadal miRNomes and transcriptomes in infected fish reveal sexually dimorphic patterns of the immune response

doi:10.21203/rs.3.rs-5306100/v1

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Gonadal miRNomes and transcriptomes in infected fish reveal sexually dimorphic patterns of the immune response

https://doi.org/10.21203/rs.3.rs-5306100/v1

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Background

Fish disease outbreaks caused by bacterial burdens are responsible for decreasing productivity in aquaculture. Unraveling the molecular mechanisms activated in the gonads after infections is pivotal for enhancing husbandry techniques in fish farms, ensuring disease management, and selecting the most resistant phenotype.

Methods

Here, an experiment with European sea bass (Dicentrarchus labrax), an important commercial species in Europe, was conducted to study the miRNome and transcriptome through sequencing analysis 48 hours after an intraperitoneal infection with Vibrio anguillarum.

Results

The findings indicate that following infection, testes exhibited more pronounced alterations in both the miRNome and transcriptome. Specifically, males showed approximately 26% more differentially expressed genes in testicular genes compared to females (2,624 vs. 101 DEGs). Additionally, four miRNAs (miR-183-5p, miR-191-3p, miR-451-5p, and miR-724-5p) were significantly expressed post-infection in males, while none were identified in females. Interestingly, upon deep analysis of sexual dimorphic gene modules, a larger number of miRNAs were identified in infected females targeting genes related to the immune system compared to infected males. These results suggest that fish ovaries demonstrate greater resilience in response to infections by suppressing genes related to the immune system through a post-transcriptional mechanism performed by miRNAs. In contrast, testes activate genes related to the immune system and repress genes related to cellular processes to cope with the infection. In particular, the crosstalk between the miRNome and transcriptome in infected males revealed a pivotal gene, namely, insulin-like growth factor binding protein (igfbp), acting as a gene network hub in which miR-192-3p was connected.

Conclusions

The current study elucidated the need to comprehend the basic immune regulatory responses associated with miRNAs and gene regulation networks that depend on fish sex. The data reveal the importance of considering sex as a factor in interpreting the immune system in fish to generate efficient protocols to prevent outbreaks in fish farms.

aquaculture

infection

marker

immune

reproduction

epigenetic

Dicentrarchus labrax

Vibrio anguillarum

This study investigates how European sea bass (Dicentrarchus labrax) respond to infections caused by the bacteria Vibrio anguillarum, with a focus on understanding the molecular mechanisms in the gonads. Fish disease outbreaks are a major issue in aquaculture, and knowing how fish resist infections can improve disease management. Researchers analyzed changes in the miRNome (microRNAs) and transcriptome in male and female European sea bass 48 hours after infection. The results showed that males had more significant changes in gene expression compared to females, with 2,624 differentially expressed genes in testes vs. 101 in ovaries. Four specific miRNAs were active in males post-infection, but none were found in females. However, females exhibited a higher number of miRNAs targeting immune-related genes, suggesting that ovaries may suppress immune responses better than testes. Males, on the other hand, activated immune-related genes while suppressing cellular processes to fight the infection. A key gene, insulin-like growth factor binding protein (igfbp), was identified as important in male immune response, connected to miR-192-3p. This study highlights the importance of considering fish sex as a relevant factor when studying immune responses, as it could help develop better disease prevention strategies in aquaculture.

Testes showed more molecular changes at the microRNome and transcriptome levels in response to infection than ovaries
In testes of infected fish, miR-183-5p and miR-191-3p were upregulated while miR-451-5p and miR-724-5p were downregulated
Larger number of target genes were inhibited in females which were mostly related to the immune system
Importance of considering sexual dimorphism in immune responses

Aquaculture is a crucial industry for fish supply around the world. To achieve high production rates, fish could be subjected to a variety of stressful conditions, including high stocking density, poor water quality, and inadequate nutrition, all of which have the potential to cause the emergence of pathogenic diseases. Disease outbreaks are a frequent problem in aquaculture and affect the welfare as well as nutritional and commercial yield, among others (FAO, 2022). A common pathogen in aquaculture is Vibrio anguillarum, a Gram-negative bacterium that causes vibriosis in crustaceans, bivalves and fish, inducing symptoms such as haemorrhages, ulceration and necrosis, appetite loss among others (Frans et al., 2011; Hickey and Lee, 2018). This pathogen is responsible for significant losses in aquaculture production worldwide being one of the most common aquacultural diseases in Asia (Ina-Salwany et al., 2019) but also affecting Mediterranean aquaculture farms (Muniesa et al., 2022). In particularly, European sea bass (Dicentrachus labrax) together with other important commercial species in Europe such as the gilthead sea bream (Sparus aurata), are threatened by these bacterial infections (Muniesa et al., 2020).

Micro RNAs (miRNAs) are short (17–22 nucleotides) non-coding RNAs that suppress protein synthesis post-transcriptionally by binding to complementary regions in their target mRNA sequence (Ambros, 2004). Since their initial detection, miRNAs have served as valuable instruments in a wide array of applications in human health diagnostics, particularly in tumor identification in cancer (Hamam et al., 2017). The notable characteristic that makes miRNAs valuable markers for long-lasting molecular processes is their substantial preservation of both structure and function across evolutionary time (Niwa and Slack, 2007), paving the way for their discovery in many species. In fish, following the initial discovery of miRNAs in zebrafish (Lim et al., 2003), numerous investigations have been conducted to characterize the miRNA repertoire in other teleost species. In aquaculture, the collection of miRNAs, along with the currently described patterns of miRNA expression, has placed miRNAs at the forefront of promising indicators for enhancing productivity. These indicators include refining selection for breeding programs. For example, recently in rainbow trout (Onchorynchus mykiss), in which circulating miRNAs in the bloodstream were recognized as a non-invasive method to elucidate metabolic and reproductive conditions (Cardona et al., 2021).

