Chlorogenic acid on the in vitro germination, invasion and intracellular proliferation of Ameson portunus (Microsporidia)

Chlorogenic acid on the in vitro germination, invasion and intracellular proliferation of Ameson portunus (Microsporidia) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chlorogenic acid on the in vitro germination, invasion and intracellular proliferation of Ameson portunus (Microsporidia) Jiu-Yang Chen, Ya-Li Xu, Zhi-Qiang Zhu, Jin-Yong Zhang, Zhao-Zhe Xin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6226867/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ameson portunus , the causative agent of "toothpaste disease" in Portunus trituberculatus and "slurry-like syndrome" in Scylla paramamosain , has caused substantial economic losses in Chinese aquaculture. Currently, there are no effective strategies to control this pathogen. Chlorogenic acid, known for its antioxidant, anti-inflammatory, antibacterial, and antiviral properties, is widely used in aquaculture due to its safety and efficacy. This study explored its impact on the in vitro germination, invasion, and intracellular proliferation of A. portunus spores through molecular and omics analyses. Results showed that chlorogenic acid significantly inhibited spore germination in a dose-dependent manner and effectively blocked invasion and intracellular proliferation at a safe dose of 100 µg/ml. Transcriptome analysis comparing 1 µg/ml (PO), 100 µg/ml (PH), and 0 µg/ml (PZ) groups revealed that the drug suppressed spore DNA replication and key signaling pathways, including cAMP, Rap1, Ras, Wnt, ErbB, Notch, and JAK/STAT. These pathways are critical for spore proliferation, differentiation, and development. The 100 µg/ml dose demonstrated significantly greater inhibition of spore germination and invasion compared 0 to 1 µg/ml. The Ras protein in the Rap1 and Ras pathways emerged as a potential drug target. Additionally, spores upregulated genes related to ribosome, proteasome, peroxisome, and metabolic processes as a survival response to drug exposure. These findings highlight chlorogenic acid's potential as a therapeutic agent against A. portunus. Ameson portunus Chlorogenic acid In vitro germination Intracellular proliferation Comparative transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Microsporidia are a group of unicellular eukaryotic organisms that have evolved a specialised form of intracellular parasitism [ 16 , 51 ]. Since the initial discovery and naming of microsporidia in the mid-19th century, over 1,700 species of microsporidia have been identified and characterised globally [ 2 , 17 , 41 ]. Microsporidia exhibit a broad host range, parasitising diverse animal hosts from vertebrates to invertebrates. This has resulted in significant harm to sericulture, aquaculture, animal husbandry, and human health. More than half of the microsporidia infested aquatic animals [ 15 , 42 ]. To date, over 30 microsporidia species have been identified as pathogens affecting more than 20 aquaculture species globally [ 22 ]. A total of over 50 different genera have been identified as potential pathogens affecting crustaceans. Of these, 11 to 12 species of microsporidia infesting crabs belong to nine genera, including Abelspora, Ameson, Areospora, Enterospora, Hepatospora, Nadelspora, Nosema, Ormieresia , and Thelohania [ 49 ]. Previous research has indicated that Ameson portunus is the recently identified causative agent responsible for "toothpaste disease" in Portunus trituberculatus and "slurry-like syndrome" in Scylla paramamosain [ 49 , 54 ]. The disease is prevalent in regions where economic crab culture is practiced, resulting in significant economic losses. Currently, there is a lack of effective prevention and treatment measures. Spore germination is defined as the process by which spores release infectious elements into host cells under specific conditions [ 15 , 47 ]. The mechanisms and chemical factors that are essential for spore germination remain poorly understood. Currently, a number of stimuli, including pH, cations and anions, have been identified as triggers for germination [ 3 , 4 , 43 ]. For example, the spores of Nosema bombycis , a parasite found in insects, require treatment with a strong alkaline solution in order to induce germination [ 46 ]. The permeability of spores was found to be significantly altered by strongly alkaline solutions, which permitted the influx of greater numbers of external ions into the cell. It can be reasonably deduced that external ions compete with spore membrane ions for binding sites, which ultimately results in significant damage to the microsporidian membrane structure. There are three known modes of infestation of microsporidia. The first is the delivery of sporoplasm into the host cell by a pole tube piercing the host cell wall [ 44 ]. The second one is to swallow the spores into the cell by endocytosis, and then the pole tube punctures the phagocytic vesicles to inject the sporoplasm into the cell to complete the invasion [ 11 ]. The third one is that the pole tube protein interacts with the host cell receptor, induces its surface to be concave, and subsequently phagocytoses the sporoplasm by cytophagy to complete the invasion [ 16 ]. In recent decades, microsporidiosis has caused serious harm to sericulture, aquaculture, animal husbandry and human health, and the search for drugs that are effective against microsporidiosis has become a focus of attention recently. Currently, only a few medications are available for the treatment of microsporidiosis. Albendazole acts by binding to the microsporidian β-tubulin protein, thereby inhibiting its polymerisation and acting as a treatment for Encephalitozoon intestinalis infections [ 13 , 8 ]. Fumaricin, a crystalline antibiotic, has been shown to be effective against Encephalitozoon protozoa [ 34 ]. The nifedipine, metronidazole, and nitric oxide donors have been shown to significantly inhibit germination and infection of E. hellem and E. intestinalis [ 19 ] recently been demonstrated that the combination vinpocetine and albendazole is markedly more efficacious in the eradication of E. intestinalis [ 39 ]. Chlorogenic acid has attracted attention for its wide range of biological activities such as antioxidant, anti-inflammatory, antibacterial and antiviral [ 56 ]. It is non-toxic and has no side effects and is widely used in aquaculture [ 24 , 50 ]. Here, we initially sought to ascertained the impact of aforementioned drug in question on the in vitro germination, invasion and intracellular proliferation of A. portunus . Firstly, the drug concentrations of 0 µg/ml, 1 µg/ml, 10 µg/ml, 100 µg/ml, 1000 µg/ml, and 10000 µg/ml were employed for the treatment of the spores of A. portunus , followed by induction of germination. Subsequently, once the safe dose range of the drug had been established, experiments were conducted to determine the inhibitory effect of the drug on spore invasion and intracellular proliferation in RK13 cells. Finally, the spores treated with varying drug concentrations 0 µg/ml (PZ), 1 µg/ml (PO), 100 µg/ml (PH)) underwent comparative transcriptional analysis (PO/PZ, PH/PZ, and PH/PO) to elucidate the distinctions between the pre- and post-treatment stages. The methodologies and findings presented here can serve as a basis for future research into the development of drugs for the treatment of aquatic microsporosis. Materials and Methods Sample collection and processing The swimming crabs were obtained from farms in Lianyungang (Jiangsu, China) in October 2023. The specimens were expeditiously conveyed to the laboratory for subsequent dissection. qPCR experiments were performed using SSU rRNA-specific primers tailored for A. portunus to determine the presence of A. portunus infection in swimming crabs. Purification and isolation of spores of A. portunus The crab muscles were isolated using sterilized tweezers, followed by the addition of 3 ml of PBS and thorough grinding using a metal grinder. Subsequently, the filtrate was subjected to centrifugation at 3000 rpm for a period of five minutes, with the objective of removing the supernatant. This was followed by filtration through a cell strainer, with the intention of removing any residual large tissue particles. Subsequently, the filtrate was subjected to centrifugation using discontinuous density gradient percoll separation solutions (25%, 50%, 75%, and 100%) for 30 minutes at 4 degrees celsius and 8000 g, with the objective of isolating purified spores. Following this, the spores were enumerated using a hemocytometer. The aforementioned purification process was repeated to obtain twelve batches of spores in test tubes. Drug treated and in vitro germination The drug was dissolved in sterile distilled water and then diluted in gradients to 0 µg/ml (ctrl), 1 µg/ml, 10 µg/ml, 100 µg/ml, 1000 µg/ml, and 10000 µg/ml. Different concentrations of drugs were added to six tubes of purified spores, which were placed in an incubator at 27°C for overnight treatment for 12 h. Each group was subsequently washed three times with PBS. Finally, each group was incubated at 27°C for one hour with 0.01 mol/L KOH, and the number of germinated spores was counted under a microscope at 100x magnification (Zeiss, AxioScope.A1). The analysis of germination rate ware generated using the SPSS (IBM SPSS Statistics) and GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA). Drug cytotoxicity assay The adherent RK13 cells were digested with trypsin and collected by centrifugation, and resuspended by adding complete medium. The cells were inoculated into 96-well cell culture plates and incubated overnight at 37°C 5% CO 2 incubator until the cells spread to the bottom of the wells. The culture fluid was aspirated, washed with sterile PBS, and medium containing different concentrations of the drug was added with concentration gradients of 0 µg/ml, 1 µg/ml, 10 µg/ml, 100 µg/ml, 1000 µg/ml, and 10,000 µg/ml. It was incubated in the incubator for 48 h. The medium was aspirated and washed once with PBS. To each group, 90µl of fresh medium without drug and 10µl of CCK-8 were added and incubated in the incubator for 2 h. Absorbance was detected in the enzyme marker and calculated. Based on the cytotoxicity assay experiments, we determined the concentrations of the drug for anti- A. portunus invasion and intracellular proliferation assays to be 1 µg/ml,10 µg/ml, and 100 µg/ml, respectively. The analysis of drug cytotoxicity assay ware generated using the GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA). Experiments