The miRNAs are associated with numerous biological processes, such as cell division and proliferation, embryonic growth, apoptosis, metabolism, immunity, and gametogenesis, among others (Niwa and Slack, 2007; Vidigal and Ventura, 2015; Avital et al., 2018). In mammals, miRNAs play significant roles during infection by a variety of pathogens, such as viruses, parasites, and bacteria (Eulalio et al., 2012; Staedel and Darfeuille, 2013). Similarly in teleosts, numerous studies exposed the role of miRNAs in both innate and adaptive immune responses. To date, many studies documented the contribution of miRNAs to pathogenic bacteria responses in commercial fish species, for example: V. anguillarum in turbot (Scophthalmus maximus) (Gao et al., 2019a), Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus) (Gao et al., 2019b), V. parahaemolyticus in zebrafish (Danio rerio) larvae (Ji et al., 2019), Staphylococcus aureus in zebrafish adults (Zhang et al., 2019), and Aeromonas salmonicida subsp. salmonicida in rainbow trout (Cao et al., 2019). In Atlantic salmon (Salmon salar) macrophages infected with A. salmonicidam, revealed the alteration of three miRNAs (miR-21-5p, 223-3p, and 223-5p) which functions appear to be connected to pathogenesis, stress response, and allosteric exchange of histones (Hairul-Islam et al., 2014). Additionally, miR-21-5p, miR-202-5p, miR-451, miR-30a-5p contributed to versatile functions, including immune regulation in teleosts across evolution (Qiu et al., 2018; Liyanage et al., 2020).

Fish reproduction involves complex processes regulated by numerous molecules and signaling networks at the transcriptional and post-transcriptional level in which miRNAs play significant role in gonadal development in fish (Alvi et al., 2021; Bhat et al., 2021). In the last years of research, numerous sex-biased miRNAs have been found in aquaculture species: spotted knifejaw (Oplegnathus punctatus) (Du et al., 2018), sturgeon (Acipenser schrenckii) (Zhang et al., 2018), silver fish (Trachinotus ovatus) (He et al., 2019), Odontobutis potamophila (Zhao et al., 2017) and European sea bass (Sarropoulou et al., 2019). For example, miR-21-5p, and miR-462-5p were highly expressed in the ovary in yellow catfish (Pelteobagrus fulvidraco) (Jing et al., 2014), whereas others (e.g., miR-9-3p, miR-103b-3p, and miR-7b) were primarily expressed in the testis (Wang et al., 2018). Meanwhile, several miRNAs, including miR-29a, miR-34 or miR-143, showed tissue specificity and sexually dimorphic expression patterns in Atlantic halibut (Hippoglossus hippoglossus) (Bizuayehu et al., 2012). These investigations contribute to clarify the reproduction-related roles played by miRNAs in the gonadal tissue in fish. However, to our understanding, to date, no research has yet paid attention to investigate how miRNAs regulate the gonadal immune function during infections.

Exploring the interaction between the immune and reproductive systems holds significant value. It not only aids in controlling pathogen transmission in fish farms but also sheds light on how fish combat infections in their reproductive organs. These organs enjoy immune privilege, shielding germ cells responsible for passing on parental genetic material from pathogens. Nevertheless, sometimes, infections in the gonads result in transferring either the pathogen, for example that described in zebrafish after parasitic infection (Caballero-Huertas et al., 2021), viral infections in Gilthead sea bream (Sparus aurata) or European sea bass (Padrós et al 2022) or transferring immune-epigenetic memory (Beemelmanns and Roth, 2017; Pierron et al., 2021). The gain of the knowledge of the miRNA plethora and their post-transcriptional regulatory mechanisms in the infected gonads may advance our understanding of host-pathogen interactions. Further, the research on this field will help to development immunological strategies for disease management and prevention in cultured fish and to prevent vertical transmission from broodstock to offspring. The purpose of the current study was to investigate the alterations of the miRNome and the transcriptome in the gonads of a relevant commercial Mediterranean species, the European sea bass, subjected to a bacterial infection commonly found in fish farms.

Experimental fish

Unvaccinated European Sea bass fry (3.9 g) were obtained from a commercial Spanish hatcheryand raised during three years at the Institute of Agrifood Research and Technology (IRTA, Tarragona, Spain), to provide a broodstock of 32 months of age. Animals (N = 104) fish with mean weight of 663 ± 162 g were chipped and sexed. Throughout the period of rearing, commercial feed (Skretting) was provided to all the fish in accordance with feeding tables. Fish were held in tanks (2000–10000 L) connected to an IRTAmar® water recirculation aquaculture system (RAS), where the fish were maintained in flowthrough or RAS mode until the challenge experiment. Photoperiod was natural from natural daylight. The water temperature was natural (10–22 ºC) and dissolved oxygen (6.4 ± 0.6 mg L − 1; OXI330, Crison Instruments) were daily measured, and pH (7.4 ± 0.2; pHmeter 507, Crison Instruments), salinity (36‰; MASTER-20 T, ATAGO Co. Ltd), ammonia (0.14 ± 0.1 mg NH4 + L − 1) and nitrite (0.2 ± 0.1 mg NO2 − L−1) levels (HACH DR9000 Colorimeter, Hach, Spain) were monitored weekly. In February, six weeks before the challenge, the breeders were sexed and individually marked for identification with a PIT (passive integrated transponder) tag (TROVAN, Madrid, Spain).

Vibrio anguillarum infection and sample collection

To conduct the bacterial challenge experiment, 40 healthy fish (20 per sex) were chosen at random from the stock. On the 5th April, the fish were transferred to IRTA’s biosafety challenge room, into two cylindrical tanks (2000 L) connected to a RAS unit (IRTAmar®) equipped with real-time control of oxygen and temperature, mechanical filtration, biofiltration, and ultraviolet disinfection of the water. The outflow water was chlorinated, followed by ozone treatment before being discharged. The photoperiod was natural and water quality conditions in terms of temperature and salinity were 14.1 ± 1.1°C and 32.3 ± 0.4 ppt, respectively. Fish were given 100 ppm tricaine methane-sulfonate (MS-222, Sigma-Aldrich, Madrid, Spain) anesthesia before being intraperitoneally (IP) injected with sublethal dose (10E + 05 CFU/fish) of V. anguillarum. Phosphate Buffer Saline (PBS) was IP injected as a control. Fish were divided into two tanks, one for infected animals (ten males and ten females) and one for uninfected animals (ten males and ten females). Daily observations of fish were made to monitor the wellbeing of the fish. Over the duration of the experiment, no mortalities were found. After 48h, fish were euthanized under an overdose of MS-222 and assessed for biometry (standard length and weight). For each gonadal tissue, the gonads were cut into three pieces, two for sequencing analysis which were flash frozen in liquid nitrogen and kept at -80ºC, and the other one for histological purposes. Further, visual assessment of the gonadal maturation was identified to indicate the maturation stages of the gonads (Carrillo et al., 2015).

Microbiological postmortem analyses

Microbiological postmortem sampling of head kidney and gonad was conducted to assess V. anguillarum presence in both tissue compartments. Sterile swaps were soaked in each of the tissue of freshly dead fish, and plated onto general media TSA- ClNa (Trypto – Casein Soy Agar, Difco laboratories) supplemented with 0.5% of ClNa) and onto Vibrionaceae selective media TCBS (Thiosulfate-Citrate-Bile Salts-Sucrose, DIFCO laboratories, also supplemented with ClNa) to a final concentration of 1% (w/v) agar plates, to asses viable bacterial recovery. Additionally, PCR to determine the presence of V. anguillarum (Hong et al 2007) was performed on DNA extracted by following the manufacturer's instructions (Dneasy Blood & Tissue Kit, QIAGEN) from the same tissue samples. Plates were incubated for 96 hours at 23ºC, and were checked every 24 h for growth recording.

Histological analysis

For histological study, ovaries and testes of all sampled fish (N = 40) were collected and fixed for 24 hours at room temperature in 4% paraformaldehyde (PAF) in PBS, pH 7.4. After incubations, PAF was washed for 24 hours in PBS and dehydrated in an ascending sequence of ethanol concentrations. Finally, tissues were cleaned in xylene, and then embedded in paraffin. Sections (7 µm thick) were cut using a microtome (Reichert-Jung 2040), float mounted on glass slides, stained with haematoxylin-eosin, and mounted with DPX. Photographs were taken under a microscope objective 10x for testis and 4X for ovaries with the stereoscope (Leica M205 C).

RNA and miRNA gonadal extraction

Total RNA from four fish per group and sex (total of 16), was isolated from gonadal samples by using an RNA isolation kit (RNeasy mini kit, Qiagen) according to the manufacturer’s instructions. Quality of the samples was assessed by Nanodrop (Agilent Technologies) with ratio 260/280 = 2.11 ± 0.05 and 260/230 = 1.89 ± 0.51) and by the RNA Integrative Number (RIN) measured with a Bioanalyzer (Agilent Technologies, USA) with values > 8.3 for testes. In ovary, RIN numbers were not considered due to naturally high levels of 5s rRNA and tRNA (Mazabraud et al., 1975; Bir et al., 2023).

Library preparations and sequencing

Libraries were prepared at BGI genomics in Hong Kong for DNBSEQ Eukaryotic Strand-specific mRNA sequencing. DNBseq with paired-end mode and read length 100 bp was performed. Filtering was done using SOAPNuke software by BGI Genomics (Chen et al., 2018). For small RNA sequencing, 16 libraries were constructed from European sea bass gonad samples. Library preparation was performed by NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible) kit following manufacture instructions, using sequencing Lane (1x50, 178 v4, HiSeq) single-end mode with a read length of 50 bp at a BGI. After sequencing, data filtering was performed to removed adaptor sequences and low-quality reads from raw reads. Resulted reads were aligned to the D. labrax genome (version dlabrax2021) from ENSEMBL using Hisat2 (Kim et al., 2019) and counted using Featurecounts (Liao et al., 2014). miRNAs were aligned using Prost! (Desvignes et al., 2019) to the D. labrax genome and annotated using medaka (Oryzias latipes) genome GTF file.

Statistical Analysis

Mean weight and size of the fish was calculated using R software. Data was expressed as mean ± Standard Deviation (SD) and the differences were considered significant when p < 0.05. Homogeneity and normality of data were analyzed by Levene test and Shapiro-Wilk normality test, respectively. Differences between control and infected groups were done by non-parametric Kruskal Wallis test. All the graphs were generated by using the ggplot2 package (v. 4.1.3) in R Studio (v. 4.1.3).

Bioinformatics and statistical analysis

All bioinformatic analyses were done with R (R Core team, 2023). Raw reads of RNA and miRNA sequencing were filtered by retaining genes and miRNAs with at least ten reads in at least four samples. In order to visualize similarities in miRNA and transcript expression patterns between samples, principal component analyses (PCA) were performed using the plotPCA function of the DESeq2 package (version 1.12.3) (Love et al., 2014).

Differential expression analysis was done using the DESeqDataSetFromMatrix, DESeq and results functions of the DESeq2 package, upholding a criterion of adjusted p-value < 0.05. Differential expression (DE) was visualized with heatmaps using the Pheatmap package (version 1.0.12). For annotation, gene names, Gene Ontology (GO) terms and GO slim terms were assigned to genes using the getBM function of the biomaRt package (version 3.18) (Durinck et al., 2005, 2009). Enriched GO terms were identified using the enricher function of the clusterProfiler package (version 3.0.4) (Yu et al., 2012; Wu et al., 2021) and GO terms with an adjusted p-value < 0.05 were considered significantly enriched. GO slim terms of miRNA targets were identified using BiomaRt and plotted in circular plots using the Circlize package (version 0.4.15) (Gu et al., 2014). Gene names missing from the ENSEMBL database were supplemented via the NCBI gene annotation database using BLAST+ (Camacho et al., 2009).