on drug inhibition of invasion of A. portunus The RK13 cells were digested with trypsin and collected by centrifugation, and resuspended by adding complete medium. The cells were inoculated into 12-well cell culture plates with 10 5 cells per well and incubated overnight at 37°C 5% CO 2 incubator until the cells spread over the bottom of the wells. After PBS washing, fresh medium containing different concentrations of drugs and purified spores was added and incubated for 6 h, 12 h and 24 h. After PBS washing for 2 times to remove spores that did not successfully invade the RK13 cells, the medium was finally added and incubated in the incubator at 37°C for 1 day. Cells were digested in each well using trypsin and collected. The analysis of the experiments on drug inhibition of invasion of A. portunus ware generated using the GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA). Experiments on drug inhibition of intracellular proliferation of A. portunus The RK13 cells were digested with trypsin and collected by centrifugation, and resuspended by adding complete medium. The cells were inoculated into 12-well cell culture plates with 10 5 cells per well and incubated overnight at 37°C 5% CO 2 incubator until the cells spread over the bottom of the wells. After washing with PBS, fresh medium containing spores was added, controlled to 10 8 spores per well, and incubated in the incubator for 24 h. After washing with PBS twice to remove spores that did not successfully invade the RK13 cells, medium containing different concentrations of the drug, 0 µg/ml, 1 µg/ml, 10 µg/ml, and 100 µg/ml, was added; and incubated in the incubator at 37°C for 6 days. Cells were digested in each well using trypsin and collected. Finally, qRT-PCR experiments were performed using SSU rRNA-specific primers designed for A. portunus to quantify microsporidian invasion and intracellular proliferation in RK13 cells. Based on the above experimental results, we selected 0 µg/ml, 1 µg/ml and 100 µg/ml for transcriptome sequencing to investigate the molecular mechanism of the drug in inhibiting spore invasion and intracellular proliferation. The analysis of the experiments on drug inhibition of intracellular proliferation of A. portunus ware generated using the GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA). Total RNA extraction, library preparation for transcriptome sequencing Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA). Total amount and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bio analyzer 2100 system. All nine samples had RNA integrity values greater than seven and could be used for subsequent analysis. High quality sequencing libraries were obtained using the NEB Next Ultra™ RNA Library Prep Kit for Illumina (NEB, USA). Quality control and gene expression level analysis Reads containing adapters, poly-N sequences, and low-quality reads were filtered from the raw data to obtain clean reads. The clean reads were assembled using Trinity [ 12 ] to generate the reference sequence. Subsequently, we conducted functional annotation and gene analysis using seven databases: NR, NT, KO, Swiss-Prot, Pfam, GO, and KOG. Differential expression analysis and functional enrichment Differential expression analysis between PO/PZ, PH/PZ, and PH/PO groups was conducted by calculating the expression level of each gene using the FPKM method [ 45 ] based on the DESeq2 software [ 27 ]. The resulting P-values were adjusted using the Benjamini and Hochberg method to control the false discovery rate. The padj 1 were set as the threshold for DEGs. GOseq [ 55 ] and KOBAS [ 29 ] were used for gene Ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, respectively. Experimental validation using qRT-PCR Ten DEGs were randomly selected based on the enrichment results of GO and KEGG for qRT-PCR to validate transcriptome sequencing results. Primer Premier 5.0 was used to design primers, and the 18S rRNA was used as internal reference gene The oligonucleotide primer pairs listed in Table S1 were used for the qRT-PCR assays. We reversely transcribed total RNA to cDNA using the Prime Script™ RT kit (Takara, China). The qRT-PCR experiment was performed with Hieff qPCR SYBR Green Master Mix (Yeasen, China) on the CFX Connect (BIORAD, USA). The fold­change in gene expression relative to the control was calculated using the 2 −ΔΔCt method. with the A. portunus 18S rRNA gene as the reference gene. All qRT-PCR experiments were performed in triplicate. All figures were produced utilizing Adobe Illustrator software. Results The effect of drugs on spore germination After the spores were treated with different concentrations of drugs and then germinated in vitro (Fig. 1 A). At a drug concentration of 1 µg/ml, the spore germination rate was observed to be 45.99%, a figure that represents a decrease of 21.14% in comparison to the control group. When the drug concentration was 10 µg/ml, the spore germination rate was 40.61%, compared with the control group, the decrease rate was 30.37%. When the drug concentration was 100 µg/ml, the spore germination rate was 32.26%, which was 44.68% lower than that of the control group. At a drug concentration of 1000 µg/ml, the spore germination rate was observed to be 27.07%, a figure that represents a decrease of 53.58% in comparison to the control group. When the drug concentration was 10000 µg/ml, the spore germination rate was 17.61%, compared with the control group, the decrease rate was 69.80%. The detection of drug toxicity to RK13 cells The cytotoxicity of the drug on RK13 cells was assessed using the CCK-8 assay (Fig. 1 B). The viability of RK13 cells was found to be largely unaffected by drug concentrations of 1 µg/ml, 10 µg/ml, and 100 µg/ml, with a maintained viability of approximately 98%. While at 1000 µg/ml concentration, the cell viability was only 10.99%, while at 10000 µg/ml, it was only 4.60%. Therefore, within the range of safe doses, we conducted subsequent experiments and analysis to determine the drug's inhibition of invasion and intracellular proliferation of spores. The experiment of drug inhibition of spore invasion and intracellular proliferation We infected RK13 cells with different concentrations of the drug mixed with fresh medium containing spores and incubated them for 6 h, 12 h and 24 h (Fig. 1 C). Quantification of spores in the RK13 cells by qRT-PCR found that at 6 h and 12 h, the number of spores in the RK13 cells decreased at 1 µg/ml and 10 µg/ml. In addition, at 12 h of incubation, the number of spores in RK13 cells at 100 µg/ml drug concentration was less than 1 µg/ml. After the spores infected RK13 cells, medium containing different concentrations of drugs was added and incubated at 37°C incubator for 6 days (Fig. 1 D). The spores in R13 cells were quantified by qRT-PCR, and it was found that the number of spores in the RK13 cells decreased under the treatment of different concentrations of the drug. Sequencing analysis of the A. portunus transcriptome After filtering and quality control of the raw reads, an average of 21,960,342 clean reads and a sequence count of 6.8G were obtained, with average Q20 and Q30 ratios of 98.35% and 95.22%, respectively, and a GC content of 40.43%. These results indicated that the data were of high quality and suitable for subsequent bioinformatic analysis. The statistical results of transcriptome sequencing data are available in Table S2. Functional annotation of genes and differentially expressed genes analysis The annotation of unigenes in different gene database are available in Table S3. In total, 118,762 unigenes were annotated in the public database, with 78,098 (56.15%) in NR, 81,424 (58.54%) in NT, 52,475 (37.73%) in KO, 75,562 (54.33%) in Swiss-Prot, 60,960 (43.83%) in Pfam, 59,999 (43.14%) in GO, and 39,444 (28.36%) in KOG, of which 118,726 (85.36%) unigenes were annotated in at least one database. The padj 1 was used as criteria to identify differentially expressed up- and down-regulated genes. A total of 2,049 DEGs between PO and PZ, with 4 up-regulated and 2,045 down-regulated DEGs (Fig. 2 A), and a total of 5,703 DEGs between PH and PZ, with 1,114 up-regulated and 4,589 down-regulated DEGs (Fig. 2 B), and a total of 6,492 DEGs between PH and PO, with 4,614 up-regulated and 1,878 down-regulated DEGs (Fig. 2 C), were identified, respectively. Functional enrichment analysis of DEGs The most enriched pathway terms are available in Table S4. GO analysis includes three levels, namely biological process (BP), cellular component (CC) and molecular function (MF). Between PO and PZ, the most enriched up-regulated DEGs were associated with protein modification process, catalytic activity, and transferase activity, and the most enriched down-regulated DEGs were associated with signaling, DNA replication , endoplasmic reticulum, immune system process (Fig. 2 D) (padj < 0.05). Between PH and PZ, the most enriched up-regulated DEGs were associated with carbohydrate metabolic process, ribosome biogenesis, transmembrane transport, and cellular amino acid metabolic process, and the most enriched down-regulated DEGs were associated with signaling, DNA replication , reproductive process, transcription, DNA-templated, meiotic nuclear division (Fig. 2 E) (padj < 0.05). Between PH and PO, the most enriched up-regulated DEGs were associated with carbohydrate metabolic process, transmembrane transport, lipid metabolic process, cellular amino acid metabolic process, and ribosome biogenesis, and the most enriched down-regulated DEGs were associated with cellular amino acid metabolic process, detoxification, and ribosome biogenesis (Fig. 2 F) (padj < 0.05). Between PO and PZ, KEGG analysis showed that significantly upregulated DEGs were mainly enriched in digestive system, including carbohydrate digestion and absorption, mineral absorption, gastric acid secretion, protein digestion and absorption, salivary secretion, bile secretion, and pancreatic secretion; excretory system , including proximal tubule bicarbonate reclamation, aldosterone-regulated sodium reabsorption, and endocrine and other factor-regulated calcium reabsorption; endocrine system, including insulin secretion and thyroid hormone synthesis (Fig. 3A). Significantly downregulated DEGs were mainly enriched in various signal transduction pathway, such as cAMP signaling pathway, Rap1 signaling pathway, Ras signaling pathway, and PI3K-Akt signaling pathway (Fig. 3B). Between PH and PZ, significantly upregulated DEGs were mainly enriched in carbohydrate metabolism, including propanoate metabolism, citrate cycle (TCA cycle), glyoxylate and dicarboxylate metabolism, starch and sucrose metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, galactose metabolism, amino sugar and nucleotide sugar metabolism, and pentose and glucuronate interconversions; amino acid metabolism, including beta-Alanine metabolism, valine, leucine and isoleucine degradation, alanine, aspartate and glutamate metabolism, valine, leucine and isoleucine biosynthesis, histidine metabolism, lysine biosynthesis; lipid metabolism, including alpha-Linolenic acid metabolism, fatty acid degradation, biosynthesis of unsaturated fatty acids. Ribosome, proteasome, and peroxisome (Fig. 3C). Significantly downregulated DEGs were also mainly enriched in various signal transduction pathway such as Wnt signaling pathway, ErbB signaling pathway, JAK-STAT signaling pathway, Notch signaling pathway, cAMP signaling pathway, Rap1 signaling pathway, and Ras signaling pathway (Fig. 3D). Between PH and PO, the enrichment of significantly up-regulated DEGs was similar to that in the PH and PZ groups, mainly focusing on carbohydrate metabolism, amino acid metabolism, lipid metabolism, ribosome, proteasome, and peroxisome (Fig. 3E). Significantly downregulated DEGs were also mainly enriched in amino acid metabolism (Fig. 3F). Figure 3 The KEGG enrichment of differentially expressed genes between PO and PZ group ( A , B ), PH and PZ group ( C, D ), PH and PO group ( E, F ) Validation of transcriptome data by qRT-PCR To verify the accuracy of the transcriptome sequencing results, we randomly selected ten DEGs for qRT-PCR based on the enrichment results of GO and KEGG, including five up-regulated genes and five down regulated genes. The expression trend of qRT-PCR was consistent with that of transcriptome sequencing (Fig. 4 ). These results demonstrated the reliability of the transcriptome sequencing data. Discussion Following the treatment of spores with varying concentrations of drugs and then germination in vitro, it was observed that the drugs exerted a pronounced inhibitory effect on the germination of spores. The germination rate of spores was found to decline significantly with the increase in drug concentration (Fig. 1 A), and the rate of decrease was significantly increased compared to the control. The growth of RK13 cells was found to be inhibited in a dose-dependent manner as the concentration of the drug increased (Fig. 1 B). At concentrations below 100 µg/ml, the drug was not observed to exert a toxic effect on RK13 cells. The viability of the cells is maintained within a stable range. Whereas, at 1000 µg/ml and 10000 µg/ml, the cell viability was significantly reduced, and the toxicity of the drug to the cells increased significantly. The experimental results in the safe dose range showed that the drug could inhibit the invasion and the intracellular proliferation of the spores of A. portunus (Fig. 1 C and D). The most enriched pathway terms DNA replication is essential for the reproduction of life and the development of complex organisms [ 48 ]. DNA replication is the basis of cell proliferation, and only accurate DNA replication can ensure the healthy growth and division of cells [ 33 , 53 ]. Genes associated with DNA replication were found to be significantly down-regulated at drug concentrations of 1 µg/ml and 100 µg/ml (Fig. 2 D and E), which suggests that this drug could significantly inhibit the DNA replication process of spores, thereby affecting their proliferation, differentiation, growth and development. In addition, we found that the number of down-regulated genes associated with DNA replication was significantly higher under 100 µg/ml drug treatment than under 1 µg/ml drug treatment (83 vs 33). This may be related to the different effects of different drug concentrations on in vitro germination, invasion and intracellular proliferation of spores. This also explains why the inhibitory effect of 100 µg/ml drug on the in vitro germination and the invasion of spores was significantly higher than that of 1 µg/ml (Fig. 1 A and C). Under both 1 µg/ml and 100 µg/ml drug treatments, the significantly down-regulated genes were concurrently enriched in cAMP signaling pathway, Rap1 signaling pathway, and Ras signaling pathway (Fig. 3B and D). The cAMP signaling pathway affects cellular metabolic activities and physiological processes by regulating intracellular cAMP levels, which plays important roles in differentiation, proliferation, osmoregulation, oxidative stress, and community sensing [ 35 ]. Research suggests that adenylyl cyclases (ACs) and phosphodiesterases (PDEs) in the cAMP signaling pathway may be promising new drug targets for the treatment of certain parasitic infections such as malaria, toxoplasmosis, leishmaniasis and giardiasis [ 21 , 25 , 28 , 37 , 38 ]. In our study, the genes encoding these two enzymes showed no significant changes in expression in response to 1 µg/ml and 100 µg/ml drug stimulation. Therefore, these two enzymes may not be able to be used as drug targets for the treatment of A. portunus infesting P. trituberculatus and S. paramamosain . Nevertheless, we found that the expression of many genes in the cAMP signaling pathway was significantly down-regulated under both concentrations of drug treatment (Fig. 5 and Table S4). This suggests that this drug affects the cAMP signaling pathway in spores, which in turn may affect processes such as spore proliferation and differentiation, which is consistent with our experimental results. Moreover, the number of genes down-regulated at 100 µg/ml drug concentration was significantly more than 1 µg/ml, and, the genes significantly down-regulated under the stimulation of 100 µg/ml drug concentration spread all over the cAMP signaling pathway, which suggests that the 100 µg/ml drug stimulation had a greater effect than 1 µg/ml on the proliferation and differentiation aspect of spores. This is in line with the conclusion drawn from the DNA replication discussed above. The Rap1 signaling pathway plays a role in a variety of cellular processes, including cell adhesion, cell junction, cell migration, polarisation, proliferation and survival [ 5 ]. Similarly, in this signaling pathway, the number of genes significantly down-regulated by 100 µg/ml drug stimulation was significantly higher than 1 µg/ml, and the significantly down-regulated genes were spread throughout the signaling pathway (Fig. 6 and Table S4). This is consistent with the above findings and further validates our inference. In addition, we found that the expression of genes encoding rap1 and ras was significantly down-regulated under 100 µg/ml drug stimulation. Rap1 and ras are two GTPases [ 40 ]. These two enzymes act as molecular switches that regulate cellular responses to external stimuli by cycling between an inactive state of GDP binding and an active state of GTP binding [ 20 , 31 ]. As a key enzyme in the ras signaling pathway, the expression of the gene encoding ras was significantly down-regulated by both 1 µg/ml and 100 µg/ml drug stimulation (Fig. 7 and Table S4). Therefore, ras is promising as a drug target for the treatment of A. portunus . However, this requires further experimental verification. In addition, consistent with the two signaling pathways described above, the number of genes down-regulated in the ras signaling pathway at 100 µg/ml drug concentration was significantly greater than 1 µg/ml, and the significantly down-regulated genes were spread throughout the signaling pathway. The results of the enrichment of differentially expressed genes in various signaling pathways provide further insight into the mechanism by which 100 µg/ml drug exhibited a significantly greater inhibitory effect on the in vitro germination and spore invasion than 1 µg/ml. In addition to the several signalling pathways mentioned above, some genes were found to be significantly enriched in other signalling pathways under 100 µg/ml drug treatment, e.g., Wnt signal pathway, ErbB signal pathway, Notch signal pathway, and JAK/STAT signaling pathway. The Wnt signal pathway, is a highly conserved pathway that regulates various cellular processes, such as cell proliferation, differentiation, apoptosis, and cell renewal [ 61 ]. ‌ErbB signaling pathway plays an important role in cell proliferation, migration, differentiation, apoptosis and cell migration [ 18 ]. The Notch signaling pathway affects several biological processes, including cell growth, development, differentiation, apoptosis, proliferation and cell boundary formation and tissue repair [ 60 ]. The JAK/STAT signaling pathway is a series of chain reactions of intracellular protein interactions that are involved in key biological processes such as cell proliferation, differentiation, apoptosis and immune regulation [ 52 ]. These several signaling pathways all play a role in cell proliferation and differentiation. This further suggests that 100 µg/ml has a greater effect on spore in vitro germination, invasion and intracellular proliferation, consistent with the above results. The spores showed stress response under different concentrations of drug treatments. Compared with 1 µg/ml and control group, the spore stress response was significantly increased at 100 µg/ml concentration, and the expression levels of genes related to ribosomal biosynthesis, ribosome, proteasome and peroxisome were significantly up-regulated (Fig. 2 E, F and Fig. 3C, E). Functionally, ribosomes play a pivotal role in regulating a multitude of cellular activities, including transcription, translation, protein synthesis, cell proliferation, differentiation, apoptosis, development, and transformation [ 23 ]. Ribosomes also play an important role in the cellular response to stress [ 30 ]. Under stress conditions, the proteasome helps cells adapt to environmental changes and maintains the stability of the intracellular environment by regulating protein degradation and synthesis [ 1 , 10 ]. Oxidoreductases, particularly peroxidases, are crucial for cell metabolism and resistance to cytotoxicity [ 6 , 32 , 58 ]. Furthermore, compared with 1 µg/ml and control group, genes linked to amino acid, carbohydrate and lipid metabolism exhibited a notable increase in expression (Fig. 3C, E). Amino acids serve as essential building blocks for protein, enzyme, and nucleic acid synthesis, and they also contribute to immune regulation [ 7 ]. Additionally, they play crucial roles in energy metabolism, cellular signal transduction, and gene expression [ 59 ]. The lipid and carbohydrate metabolic pathways, such as fatty acid