Weighted Correlation Network Analysis

Weighted Correlation Network Analysis (WGCNA) analysis was done using the method proposed by Sánchez-Baizán et. al. (2022) (Sánchez-Baizán et al., 2022). Firstly, raw reads were filtered using the GoodSamplesGenes function of the WGCNA package (Langfelder and Horvath, 2008, 2012). Then, reads were normalized using the dds function of the DESeq2 package and filtered by retaining genes and miRNAs with at least ten reads in at least four samples. The power was chosen using the PickSoftThreshold function of the WGCNA package with a scale free topology fit signed R² > 0.85. Subsequently, BlockwiseModules function was used to create a Topology Overlap Matrix (TOM) and the modules were plotted in a dendrogram using the PlotDendroAndColors function. The “modules to trait correlations” were performed of the following conditions: 1) male control vs. male infected (M inf), 2) female control vs. female infected (F inf) and 3) male control vs. female control (MvF ctrl) and 4) male infected to male control (M inf). Further studies of sexual dimorphism, the comparison of male infected vs. female infected (MvF inf) was also considered.

For correlation, Pearson analysis was used (Freedman et al., 2007) and significance was calculated by Student’s t-test (Mishra et al., 2019). Hub genes per module were identified with the option TopHubInEachModule function. To visualize the “module membership to gene significance”, firstly the Pearson’s correlation and significance by Student’s t -test between the normalized reads and the module eigengenes were calculated, and, secondly, between the normalized reads and the conditions (1), (2) or (3). Finally, each module to each condition was plotted in a scatterplot using the function VerboseScatterplot, following the method proposed in Sánchez-Baizán et. al. (2022).

Individual module gene networks were constructed by recalculating the TOM values and filtering by only keeping previously identified differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) for each condition. Then, genes without annotation were removed. Networks were constructed using the function Graph_from_adjacency_matrix from the Igraph package (Csárdi et al., 2023). Subsequently, in silico targets of chosen miRNAs were identified by predicting targets and calculating context scores using the Perl script from Targetscan (version 6.0).

Biometry

The mean weight of the female fish for the infected and control groups was 709.04 g ± 73.80 g and 698.40 g ± 69.50 g, respectively, whereas the mean weights of the male fish for the infected and control groups was 683.93 g ± 36.66 g, and 677.06 g ± 35.00 g, respectively (Supplementary Figure S1A). Infected and control fish had mean lengths of 34.05 cm ± 1.21 cm and 34.05 cm ± 1.04 cm for female. The infected male had a mean length of 33.65 cm ± 0.47 cm while the control represented of 34.05 cm ± 0.72 cm (Supplementary Figure S1B). Both the weight and size of the male and female fish in the treatment and control groups did not significantly differ from one another, indicating that the findings were due to the treatment and not due to biometry differences among groups.

Postmortem results by qPCR

V. anguillarum was detected in two testes and two ovaries, while in the head kidney, bacteria were found in one male and one female fish. The fish that tested positive for bacteria in the gonads did not show bacteria in the head kidney. Overall, the results indicated that all infected groups tested positive for V. anguillarum, confirming the success of the infection in the present experiment.

Histological analysis

In ovaries, four stages of oocyte maturation were identified: I) primary growth stage, II) cortical alveolus stage, III) early-vitellogenic stage and IV) late-vitellogenic stage. All the fish ovaries were of similar maturation independently of the treatment (Supplementary Figure S2A, B). In testes, maturation was determined based on the presence or absence of spermatozoids. All male fish, independently of the treatment, were mature as presence of spermatozoids were found inside the seminiferous tubules (Supplementary Figure S2C, D).

RNA sequencing data

After filtering, sequencing yielded an average of 34 million reads per sample. All samples had a QC20 of > 98% and a GC count around 49%. The minimum and maximum alignment between all the sequenced samples was 93.9% and 98.3% which was aligned to the European sea bass genome and mapping with Featurecounts (Liao et al., 2014). The resulted alignment ranged in all samples between 57.3–68.6%. In total, 24,969 genes were identified of which 11,915 were named using ENSEMBL biomaRt (Durinck et al., 2005). Furthermore, blasting coding DNA sequences (CDS) to the NCBI D. labrax database yielded 16,445 named genes, with 4,953 genes not annotated by ENSEMBL biomaRt. Thus, in total, 16,868 genes were named.

Transcriptomic alterations in the gonads after infection

PCA analysis clustered the samples in two groups in males, one for control and the other for infected explaining the 82% of the variance for PC1 and 10% for PC2 (Supplementary Figure S3A) while in females, PC1 explained 32% and PC2 24% (Supplementary Figure S3B). DE analysis of infected testes compared to control testes resulted in 2,624 DEGs, among which 1,050 were upregulated and 1,574 were downregulated genes (Fig. 1A, Dataset 1). In ovaries, 101 DEGs were identified, among which 37 were upregulated and 64 were downregulated (Fig. 1B, Dataset 1).

GO term enrichment analysis of testicular upregulated genes identified genes mainly related to RNA synthesis processes, such as “RNA processing”, “RNA helicase activity” and being “extracellular region” and “methyltransferase activity” with more enrichment (Fig. 2A, Dataset 2). Notably, “immune response” was also identified as an enriched GO term. Testicular downregulated genes pertained mainly to microtubule activity, being “microtubule binding” and “microtubule-based movement” the two top enriched GO terms together with “cilium assembly” or “dynein complex” (Fig. 2B). No enriched GO terms were identified in ovarian DEGs.

MicroRNome modifications in the gonads after infection

After filtering, UMI small RNA library DNBseq, resulted in an average of 25 million reads per sample with an average read length of 26 bp. A total of 212 mature miRNAs were identified (Dataset 3). PCA analysis clustered the samples in two groups in males, one for control and the other for infected, explaining 33% of the variance for PC1 and 19% for PC2 (Supplementary Figure S3C). Similarly, in females, two clusters were observed for each control and infected fish, explaining the 28% and 24% of the variance for the PC1 and PC2, respectively (Supplementary Figure S3D). In testes, four miRNAs were found to be DE in infected males two days after the infection with V. anguillarum, namely miR-183-5p, miR-191-3p, miR-451-5p and miR-724-5p (Dataset 4). In ovaries, no DEMs were found.

Identification of miRNA targets in the infected testes

The identified four DEMs in infected testes were further inspected by identifying potential targets. Targets were obtained by 1) negative correlation of miRNA-transcript expression by Pearson’s analysis with a p-value of < 0.05, and 2) in silico binding as determined by Targetscan. There were 1,693; 79; 28 and 5 potential target genes that comply with the aforementioned criteria for miR-724, miR-191, miR-451 and miR-183, respectively (Dataset 5). In total, 128 GO slim terms were assigned to the target genes, of which most genes pertained to “catalytic activity”, “organelle”, “nucleus”, “hydrolase activity”, and “transferase activity” (Dataset 6). As shown in Fig. 3, an interesting large number of potential targets of miR-451, miR-191 and miR-724 were related to the immune system by the GO terms: “immune system process”, “defense response to other organisms” and “inflammatory response”. Furthermore, some GO related to the reproduction system were identified: “reproductive process” and “cillium organization”, and to cell division: “cell differentiation process” and “transcription regulator activity”.

Crosstalk between miRNA and genes altered after infections in the gonads

WGCNA was performed in order to identify gene and miRNA interactions and relate gene-miRNA networks to infected fish. The power was set at 14 based on the scale free topology model fit (Supplementary Figure S4A) and mean connectivity (Supplementary Figure S4B). The dendrogram resulting from the WGCNA provides a comprehensive view of the relationships between genes based on their expression patterns (Fig. 4A). The hierarchical clustering depicted in the dendrogram organizes genes into the following modules, named after colors: yellow (519 genes), turquoise (6,294 genes), red (826 genes), blue (2,393 genes), green (8,849 genes) and brown (185 genes) (Fig. 4B). In M inf, module brown and module turquoise were most significantly correlated (correlation value 0.97, p-value 2.24E-10 and correlation value 0.71, p-value 0.002, respectively). Modules pertaining to inherent sexual dimorphism trait, i.e. significant for both MvF ctrl and MvF inf, were turquoise (correlation value 0.98, p-value 2.50E-11 in MvF ctrl and correlation value 1, p-value 1.07E-20 in MvF inf) and green (correlation value 0.95, p-value 9.64E-09 in MvF ctrl and correlation value 1, p-value 6.52E-17 in MvF inf, respectively). Furthermore, sexual dimorphism expression patterns due to infection were identified in three modules: red, brown and blue (significant for MvF inf, p-value 7.61E-13, 2.02E-12, 2.68E-05, respectively, with correlation values 0.99, 0.99 and 0.85, respectively). No modules were significantly associated with female infected against female control (F inf).

To further highlight the significant modules related to infection, module membership vs. gene significance was calculated for M inf and MvF inf significant modules MvF inf. M inf for the brown module resulted in a correlation coefficient of 0.72 and p-value 7.5E-31 (Supplementary Figure S5A). For MvF inf modules, the blue module resulted in a correlation coefficient of 0.38 and p-value 4.6E-83 (Supplementary Figure S5B), the red module resulted in a correlation coefficient of 0.82 and p-value < 1E-200 (Supplementary Figure S5C) and the brown module in a correlation coefficient of 0.83 and p-value 2.8E-48 (Supplementary Figure S5D).

Connections in infected testes

With the aim of finding correlation between alteration in the transcriptome and the miRNome in the infected gonads, a network analysis was performed in the brown module in the M inf group, which based on the WGCNA, had the highest significance. After filtering by DEGs of infected male vs control male, removing unnamed genes and passing a TOM threshold of > 0.3, a total of 54 genes and one miRNA, namely miR-191-3p, remained in the brown module. The hub node of the brown module, being the major player in the module, was insulin like growth factor binding protein 1a (igfbp1a) (Fig. 5).

Sexual dimorphism in the immune response

In order to describe differences between males and females in managing the infection, several strategies were performed. First, DEGs between sexes were identified (Supplementary Figure S6A). To discard the inherent sexual dimorphism, DE between control male and control female was assessed, as well as between infected male and infected female. Then, DEGs between the two control and two infected sexual groups were compared and overlapping genes were discarded from further analysis. In total, 1,347 genes were DE between control males and control females but not in infected males and infected females, whereas 2,768 genes were DE between infected males and infected females but not between the two control groups, making a total of 4,115 unique DEGs (Supplementary Figure S6B). Of these unique DEGs in the control group, 906 DEGs were male-skewed whereas 441 were female-skewed. In infected groups, 1,577 DEGs were male-skewed whereas 1,191 were female-skewed (Dataset 1).

Secondly, to decipher transcriptome and miRNAome immune networks that differ due to the sexual dimorphism, the modules that were significant for MvF inf but not significant for MvF ctrl (i.e., modules red, blue and brown Fig. 4B) were selected. Significant genes in the modules were filtered based on DEGs and a network with a threshold of > 0.45 was created to visualize the interactions between genes and miRNAs (Supplementary figure S6C). No single gene played a central role in all the connections in the network, but rather was formed by two big clusters, one with neuropeptide FF receptor 2 (npffr2a) and sperm autoantigenic protein 17.1 (spa17.1) genes, and protein arginine methyltransferase 9 (prmt9) gene in the other cluster, all three showing higher connectivity in the network. Notably, no miRNAs passed the threshold.

Additionally, differences in miRNA expression between infected males and females were identified as well as between control males and females (Supplementary Figure S7). DEMs between the two control and the two infected groups between male and females resulted in 49 and 72 miRNAs, respectively (Dataset 4). The overlapping 29 miRNAs shown in the Venn diagram were discarded from further analysis since they were DEMs founds in both sexes regardless of being control or infected (Fig. 6A). Thus, 20 miRNAs were DEM between male control and female control being 12 miRNAs upregulated in females and 8 miRNAs in males. In infected fish, 43 miRNAs were DE being 22 upregulated in infected females and 21 upregulated in infected males.