metabolism, glycolysis/gluconeogenesis, and pentose phosphate pathway, are also involved in energy provision [ 9 , 26 , 36 , 57 ]. Therefore, we hypothesised that the up-regulation of these pathways is related to the stress response generated by spores in response to drug stimuli. They provide sufficient energy and raw materials for spores to resist the drug stimulation process. Conclusions Chlorogenic acid is a safe drug that is being investigated for the treatment of a variety of diseases due to its wide range of biological activities, but there is no information on its effects on microsporidiosis. In the present study, we found that this drug could significantly inhibit the in vitro germination, invasion and intracellular proliferation of the spores of A. portunus , which in turn affected spore proliferation, differentiation, growth and development. The inhibition of spore germination and invasion by the 100 µg/ml drug concentration was significantly greater than that observed at the 1 µg/ml concentration. The ras protein in the Rap1 and Ras signaling pathway is promising as a drug target. The spores also demonstrated a range of survival strategies in response to varying concentrations of drugs. To the best of our knowledge, this is the inaugural study to examine the microsporicidal activity of chlorogenic acid on aquatic microsporidia. Nevertheless, further research is imperative to elucidate the mechanism of action and to corroborate these findings in vivo conditions. Declarations CRediT authorship contribution statement Jiu - Yang Chen : Conceptualization , Methodology, Formal analysis, Investigation, Data Curation, Writing-Original Draft, Writing-Review and Editing; Ya-Li Xu and Zhi-Qiang Zhu: Formal analysis, Investigation, Data Curation, Writing-Original Draft; Jin-Yong Zhang: Conceptualization, Resources, Supervision, Project administration, Funding acquisition. Zhao-Zhe Xin: Conceptualization, Resources, Writing-Review and Editing, Supervision, Project administration, Funding acquisition. All authors have read and agreed to the publication of this manuscript. Acknowledgments This study was supported by the Natural Science Foundation of Shandong Province (no. ZR2022QC250), the Special Support Program of Qingdao Agricultural University awarded to ZZ Xin, National Natural Science Foundation of China (no. 32173019), Young experts of Taishan Scholars in Shandong Province (no. tsqn201909133), Initiative grant for high-level personnel recruitment in Qingdao Agricultural University awarded to JY Zhang, the “First Class Fishery Discipline" Programme [(2020)3] in Shandong Province, China, the Talent plan “One Thing One Decision (Yishi Yiyi)” in Shandong Province, China. Declaration of competing interest All authors have read and approved the contents of this manuscript. 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. Ethical Statement In this experiment, the collection and handling of crabs were performed in accordance with the guidelines of the Qingdao Agricultural University Animal Care and Use Committee (Qingdao, China) References An H, Ordureau A, Korner M, Paulo JA, Harper JW. Systematic quantitative analysis of ribosome inventory during nutrient stress. Nature. 2020;583(7815):303-309. https://doi.org/10.1038/s41586-020-2446-y. Bojko J, Reinke AW, Stentiford GD, Williams B, Rogers MSJ, Bass D. Microsporidia: a new taxonomic, evolutionary, and ecological synthesis. Trends Parasitol. 2022;38(8):642-659. https://doi.org/10.1016/j.pt.2022.05.007. Cali A, Takvorian PM. Developmental Morphology and Life Cycles of the Microsporidia. 2nd ed. Microsporidia; 2014. https://doi.org/10.1002/9781118395264.ch2. Cali A, Weiss LM, Takvorian PM. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6226867","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":437007946,"identity":"2e4866c3-afc0-4ab5-9622-fa184fa933ad","order_by":0,"name":"Jiu-Yang Chen","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiu-Yang","middleName":"","lastName":"Chen","suffix":""},{"id":437007947,"identity":"d2f0c724-310b-4ee0-902d-901f8b31a543","order_by":1,"name":"Ya-Li Xu","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ya-Li","middleName":"","lastName":"Xu","suffix":""},{"id":437007948,"identity":"39911854-dcdd-4346-a261-1410ab6fa056","order_by":2,"name":"Zhi-Qiang Zhu","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Qiang","middleName":"","lastName":"Zhu","suffix":""},{"id":437007949,"identity":"5af21230-439f-4588-a087-310021e291b9","order_by":3,"name":"Jin-Yong Zhang","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jin-Yong","middleName":"","lastName":"Zhang","suffix":""},{"id":437007950,"identity":"0b020a65-faec-4ae3-87e6-582706ceed63","order_by":4,"name":"Zhao-Zhe Xin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDACZjBiYDBgYD5Asha2BJIsAmnhMSBOucFx3sOfC2ps7M3Zez7eeMNgJ6fbQECLZDNfgvGMY2nMlj1nN1vOYUg2NjtAQAs/M49BMg/bYTaDG7nbpHkYDiRuI6SFDajlMM+/wzwG9988I04L0BbDZt62wxIGN3jYiNMi2cxjzMzbl2ZgcCbN2HKOARF+MTh/xvgzzzcbe4Pjhx/eeFNhJ0dQCwqQIDZqkLWQqmMUjIJRMApGBAAAd0s4qTVQBfEAAAAASUVORK5CYII=","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Zhao-Zhe","middleName":"","lastName":"Xin","suffix":""}],"badges":[],"createdAt":"2025-03-14 13:38:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6226867/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6226867/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80016789,"identity":"0dce29c6-021b-4831-bd47-544dfcda5cb6","added_by":"auto","created_at":"2025-04-07 03:34:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":187148,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different concentrations of drugs on the in vitro germination, invasion and intracellular proliferation of spores. Effect of different concentrations of drugs on the in vitro germination of spores (\u003cstrong\u003eA\u003c/strong\u003e). The detection of drug toxicity to RK13 cells (\u003cstrong\u003eB\u003c/strong\u003e). Effect of drugs in the safe dose range on the spore invasion (\u003cstrong\u003eC\u003c/strong\u003e). Effect of drugs in the safe dose range on the intracellular proliferation of spores (\u003cstrong\u003eD\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/9424dc14b75bf08a5b600d82.jpg"},{"id":80015844,"identity":"28234baf-8996-4654-af73-8b3b7365c5db","added_by":"auto","created_at":"2025-04-07 03:18:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2362639,"visible":true,"origin":"","legend":"\u003cp\u003eThe volcano plots and GO enrichment analysis of differentially expressed genes between PO and PZ group (\u003cstrong\u003eA, D\u003c/strong\u003e), PH and PZ group (\u003cstrong\u003eB, E\u003c/strong\u003e), PH and PO group (\u003cstrong\u003eC, F\u003c/strong\u003e). BP: biological process; CC: cellular component; MF: molecular function\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/1fd2bf7f08e7c7e45465cb00.jpg"},{"id":80016460,"identity":"4e415a41-c47f-4196-b38c-611255620c33","added_by":"auto","created_at":"2025-04-07 03:26:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1770892,"visible":true,"origin":"","legend":"\u003cp\u003eThe KEGG enrichment of differentially expressed genes between PO and PZ group (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e), PH and PZ group (\u003cstrong\u003eC, D\u003c/strong\u003e), PH and PO group (\u003cstrong\u003eE, F\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/1a69c146867e083a32ee3738.jpg"},{"id":80015843,"identity":"e868a104-71a7-4d15-a937-3a2529c336c1","added_by":"auto","created_at":"2025-04-07 03:18:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68087,"visible":true,"origin":"","legend":"\u003cp\u003eThe qRT-PCR validation of differentially expressed genes randomly selected from the three groups PO and PZ, PH and PZ, PH and PO\u003cstrong\u003e.\u003c/strong\u003e The x-axis represents gene abbreviations, the y-axis represents fold change value. Details of the gene abbreviations are as follows: RBS1: 40S ribosomal protein S1, ACoA: Acetyl-CoA synthetase, AOD: Alternative oxidase, RBS6: 40S ribosomal protein S6-B, DBPS: 3,4-dihydroxy-2-butanone 4-phosphate synthase. TSC: TSC22 domain family protein 2, CAP: CAP-Gly domain-containing linker protein 1, BCP: Bromodomain-containing protein 4, LA2: Liprin-alpha-2, ZFM: Zinc finger MYM-type protein 4\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/9bf42d138b0cc19bcc6bdabf.jpg"},{"id":80015847,"identity":"9a97ebe3-dc28-4537-aa0f-b0991a0ac770","added_by":"auto","created_at":"2025-04-07 03:18:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1607704,"visible":true,"origin":"","legend":"\u003cp\u003eThe cAMP signaling pathway with significant enrichment of differentially expressed genes in the PO and PZ group (\u003cstrong\u003eA\u003c/strong\u003e), PH and PZ group (\u003cstrong\u003eB\u003c/strong\u003e). Red boxes represent significantly down-regulated genes\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/ee17dc3931a07ced9bf916ab.png"},{"id":80015848,"identity":"442c82d2-705d-4341-8273-9db154eed0ee","added_by":"auto","created_at":"2025-04-07 03:18:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5024029,"visible":true,"origin":"","legend":"\u003cp\u003eThe Rap1 signaling pathway with significant enrichment of differentially expressed genes in the PO and PZ group (\u003cstrong\u003eA\u003c/strong\u003e), PH and PZ group (\u003cstrong\u003eB\u003c/strong\u003e). Red boxes represent significantly down-regulated genes.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/38ad7f28b6b5b647d22d6bec.jpg"},{"id":80016463,"identity":"e8426b84-f13f-4804-be4c-63cc190c80a8","added_by":"auto","created_at":"2025-04-07 03:26:05","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5769870,"visible":true,"origin":"","legend":"\u003cp\u003eThe Ras signaling pathway with significant enrichment of differentially expressed genes in the PO and PZ group (\u003cstrong\u003eA\u003c/strong\u003e), PH and PZ group (\u003cstrong\u003eB\u003c/strong\u003e). Red boxes represent significantly down-regulated genes.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/bff6b7f3ccd642b9a7f94eb3.jpg"},{"id":83235584,"identity":"6eff28f3-b197-4381-9f3d-3de33a2e1c1f","added_by":"auto","created_at":"2025-05-21 14:32:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":67226241,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/bec5ff97-53fc-4273-a696-98d882150866.pdf"},{"id":80015842,"identity":"6e77fda5-efba-4d9a-a5fc-d32962174e9d","added_by":"auto","created_at":"2025-04-07 03:18:05","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13191,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationSI.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6226867/v1/3d6f55241c0cf4730083c784.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chlorogenic acid on the in vitro germination, invasion and intracellular proliferation of Ameson portunus (Microsporidia)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrosporidia are a group of unicellular eukaryotic organisms that have evolved a specialised form of intracellular parasitism [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Since the initial discovery and naming of microsporidia in the mid-19th century, over 1,700 species of microsporidia have been identified and characterised globally [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Microsporidia exhibit a broad host range, parasitising diverse animal hosts from vertebrates to invertebrates. This has resulted in significant harm to sericulture, aquaculture, animal husbandry, and human health. More than half of the microsporidia infested aquatic animals [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To date, over 30 microsporidia species have been identified as pathogens affecting more than 20 aquaculture species globally [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A total of over 50 different genera have been identified as potential pathogens affecting crustaceans. Of these, 11 to 12 species of microsporidia infesting crabs belong to nine genera, including \u003cem\u003eAbelspora, Ameson, Areospora, Enterospora, Hepatospora, Nadelspora, Nosema, Ormieresia\u003c/em\u003e, and \u003cem\u003eThelohania\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Previous research has indicated that \u003cem\u003eAmeson portunus\u003c/em\u003e is the recently identified causative agent responsible for \"toothpaste disease\" in \u003cem\u003ePortunus trituberculatus\u003c/em\u003e and \"slurry-like syndrome\" in \u003cem\u003eScylla paramamosain\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The disease is prevalent in regions where economic crab culture is practiced, resulting in significant economic losses. Currently, there is a lack of effective prevention and treatment measures.\u003c/p\u003e \u003cp\u003eSpore germination is defined as the process by which spores release infectious elements into host cells under specific conditions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The mechanisms and chemical factors that are essential for spore germination remain poorly understood. Currently, a number of stimuli, including pH, cations and anions, have been identified as triggers for germination [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. For example, the spores of \u003cem\u003eNosema bombycis\u003c/em\u003e, a parasite found in insects, require treatment with a strong alkaline solution in order to induce germination [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The permeability of spores was found to be significantly altered by strongly alkaline solutions, which permitted the influx of greater numbers of external ions into the cell. It can be reasonably deduced that external ions compete with spore membrane ions for binding sites, which ultimately results in significant damage to the microsporidian membrane structure. There are three known modes of infestation of microsporidia. The first is the delivery of sporoplasm into the host cell by a pole tube piercing the host cell wall [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The second one is to swallow the spores into the cell by endocytosis, and then the pole tube punctures the phagocytic vesicles to inject the sporoplasm into the cell to complete the invasion [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The third one is that the pole tube protein interacts with the host cell receptor, induces its surface to be concave, and subsequently phagocytoses the sporoplasm by cytophagy to complete the invasion [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent decades, microsporidiosis has caused serious harm to sericulture, aquaculture, animal husbandry and human health, and the search for drugs that are effective against microsporidiosis has become a focus of attention recently. Currently, only a few medications are available for the treatment of microsporidiosis. Albendazole acts by binding to the microsporidian β-tubulin protein, thereby inhibiting its polymerisation and acting as a treatment for \u003cem\u003eEncephalitozoon intestinalis\u003c/em\u003e infections [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Fumaricin, a crystalline antibiotic, has been shown to be effective against \u003cem\u003eEncephalitozoon\u003c/em\u003e protozoa [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The nifedipine, metronidazole, and nitric oxide donors have been shown to significantly inhibit germination and infection of \u003cem\u003eE. hellem\u003c/em\u003e and \u003cem\u003eE. intestinalis\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] recently been demonstrated that the combination vinpocetine and albendazole is markedly more efficacious in the eradication of \u003cem\u003eE. intestinalis\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChlorogenic acid has attracted attention for its wide range of biological activities such as antioxidant, anti-inflammatory, antibacterial and antiviral [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. It is non-toxic and has no side effects and is widely used in aquaculture [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Here, we initially sought to ascertained the impact of aforementioned drug in question on the in vitro germination, invasion and intracellular proliferation of \u003cem\u003eA. portunus\u003c/em\u003e. Firstly, the drug concentrations of 0 \u0026micro;g/ml, 1 \u0026micro;g/ml, 10 \u0026micro;g/ml, 100 \u0026micro;g/ml, 1000 \u0026micro;g/ml, and 10000 \u0026micro;g/ml were employed for the treatment of the spores of \u003cem\u003eA. portunus\u003c/em\u003e, followed by induction of germination. Subsequently, once the safe dose range of the drug had been established, experiments were conducted to determine the inhibitory effect of the drug on spore invasion and intracellular proliferation in RK13 cells. Finally, the spores treated with varying drug concentrations 0 \u0026micro;g/ml (PZ), 1 \u0026micro;g/ml (PO), 100 \u0026micro;g/ml (PH)) underwent comparative transcriptional analysis (PO/PZ, PH/PZ, and PH/PO) to elucidate the distinctions between the pre- and post-treatment stages. The methodologies and findings presented here can serve as a basis for future research into the development of drugs for the treatment of aquatic microsporosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eSample collection and processing\u003c/p\u003e \u003cp\u003eThe swimming crabs were obtained from farms in Lianyungang (Jiangsu, China) in October 2023. The specimens were expeditiously conveyed to the laboratory for subsequent dissection. qPCR experiments were performed using SSU rRNA-specific primers tailored for \u003cem\u003eA. portunus\u003c/em\u003e to determine the presence of \u003cem\u003eA. portunus\u003c/em\u003e infection in swimming crabs.\u003c/p\u003e \u003cp\u003ePurification and isolation of spores of \u003cem\u003eA. portunus\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe crab muscles were isolated using sterilized tweezers, followed by the addition of 3 ml of PBS and thorough grinding using a metal grinder. Subsequently, the filtrate was subjected to centrifugation at 3000 rpm for a period of five minutes, with the objective of removing the supernatant. This was followed by filtration through a cell strainer, with the intention of removing any residual large tissue particles. Subsequently, the filtrate was subjected to centrifugation using discontinuous density gradient percoll separation solutions (25%, 50%, 75%, and 100%) for 30 minutes at 4 degrees celsius and 8000 g, with the objective of isolating purified spores. Following this, the spores were enumerated using a hemocytometer. The aforementioned purification process was repeated to obtain twelve batches of spores in test tubes.\u003c/p\u003e \u003cp\u003eDrug treated and in vitro germination\u003c/p\u003e \u003cp\u003eThe drug was dissolved in sterile distilled water and then diluted in gradients to 0 \u0026micro;g/ml (ctrl), 1 \u0026micro;g/ml, 10 \u0026micro;g/ml, 100 \u0026micro;g/ml, 1000 \u0026micro;g/ml, and 10000 \u0026micro;g/ml. Different concentrations of drugs were added to six tubes of purified spores, which were placed in an incubator at 27\u0026deg;C for overnight treatment for 12 h. Each group was subsequently washed three times with PBS. Finally, each group was incubated at 27\u0026deg;C for one hour with 0.01 mol/L KOH, and the number of germinated spores was counted under a microscope at 100x magnification (Zeiss, AxioScope.A1). The analysis of germination rate ware generated using the SPSS (IBM SPSS Statistics) and GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA).\u003c/p\u003e \u003cp\u003eDrug cytotoxicity assay\u003c/p\u003e \u003cp\u003eThe adherent RK13 cells were digested with trypsin and collected by centrifugation, and resuspended by adding complete medium. The cells were inoculated into 96-well cell culture plates and incubated overnight at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e incubator until the cells spread to the bottom of the wells. The culture fluid was aspirated, washed with sterile PBS, and medium containing different concentrations of the drug was added with concentration gradients of 0 \u0026micro;g/ml, 1 \u0026micro;g/ml, 10 \u0026micro;g/ml, 100 \u0026micro;g/ml, 1000 \u0026micro;g/ml, and 10,000 \u0026micro;g/ml. It was incubated in the incubator for 48 h. The medium was aspirated and washed once with PBS. To each group, 90\u0026micro;l of fresh medium without drug and 10\u0026micro;l of CCK-8 were added and incubated in the incubator for 2 h. Absorbance was detected in the enzyme marker and calculated. Based on the cytotoxicity assay experiments, we determined the concentrations of the drug for anti- \u003cem\u003eA. portunus\u003c/em\u003e invasion and intracellular proliferation assays to be 1 \u0026micro;g/ml,10 \u0026micro;g/ml, and 100 \u0026micro;g/ml, respectively. The analysis of drug cytotoxicity assay ware generated using the GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA).\u003c/p\u003e \u003cp\u003eExperiments on drug inhibition of invasion of \u003cem\u003eA. portunus\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe RK13 cells were digested with trypsin and collected by centrifugation, and resuspended by adding complete medium. The cells were inoculated into 12-well cell culture plates with 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e incubator until the cells spread over the bottom of the wells. After PBS washing, fresh medium containing different concentrations of drugs and purified spores was added and incubated for 6 h, 12 h and 24 h. After PBS washing for 2 times to remove spores that did not successfully invade the RK13 cells, the medium was finally added and incubated in the incubator at 37\u0026deg;C for 1 day. Cells were digested in each well using trypsin and collected. The analysis of the experiments on drug inhibition of invasion of \u003cem\u003eA. portunus\u003c/em\u003e ware generated using the GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA).\u003c/p\u003e \u003cp\u003eExperiments on drug inhibition of intracellular proliferation of \u003cem\u003eA. portunus\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe RK13 cells were digested with trypsin and collected by centrifugation, and resuspended by adding complete medium. The cells were inoculated into 12-well cell culture plates with 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e incubator until the cells spread over the bottom of the wells. After washing with PBS, fresh medium containing spores was added, controlled to 10\u003csup\u003e8\u003c/sup\u003e spores per well, and incubated in the incubator for 24 h. After washing with PBS twice to remove spores that did not successfully invade the RK13 cells, medium containing different concentrations of the drug, 0 \u0026micro;g/ml, 1 \u0026micro;g/ml, 10 \u0026micro;g/ml, and 100 \u0026micro;g/ml, was added; and incubated in the incubator at 37\u0026deg;C for 6 days. Cells were digested in each well using trypsin and collected. Finally, qRT-PCR experiments were performed using SSU rRNA-specific primers designed for \u003cem\u003eA. portunus\u003c/em\u003e to quantify microsporidian invasion and intracellular proliferation in RK13 cells.\u003c/p\u003e \u003cp\u003eBased on the above experimental results, we selected 0 \u0026micro;g/ml, 1 \u0026micro;g/ml and 100 \u0026micro;g/ml for transcriptome sequencing to investigate the molecular mechanism of the drug in inhibiting spore invasion and intracellular proliferation. The analysis of the experiments on drug inhibition of intracellular proliferation of \u003cem\u003eA. portunus\u003c/em\u003e ware generated using the GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA).\u003c/p\u003e \u003cp\u003eTotal RNA extraction, library preparation for transcriptome sequencing\u003c/p\u003e \u003cp\u003eTotal RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA). Total amount and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bio analyzer 2100 system. All nine samples had RNA integrity values greater than seven and could be used for subsequent analysis. High quality sequencing libraries were obtained using the NEB Next Ultra\u0026trade; RNA Library Prep Kit for Illumina (NEB, USA).\u003c/p\u003e \u003cp\u003eQuality control and gene expression level analysis\u003c/p\u003e \u003cp\u003eReads containing adapters, poly-N sequences, and low-quality reads were filtered from the raw data to obtain clean reads. The clean reads were assembled using Trinity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] to generate the reference sequence. Subsequently, we conducted functional annotation and gene analysis using seven databases: NR, NT, KO, Swiss-Prot, Pfam, GO, and KOG.\u003c/p\u003e \u003cp\u003eDifferential expression analysis and functional enrichment\u003c/p\u003e \u003cp\u003eDifferential expression analysis between PO/PZ, PH/PZ, and PH/PO groups was conducted by calculating the expression level of each gene using the FPKM method [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] based on the DESeq2 software [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The resulting P-values were adjusted using the Benjamini and Hochberg method to control the false discovery rate. The padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2(fold change)| \u0026gt;1 were set as the threshold for DEGs. GOseq [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and KOBAS [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] were used for gene Ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, respectively.\u003c/p\u003e \u003cp\u003eExperimental validation using qRT-PCR\u003c/p\u003e \u003cp\u003eTen DEGs were randomly selected based on the enrichment results of GO and KEGG for qRT-PCR to validate transcriptome sequencing results. Primer Premier 5.0 was used to design primers, and the 18S rRNA was used as internal reference gene The oligonucleotide primer pairs listed in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e were used for the qRT-PCR assays. We reversely transcribed total RNA to cDNA using the Prime Script\u0026trade; RT kit (Takara, China). The qRT-PCR experiment was performed with Hieff qPCR SYBR Green Master Mix (Yeasen, China) on the CFX Connect (BIORAD, USA). The fold\u0026shy;change in gene expression relative to the control was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. with the \u003cem\u003eA. portunus\u003c/em\u003e 18S rRNA gene as the reference gene. All qRT-PCR experiments were performed in triplicate. All figures were produced utilizing Adobe Illustrator software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe effect of drugs on spore germination\u003c/p\u003e \u003cp\u003eAfter the spores were treated with different concentrations of drugs and then germinated in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). At a drug concentration of 1 \u0026micro;g/ml, the spore germination rate was observed to be 45.99%, a figure that represents a decrease of 21.14% in comparison to the control group. When the drug concentration was 10 \u0026micro;g/ml, the spore germination rate was 40.61%, compared with the control group, the decrease rate was 30.37%. When the drug concentration was 100 \u0026micro;g/ml, the spore germination rate was 32.26%, which was 44.68% lower than that of the control group. At a drug concentration of 1000 \u0026micro;g/ml, the spore germination rate was observed to be 27.07%, a figure that represents a decrease of 53.58% in comparison to the control group. When the drug concentration was 10000 \u0026micro;g/ml, the spore germination rate was 17.61%, compared with the control group, the decrease rate was 69.80%.\u003c/p\u003e \u003cp\u003eThe detection of drug toxicity to RK13 cells\u003c/p\u003e \u003cp\u003eThe cytotoxicity of the drug on RK13 cells was assessed using the CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The viability of RK13 cells was found to be largely unaffected by drug concentrations of 1 \u0026micro;g/ml, 10 \u0026micro;g/ml, and 100 \u0026micro;g/ml, with a maintained viability of approximately 98%. While at 1000 \u0026micro;g/ml concentration, the cell viability was only 10.99%, while at 10000 \u0026micro;g/ml, it was only 4.60%. Therefore, within the range of safe doses, we conducted subsequent experiments and analysis to determine the drug's inhibition of invasion and intracellular proliferation of spores.\u003c/p\u003e \u003cp\u003eThe experiment of drug inhibition of spore invasion and intracellular proliferation\u003c/p\u003e \u003cp\u003eWe infected RK13 cells with different concentrations of the drug mixed with fresh medium containing spores and incubated them for 6 h, 12 h and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Quantification of spores in the RK13 cells by qRT-PCR found that at 6 h and 12 h, the number of spores in the RK13 cells decreased at 1 \u0026micro;g/ml and 10 \u0026micro;g/ml. In addition, at 12 h of incubation, the number of spores in RK13 cells at 100 \u0026micro;g/ml drug concentration was less than 1 \u0026micro;g/ml. After the spores infected RK13 cells, medium containing different concentrations of drugs was added and incubated at 37\u0026deg;C incubator for 6 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The spores in R13 cells were quantified by qRT-PCR, and it was found that the number of spores in the RK13 cells decreased under the treatment of different concentrations of the drug.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSequencing analysis of the \u003cem\u003eA. portunus\u003c/em\u003e transcriptome\u003c/p\u003e \u003cp\u003eAfter filtering and quality control of the raw reads, an average of 21,960,342 clean reads and a sequence count of 6.8G were obtained, with average Q20 and Q30 ratios of 98.35% and 95.22%, respectively, and a GC content of 40.43%. These results indicated that the data were of high quality and suitable for subsequent bioinformatic analysis. The statistical results of transcriptome sequencing data are available in Table S2.\u003c/p\u003e \u003cp\u003eFunctional annotation of genes and differentially expressed genes analysis\u003c/p\u003e \u003cp\u003eThe annotation of unigenes in different gene database are available in Table S3. In total, 118,762 unigenes were annotated in the public database, with 78,098 (56.15%) in NR, 81,424 (58.54%) in NT, 52,475 (37.73%) in KO, 75,562 (54.33%) in Swiss-Prot, 60,960 (43.83%) in Pfam, 59,999 (43.14%) in GO, and 39,444 (28.36%) in KOG, of which 118,726 (85.36%) unigenes were annotated in at least one database.\u003c/p\u003e \u003cp\u003eThe padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2|FoldChange|\u0026gt;1 was used as criteria to identify differentially expressed up- and down-regulated genes. A total of 2,049 DEGs between PO and PZ, with 4 up-regulated and 2,045 down-regulated DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and a total of 5,703 DEGs between PH and PZ, with 1,114 up-regulated and 4,589 down-regulated DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and a total of 6,492 DEGs between PH and PO, with 4,614 up-regulated and 1,878 down-regulated DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), were identified, respectively.