Target genes of the sex-skewed miRNAs (i.e., 20 for control and 43 for infected, Fig. 6A) were identified following the same criteria as that for identifying the four DEMs in infected males. After filtration, there were 474 and 804 target genes identified in infected males and females, respectively, whereas in control males and females, 133 and 263 target genes were identified, respectively.

Biological functions of the potential target genes were identified by GO slim terms (Fig. 6B). Notably, larger number of potential targets were found in infected females followed by infected males. The highest number of GOs were related to “catalytic activity, signaling, organelle and anatomical structure development (Dataset 7). Interestingly, infected female-skewed miRNAs mostly targeted immune-related transcripts, i.e. 666 genes related to immune system process, 482 to programmed cell death, 375 to receptor ligand activity, 212 to autophagy, 172 to defense response to other organism, 135 related to wound healing, 115 to lysosome and 84 to inflammatory response. On the other hand, miRNAs upregulated in infected males compared to females mostly targeted mitosis and DNA processes-related genes, i.e., 1067 to RNA binding, 435 to cytoskeleton, 327 to DNA repair, 274 to protein catabolic process and 260 to mitotic cell cycle.

The reproduction and the immune system are interconnected and are responsible for the immune defense in the gonads, but also can explain the sexual dimorphism in the immune response. In vertebrates, sex plays a crucial role in shaping the immune response, resulting in differing susceptibilities to diseases between males and females (Nunn et al., 2009; Caballero-Huertas, M., Salazar, M., & Ribas, 2023). In particularly in fish, the differing immune responses between sexes have not been fully explored, and their broader implications have often been overlooked. In this study, we wanted to shed light, on one site, on the molecular immune responses after infection in the ovaries and the testes and, one the other site, on the sexual dimorphism of the immune response in European sea bass, a high valuable cultured fish.

Here it is reported that testes showed higher alteration of both the miRNAome and the transcriptome after infection. In concrete, males altered the testicular genes in ~ 26% more than that in females (2,624 vs. 101 DEGs) and four miRNA were significantly expressed after infection while none were identified in females. Resulting data imply that fish ovaries were more robust in front of infections, indicating a faster clearance of the pathogen. From evolutionary point of view, females are generally less susceptible to infections (Moore and Wilson, 2002; Vincze et al., 2022). For example, in humans, in which sexual dimorphic differences in the immune system have been addressed, it is known that large number of immune system genes are located on the sexual chromosome X, explaining why women present more active immune system against pathogens, but, in contrast, women are more sensitive to autoimmune diseases (Mauvais-Jarvis et al., 2020). In fish, several factors determine the sex prevalence in front of a diseases, such as behaviour, social hierarchies, size and lifespan. For example, fish personality shaped the immune system, where aggressive fish have higher expression levels of genes related to innate response (MacKenzie et al., 2009). The prevalence of infection of the parasite P. neurophilia was higher in male zebrafish when compared to females, although the vertical transmission was based on oocytes, causing severe problems in zebrafish facilities around the world (Caballero-Huertas et al., 2021). In the current analysis, we observed that upregulated DEGs in the infected males were related to RNA synthesis processes and the immune response while downregulated genes were related to microtubule activity, being “cilium assembly” or “dynein complex” the most enriched, both processes being involved in the sperm motility (zur Lage et al., 2019). These results indicated that the cells in the testes invest the energy to activate the immune system while the sperm-related processes required for reproduction remain inhibited during infection.

Although sexual dimorphism manifests in infections and immune responses across evolutionary history, both in invertebrates and vertebrates (Nunn et al., 2009), scarce data on the molecular mechanisms is available to date. With the goal of discerning sexual dimorphic patterns in fish post-infection, the potential targets of the sex-biased miRNAs exhibiting negative differential expression in either infected males compared to females or control males compared to females, were examined. This mirrors the inhibitory role of the miRNAs in post-transcriptional regulation. Interestingly, this analysis revealed nearly double the number of potential targets regulated in infected females compared to infected males, with a significant portion targeting genes associated with the immune system. These findings suggest that females employed distinct miRNomic and transcriptomic pathways compared to males. Specifically, females appeared to utilize a greater number of miRNAs to inhibit the expression of immune-related genes in response to infection, probably as a mechanism to suppress the immune response after clearing the pathogen. In contrast, as previously described, males seemed to upregulate the immune-related genes in the testes as strategy to cope the infection in which large number of genes in the transcriptome were involved. Present data underscores the crucial role of sex-specific responses in shaping the host's defense mechanisms against infections in fish.

Generally, V. anguillarum has been described to be highly present in the bloodstream and in the haematopoietic tissue in fish (Frans et al., 2011) but in the fish gonads data is scarce. Here, we detected four animals with presence of V. anguillarum in the gonads and two more in the head-kidney. Recent study in European sea bass in Turkish aquaculture farm revealed that V. gigantis growth was detected in female gonads of deceased fish, whereas no bacterial growth was found in the male gonads (Yilmaz et al., 2023). In (Schmidt et al., 2017), intraperitoneal injections were executed in zebrafish and V. anguillarum presence was measured by immunohistochemistry at multiple time points, i.e. 30 min, 2 h, 8 h and 24 h, in multiple tissues (spleen, liver, swim bladder and peritoneal cavity and blood). Notably, gonads were not tested. In mollusks, Vibrio bacterial infection was monitored in male and female gonads. In live animals, higher numbers of bacteria was found in biopsies of mollusk female gonads compared to male gonads, suggesting a greater resistance to infection in male mollusks (Sainz-Hernandez and Maeda-Martinez, 2005). In all, recognizing the prevalence of sex in farmed animals is crucial for fisheries management, aquaculture practices, and conservation initiatives.

With the aim of identifying miRNAs as molecular markers of the immune response to bacterial infections in the gonads, we identified four miRNAs altered in the current study; miR-191-3p and miR-183-5p were found to be upregulated whereas miR-451-5p and miR-724-5p were downregulated 48 hours after bacterial infection in testes. The miRNA-183/96/182 cluster, of which miR-183 takes part, had a key role in macrophage regulation in mice exposed to Pseudomonas aeruginosa and lipopolysaccharide (LPS) by increasing its expression (Muraleedharan et al., 2019) as similarly found in infected testes in European sea bass in the current study. Furthermore, knockdown of the miR-183/96/182 cluster resulted in lower cytokine expression (Ichiyama and Dong, 2019; Muraleedharan et al., 2019). In T-helper 17 cells, miR-183 was responsible to increase cytokine production and pathogenicity of Th17 cells (Ichiyama et al., 2016; Wang et al., 2023). miR-191, one of the key nodes in gene regulatory network of infected testes vs. control, was reported to regulate T cell homeostasis, erythroblast enucleation, angiogenesis and T cell survival in mammals (Zhang et al., 2011; Lykken and Li, 2016; Gu et al., 2017). Targets of miR-191 include transcription factors, such as SATB1, RIOK3 (RIOkinase 3) and notably, sex gene SOX4 SRY and cell cycle regulators, such as CDK6 (cyclin-dependentkinase 6) and CCND2 (cyclin D2) (reviewed in (Nagpal and Kulshreshtha, 2014)). Scarce data is found in fish, but a recent experiment in rainbow trout also correlated miR-191 in the immune system as it was altered 48 h after hematopoietic necrosis virus (IHNV) infection in the liver (Wu et al., 2022).

The role of the miR-451 in the immune system has been deeply studied in mammals (Rosenberger et al., 2012; Chapman et al., 2017) (Wang et al., 2015). For many years it is also known that in fish miR-451 plays a crucial role in promoting erythroid maturation (Pase et al., 2009) with the aim of being an informative molecular marker of the immune system. Its expression was lower in bacterial infected in the mucus of the Chinese tongue sole (Cynoglossus semilaevis) (Zhao et al., 2021) while being upregulated 48 and 72 hours in macrophages immune stimulated by a viral mimic insult in the in Atlantic cod (Gadus morhua) (Eslamloo et al., 2018). Its role was also correlated in the early embryonic development in fish subjected to high temperature (Papadaki et al., 2022) and to toxicity (Jenny et al., 2012). Interestingly, miR-451 showed sexual dimorphism in the brain and was related to masculinization process in Atlantic halibut (Bizuayehu et al., 2012). Here, miR-451 was upregulated in the testes in European sea bass together with miR-724-5p. Similarly, miR-724-5p was highly conserved in the fish gonads, showing higher expression in males compared to females in multiple species (van Gelderen et al., 2024), for example in yellowfin seabream (Acanthopagrus latus) and salmon Atlantic salmon (Skaftnesmo et al., 2017). More evolutionary-related data indicated that miR-724 is fish-specific miRNA (Li et al., 2014; Zhao et al., 2022), highlighting its potential role as a marker in fish. Besides its role in the reproductive system, miR-724-5p was implicated in immune response pathways although little information is available. For example, miR-724 targeted immune genes in silver carp (Hypophthalmichthys molitrix) spleen (Zhao et al., 2024) and in common carp, it was upregulated in spleen due to an inflammatory damage by cadmium (Chen et al., 2020). Our data showed that European sea bass exposed to V. anguillarum exhibited downregulation of miR-724-5p in the testis.

Lastly, the network analysis of the crosstalk between microRNome and transcriptome in the infected males gave more information regarding the molecular mechanisms performed in the infected testes. The gene igfbp, which belongs to the insulin-like growth factor binding protein family is primarily expressed in the liver, but also it encodes a protein that circulates in the plasma and binds to both insulin-like growth factors (IGFs) I and II (Firth and Baxter, 2002; Lay et al., 2021). This characteristic makes, in human, to be a promising biomarker for cancers (Huang et al., 2024). Its role in the reproduction system was characterized in polycystic ovary syndrome in humans (Jin et al., 2023) and in fish, igfbp played multiple functions in metabolism, osmoregulation, reproduction, behavior, and immunity (Reindl and Sheridan, 2012). In Chinook salmon and Atlantic salmon, circulating higher levels of igfbp in plasma were related to stress response (Shimizu and Dickhoff, 2017; Breves et al., 2020). Our data manifested that igfbp had a relevant role controlling the testicular gene network but also to the post transcriptional control as the miR-192 was enriched in the network analysis.

In summary, our study highlights the intricate relationship between reproduction and the immune system, which not only defines immune defense in gonads but also contributes to sexual dimorphism in the immune response. Current findings revealed that testes exhibited a higher degree of molecular alterations at the microRNome and transcriptome level in response to infection compared to ovaries. Specifically, infected males showed a greater alteration in testicular genes and miRNA expression compared to females. Furthermore, testicular analysis of miRNA expression identified several key molecules involved in immune regulation. Notably, miR-183-5p and miR-191-3p were upregulated, while miR-451-5p and miR-724-5p were downregulated following bacterial infection in testes. In contrast, when we performed the sexual dimorphic study by subtracting only those DEM related to infection and sex, larger number of target genes were inhibited in females which were mostly related to the immune system. Overall, our study underscores the importance of considering sexual dimorphism in immune responses in fish and provides valuable insights into the molecular mechanisms underlying these differences.

Ethics approval and consent to participate

European sea bass experiments were performed in well-equipped fish facilities at the Institute of Agrifood Research and Technology (IRTA, la Ràpita, Tarragona, Spain,https://www.irta.cat/es/produccion-animal/acuicultura). All animal procedures included in this project were conducted in compliance with an experimental research protocol approved by the committee of ethics and animal experimentation of the IRTA and the Catalan Government (project nº 12197, file nº FUE-2023-03496883 and ID SLNBB6432) based on Real Decreto 53/2013 and in accordance with the guidelines of the European Union Council (2010/63/EC) for the use of laboratory animals. IRTA is an authorized biosecurity facility to work with pathogens (NBS2) certified by registers numbers: EA-01/17 and IRTA-SCRT9900002 approved by Animal Protection Section (Catalan Government). Present animal research adheres to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication

All author consent the publication of the current article.