\u003c/p\u003e \u003cp\u003eFunctional enrichment analysis of DEGs\u003c/p\u003e \u003cp\u003eThe most enriched pathway terms are available in Table S4. GO analysis includes three levels, namely biological process (BP), cellular component (CC) and molecular function (MF). Between PO and PZ, the most enriched up-regulated DEGs were associated with protein modification process, catalytic activity, and transferase activity, and the most enriched down-regulated DEGs were associated with signaling, \u003cem\u003eDNA replication\u003c/em\u003e, endoplasmic reticulum, immune system process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) (padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Between PH and PZ, the most enriched up-regulated DEGs were associated with carbohydrate metabolic process, ribosome biogenesis, transmembrane transport, and cellular amino acid metabolic process, and the most enriched down-regulated DEGs were associated with signaling, \u003cem\u003eDNA replication\u003c/em\u003e, reproductive process, transcription, DNA-templated, meiotic nuclear division (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) (padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Between PH and PO, the most enriched up-regulated DEGs were associated with carbohydrate metabolic process, transmembrane transport, lipid metabolic process, cellular amino acid metabolic process, and ribosome biogenesis, and the most enriched down-regulated DEGs were associated with cellular amino acid metabolic process, detoxification, and ribosome biogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) (padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBetween PO and PZ, KEGG analysis showed that significantly upregulated DEGs were mainly enriched in digestive system, including carbohydrate digestion and absorption, mineral absorption, gastric acid secretion, protein digestion and absorption, salivary secretion, bile secretion, and pancreatic secretion; \u003cem\u003eexcretory system\u003c/em\u003e, including proximal tubule bicarbonate reclamation, aldosterone-regulated sodium reabsorption, and endocrine and other factor-regulated calcium reabsorption; endocrine system, including insulin secretion and thyroid hormone synthesis (Fig.\u0026nbsp;3A). Significantly downregulated DEGs were mainly enriched in various signal transduction pathway, such as cAMP signaling pathway, Rap1 signaling pathway, Ras signaling pathway, and PI3K-Akt signaling pathway (Fig.\u0026nbsp;3B).\u003c/p\u003e \u003cp\u003eBetween PH and PZ, significantly upregulated DEGs were mainly enriched in carbohydrate metabolism, including propanoate metabolism, citrate cycle (TCA cycle), glyoxylate and dicarboxylate metabolism, starch and sucrose metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, galactose metabolism, amino sugar and nucleotide sugar metabolism, and pentose and glucuronate interconversions; amino acid metabolism, including beta-Alanine metabolism, valine, leucine and isoleucine degradation, alanine, aspartate and glutamate metabolism, valine, leucine and isoleucine biosynthesis, histidine metabolism, lysine biosynthesis; lipid metabolism, including alpha-Linolenic acid metabolism, fatty acid degradation, biosynthesis of unsaturated fatty acids. Ribosome, proteasome, and peroxisome (Fig.\u0026nbsp;3C). Significantly downregulated DEGs were also mainly enriched in various signal transduction pathway such as Wnt signaling pathway, ErbB signaling pathway, JAK-STAT signaling pathway, Notch signaling pathway, cAMP signaling pathway, Rap1 signaling pathway, and Ras signaling pathway (Fig.\u0026nbsp;3D).\u003c/p\u003e \u003cp\u003e Between PH and PO, the enrichment of significantly up-regulated DEGs was similar to that in the PH and PZ groups, mainly focusing on carbohydrate metabolism, amino acid metabolism, lipid metabolism, ribosome, proteasome, and peroxisome (Fig.\u0026nbsp;3E). Significantly downregulated DEGs were also mainly enriched in amino acid metabolism (Fig.\u0026nbsp;3F).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;3\u003c/b\u003e The KEGG enrichment of differentially expressed genes between PO and PZ group (\u003cb\u003eA\u003c/b\u003e, \u003cb\u003eB\u003c/b\u003e), PH and PZ group (\u003cb\u003eC, D\u003c/b\u003e), PH and PO group (\u003cb\u003eE, F\u003c/b\u003e)\u003c/p\u003e \u003cp\u003eValidation of transcriptome data by qRT-PCR\u003c/p\u003e \u003cp\u003eTo verify the accuracy of the transcriptome sequencing results, we randomly selected ten DEGs for qRT-PCR based on the enrichment results of GO and KEGG, including five up-regulated genes and five down regulated genes. The expression trend of qRT-PCR was consistent with that of transcriptome sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results demonstrated the reliability of the transcriptome sequencing data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFollowing the treatment of spores with varying concentrations of drugs and then germination in vitro, it was observed that the drugs exerted a pronounced inhibitory effect on the germination of spores. The germination rate of spores was found to decline significantly with the increase in drug concentration (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), and the rate of decrease was significantly increased compared to the control. The growth of RK13 cells was found to be inhibited in a dose-dependent manner as the concentration of the drug increased (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). At concentrations below 100 \u0026micro;g/ml, the drug was not observed to exert a toxic effect on RK13 cells. The viability of the cells is maintained within a stable range. Whereas, at 1000 \u0026micro;g/ml and 10000 \u0026micro;g/ml, the cell viability was significantly reduced, and the toxicity of the drug to the cells increased significantly. The experimental results in the safe dose range showed that the drug could inhibit the invasion and the intracellular proliferation of the spores of \u003cem\u003eA. portunus\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC and D).\u003c/p\u003e\n\u003cp\u003eThe most enriched pathway terms DNA replication is essential for the reproduction of life and the development of complex organisms [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. DNA replication is the basis of cell proliferation, and only accurate DNA replication can ensure the healthy growth and division of cells [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Genes associated with DNA replication were found to be significantly down-regulated at drug concentrations of 1 \u0026micro;g/ml and 100 \u0026micro;g/ml (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD and E), which suggests that this drug could significantly inhibit the DNA replication process of spores, thereby affecting their proliferation, differentiation, growth and development. In addition, we found that the number of down-regulated genes associated with DNA replication was significantly higher under 100 \u0026micro;g/ml drug treatment than under 1 \u0026micro;g/ml drug treatment (83 vs 33). This may be related to the different effects of different drug concentrations on in vitro germination, invasion and intracellular proliferation of spores. This also explains why the inhibitory effect of 100 \u0026micro;g/ml drug on the in vitro germination and the invasion of spores was significantly higher than that of 1 \u0026micro;g/ml (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and C).\u003c/p\u003e\n\u003cp\u003eUnder both 1 \u0026micro;g/ml and 100 \u0026micro;g/ml drug treatments, the significantly down-regulated genes were concurrently enriched in cAMP signaling pathway, Rap1 signaling pathway, and Ras signaling pathway (Fig.\u0026nbsp;3B and D). The cAMP signaling pathway affects cellular metabolic activities and physiological processes by regulating intracellular cAMP levels, which plays important roles in differentiation, proliferation, osmoregulation, oxidative stress, and community sensing [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Research suggests that adenylyl cyclases (ACs) and phosphodiesterases (PDEs) in the cAMP signaling pathway may be promising new drug targets for the treatment of certain parasitic infections such as malaria, toxoplasmosis, leishmaniasis and giardiasis [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. In our study, the genes encoding these two enzymes showed no significant changes in expression in response to 1 \u0026micro;g/ml and 100 \u0026micro;g/ml drug stimulation. Therefore, these two enzymes may not be able to be used as drug targets for the treatment of \u003cem\u003eA. portunus\u003c/em\u003e infesting \u003cem\u003eP. trituberculatus\u003c/em\u003e and \u003cem\u003eS. paramamosain\u003c/em\u003e. Nevertheless, we found that the expression of many genes in the cAMP signaling pathway was significantly down-regulated under both concentrations of drug treatment (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and Table S4). This suggests that this drug affects the cAMP signaling pathway in spores, which in turn may affect processes such as spore proliferation and differentiation, which is consistent with our experimental results. Moreover, the number of genes down-regulated at 100 \u0026micro;g/ml drug concentration was significantly more than 1 \u0026micro;g/ml, and, the genes significantly down-regulated under the stimulation of 100 \u0026micro;g/ml drug concentration spread all over the cAMP signaling pathway, which suggests that the 100 \u0026micro;g/ml drug stimulation had a greater effect than 1 \u0026micro;g/ml on the proliferation and differentiation aspect of spores. This is in line with the conclusion drawn from the DNA replication discussed above. The Rap1 signaling pathway plays a role in a variety of cellular processes, including cell adhesion, cell junction, cell migration, polarisation, proliferation and survival [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, in this signaling pathway, the number of genes significantly down-regulated by 100 \u0026micro;g/ml drug stimulation was significantly higher than 1 \u0026micro;g/ml, and the significantly down-regulated genes were spread throughout the signaling pathway (Fig.\u0026nbsp;6 and Table S4). This is consistent with the above findings and further validates our inference. In addition, we found that the expression of genes encoding rap1 and ras was significantly down-regulated under 100 \u0026micro;g/ml drug stimulation. Rap1 and ras are two GTPases [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. These two enzymes act as molecular switches that regulate cellular responses to external stimuli by cycling between an inactive state of GDP binding and an active state of GTP binding [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. As a key enzyme in the ras signaling pathway, the expression of the gene encoding ras was significantly down-regulated by both 1 \u0026micro;g/ml and 100 \u0026micro;g/ml drug stimulation (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and Table S4). Therefore, ras is promising as a drug target for the treatment of \u003cem\u003eA. portunus\u003c/em\u003e. However, this requires further experimental verification. In addition, consistent with the two signaling pathways described above, the number of genes down-regulated in the ras signaling pathway at 100 \u0026micro;g/ml drug concentration was significantly greater than 1 \u0026micro;g/ml, and the significantly down-regulated genes were spread throughout the signaling pathway. The results of the enrichment of differentially expressed genes in various signaling pathways provide further insight into the mechanism by which 100 \u0026micro;g/ml drug exhibited a significantly greater inhibitory effect on the in vitro germination and spore invasion than 1 \u0026micro;g/ml.