Competing interests

No competing interests are declared.

Funding

This study was supported by the Spanish Ministry of Science and Innovation grants 2PID2020-113781RB-I00 “MicroMet” and by the Consejo Superior de Investigaciones Científicas (CSIC) grant 02030E004 “Interomics” and Ayudas extraordinarias para la preparación de proyectos del CSIC “AEPP2024” to LR and with funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S).

Author contributions

TvG: Experiments, Bioinformatic analysis, Writing – original draft & reviewing. PD: Laboratory work, Writing – original draft. SJ: Laboratory work. EB: Experiments, Writing – reviewing. ND: Supervision, Experiments, Writing – reviewing. DF: Supervision, Writing– reviewing. LR: Experiments, Supervision, Project administration, Funding acquisition, Writing – original draft & reviewing.

Acknowledgements

We thank Josep Lluis Celades, Magda Monllaó and IRTA technicians that maintained the fish and completed lab analysis.

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No competing interests reported.

Dataset1DEGs.xlsx
Datasets All datasets can be found here: https://drive.google.com/drive/folders/1_WVcMmhoeEE03GkiF73gp6WxUBEC0JQK?usp=drive_link Dataset 1. Differentially expressed (DE) genes between the different groups resulting from mRNA-sequencing testes and ovaries of European sea bass (Dicentrarchus labrax) 48 h after intraperitoneal infected with Vibrio anguillarum. A total of four individuals per group (control and infected) were used.
Dataset2GO.xlsx
Dataset 2.Enriched GO terms of up- and downregulated DEGs in M inf vs M ctrl.
Dataset3ProstReads.xlsx
Dataset 3.miRNAs identified via miRNA-sequencing in European sea bass gonads sea bass (Dicentrarchus labrax).
Dataset4DEMs.xlsx
Dataset 4. Differentially expressed miRNAs (DEM) between the different groups resulting from miRNA-sequencing testes and ovaries of European sea bass (Dicentrarchus labrax) 48 h after intraperitoneal infected with Vibrio anguillarum. A total of four individuals per group (control and infected) were used.
Dataset5Targets.xlsx
Dataset 5.Proposed targets of the DEMs in M inf vs M ctrl identified by Targetscan and negative Pearson’s correlation.
Dataset6GOslimDEMs.xlsx
Dataset 6.Goslim terms of the potential target genes of the four DEMs in M inf vs M ctrl.
Dataset7GOslimMvF.xlsx
Dataset 7.Goslim terms of the potential target genes of the sex-skewed DEMs.
vanGelderenetalSupplementaryfigures01102024.docx
Supplementary Figures Supplementary Figure S1. Supplementary Figure S1: Mean weight (g) and length (cm) of European seabass intraperitoneal infected 48 hours of Vibrio anguillarum. Data represents mean ± SD. Similar letters in the bar indicate that data are not statistically significant (p<0.05). Supplementary Figure S2. Histological cross-section of European sea bass gonads in control (A, C) and infected (B, D) ovaries and testes, respectively, 48 h after intraperitoneal infected with Vibrio anguillarum. Several stages of oocyte maturation are indicated. Labels represent I) primary growth stage, II) cortical alveolus stage, III) early-vitellogenic stage and IV) late-vitellogenic stage. Supplementary Figure S3. Principal Component Analyses (PCA) resulting from RNA-sequencing (A, B) and miRNA-sequencing (C, D) in the testes (A, C) and ovaries (B, D), respectively, 48 h after intraperitoneal injection with Vibrio anguillarum. Supplementary Figure S4. Scale-independence with threshold R² = 0.85 (A) and mean connectivity (B) of the network in different soft-threshold powers. The left panel displays the correlation of soft threshold with scale-free fit index. Supplementary Figure S5. Scatterplots of correlation between gene significance (GS) versus module membership (MM) of the modules significant for the traits male infected to male control (M inf) and male infected to female infected (MvF inf). Modules were correlated using a Pearson’s correlation and Student’s t-test to the following modules and traits: M inf brown (A), MvF inf blue (B), MvF inf red (C), MvF inf brown (D) and (MvF inf). Supplementary Figure S6. (A) Heatmap analysis of differential expression genes obtained by RNA-sequencing of the infected testes and ovaries in European sea bass (Dicentrarchus labrax). A total of four individuals per group was used. (B) Venn diagram of genes that showed sexual dimorphism. In blue, DEGs between control males and control females, in red, DEGs between infected males and infected females. (C) Network analysis to visualize the interaction between genes responsible for the sexual dimorphism during the immune response in gonads. For the analysis, DEGs of male vs. female were used in the red, blue and brown modules from the Weighted Correlation Network Analysis (WGCNA). Supplementary Figure S7. Heatmap analysis of differential expression miRNAs after sequencing testes (A) and ovaries (B) of European sea bass (Dicentrarchus labrax) 48 h after intraperitoneal infected with Vibrio anguillarum. A total of four individuals per sex was used.

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Gonadal miRNomes and transcriptomes in infected fish reveal sexually dimorphic patterns of the immune response

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Abstract

Figures

Plain English summary

Highlights

Background

Methods

Experimental fish

Microbiological postmortem analyses

Histological analysis

RNA and miRNA gonadal extraction

Library preparations and sequencing

Statistical Analysis

Bioinformatics and statistical analysis

Weighted Correlation Network Analysis

Results

Biometry

Postmortem results by qPCR

Histological analysis

RNA sequencing data

Transcriptomic alterations in the gonads after infection

MicroRNome modifications in the gonads after infection

Identification of miRNA targets in the infected testes

Crosstalk between miRNA and genes altered after infections in the gonads

Connections in infected testes

Sexual dimorphism in the immune response

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1