\u003c/p\u003e\n\u003cp\u003eIn addition to the several signalling pathways mentioned above, some genes were found to be significantly enriched in other signalling pathways under 100 \u0026micro;g/ml drug treatment, e.g., Wnt signal pathway, ErbB signal pathway, Notch signal pathway, and JAK/STAT signaling pathway. The Wnt signal pathway, is a highly conserved pathway that regulates various cellular processes, such as \u003cem\u003ecell\u003c/em\u003e proliferation, differentiation, apoptosis, and cell renewal [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]. \u0026zwnj;ErbB signaling pathway plays an important role in cell proliferation, migration, differentiation, apoptosis and cell migration [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The Notch signaling pathway affects several biological processes, including cell growth, development, differentiation, apoptosis, proliferation and cell boundary formation and tissue repair [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. The JAK/STAT signaling pathway is a series of chain reactions of intracellular protein interactions that are involved in key biological processes such as cell proliferation, differentiation, apoptosis and immune regulation [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]. These several signaling pathways all play a role in cell proliferation and differentiation. This further suggests that 100 \u0026micro;g/ml has a greater effect on spore in vitro germination, invasion and intracellular proliferation, consistent with the above results.\u003c/p\u003e\n\u003cp\u003eThe spores showed stress response under different concentrations of drug treatments. Compared with 1 \u0026micro;g/ml and control group, the spore stress response was significantly increased at 100 \u0026micro;g/ml concentration, and the expression levels of genes related to ribosomal biosynthesis, ribosome, proteasome and peroxisome were significantly up-regulated (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE, F and Fig.\u0026nbsp;3C, E). Functionally, ribosomes play a pivotal role in regulating a multitude of cellular activities, including transcription, translation, protein synthesis, cell proliferation, differentiation, apoptosis, development, and transformation [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Ribosomes also play an important role in the cellular response to stress [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Under stress conditions, the proteasome helps cells adapt to environmental changes and maintains the stability of the intracellular environment by regulating protein degradation and synthesis [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. Oxidoreductases, particularly peroxidases, are crucial for cell metabolism and resistance to cytotoxicity [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. Furthermore, compared with 1 \u0026micro;g/ml and control group, genes linked to amino acid, carbohydrate and lipid metabolism exhibited a notable increase in expression (Fig.\u0026nbsp;3C, E). Amino acids serve as essential building blocks for protein, enzyme, and nucleic acid synthesis, and they also contribute to immune regulation [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, they play crucial roles in energy metabolism, cellular signal transduction, and gene expression [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. The lipid and carbohydrate metabolic pathways, such as fatty acid metabolism, glycolysis/gluconeogenesis, and pentose phosphate pathway, are also involved in energy provision [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]. Therefore, we hypothesised that the up-regulation of these pathways is related to the stress response generated by spores in response to drug stimuli. They provide sufficient energy and raw materials for spores to resist the drug stimulation process.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eChlorogenic acid is a safe drug that is being investigated for the treatment of a variety of diseases due to its wide range of biological activities, but there is no information on its effects on microsporidiosis. In the present study, we found that this drug could significantly inhibit the in vitro germination, invasion and intracellular proliferation of the spores of \u003cem\u003eA. portunus\u003c/em\u003e, which in turn affected spore proliferation, differentiation, growth and development. The inhibition of spore germination and invasion by the 100 \u0026micro;g/ml drug concentration was significantly greater than that observed at the 1 \u0026micro;g/ml concentration. The ras protein in the Rap1 and Ras signaling pathway is promising as a drug target. The spores also demonstrated a range of survival strategies in response to varying concentrations of drugs. To the best of our knowledge, this is the inaugural study to examine the microsporicidal activity of chlorogenic acid on aquatic microsporidia. Nevertheless, further research is imperative to elucidate the mechanism of action and to corroborate these findings in vivo conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCRediT authorship contribution statement\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJiu\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003eYang\u003c/strong\u003e\u003cstrong\u003eChen\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Conceptualization\u003cstrong\u003e,\u003c/strong\u003eMethodology, Formal analysis, Investigation, Data Curation, Writing-Original Draft, Writing-Review\u0026nbsp;and\u0026nbsp;Editing;\u0026nbsp;\u003cstrong\u003eYa-Li Xu\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;Zhi-Qiang Zhu:\u003c/strong\u003eFormal analysis, Investigation, Data Curation, Writing-Original Draft;\u003cstrong\u003e\u0026nbsp;Jin-Yong Zhang:\u003c/strong\u003e Conceptualization, Resources, Supervision, Project administration, Funding acquisition.\u0026nbsp;\u003cstrong\u003eZhao-Zhe Xin:\u003c/strong\u003e Conceptualization, Resources, Writing-Review\u0026nbsp;and\u0026nbsp;Editing, Supervision, Project administration, Funding acquisition.\u0026nbsp;All authors have read and agreed to the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of Shandong Province (no. ZR2022QC250), the Special Support Program of Qingdao Agricultural University awarded to ZZ Xin, National Natural Science Foundation of China (no. 32173019), Young experts of Taishan Scholars in Shandong Province (no. tsqn201909133), Initiative grant for high-level personnel recruitment in Qingdao Agricultural University awarded to JY Zhang, the \u0026ldquo;First Class Fishery Discipline\u0026quot; Programme [(2020)3] in Shandong Province, China, the Talent plan \u0026ldquo;One Thing One Decision (Yishi Yiyi)\u0026rdquo; in Shandong Province, China.\u003c/p\u003e\n\u003cp\u003eDeclaration of competing interest\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the contents of this manuscript. The authors declare that they\u0026nbsp;have no known competing financial interests or personal relationships that could have appeared to\u0026nbsp;influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003eEthical Statement\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this experiment, the collection and handling of crabs were performed in accordance with the guidelines of the Qingdao Agricultural University Animal Care and Use Committee (Qingdao, China)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAn H, Ordureau A, Korner M, Paulo JA, Harper JW. Systematic quantitative analysis of ribosome inventory during nutrient stress. Nature. 2020;583(7815):303-309. https://doi.org/10.1038/s41586-020-2446-y. \u003c/li\u003e\n\u003cli\u003eBojko J, Reinke AW, Stentiford GD, Williams B, Rogers MSJ, Bass D. Microsporidia: a new taxonomic, evolutionary, and ecological synthesis. 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Cancer Lett. 2022 Jan 28;525:84-96. https://doi.org/10.1016/j.canlet.2021.10.034.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ameson portunus, Chlorogenic acid, In vitro germination, Intracellular proliferation, Comparative transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-6226867/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6226867/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAmeson portunus\u003c/em\u003e, the causative agent of \"toothpaste disease\" in \u003cem\u003ePortunus trituberculatus\u003c/em\u003e and \"slurry-like syndrome\" in \u003cem\u003eScylla paramamosain\u003c/em\u003e, has caused substantial economic losses in Chinese aquaculture. Currently, there are no effective strategies to control this pathogen. Chlorogenic acid, known for its antioxidant, anti-inflammatory, antibacterial, and antiviral properties, is widely used in aquaculture due to its safety and efficacy. This study explored its impact on the in vitro germination, invasion, and intracellular proliferation of \u003cem\u003eA. portunus\u003c/em\u003e spores through molecular and omics analyses. Results showed that chlorogenic acid significantly inhibited spore germination in a dose-dependent manner and effectively blocked invasion and intracellular proliferation at a safe dose of 100 \u0026micro;g/ml. Transcriptome analysis comparing 1 \u0026micro;g/ml (PO), 100 \u0026micro;g/ml (PH), and 0 \u0026micro;g/ml (PZ) groups revealed that the drug suppressed spore DNA replication and key signaling pathways, including cAMP, Rap1, Ras, Wnt, ErbB, Notch, and JAK/STAT. These pathways are critical for spore proliferation, differentiation, and development. The 100 \u0026micro;g/ml dose demonstrated significantly greater inhibition of spore germination and invasion compared 0 to 1 \u0026micro;g/ml. The Ras protein in the Rap1 and Ras pathways emerged as a potential drug target. Additionally, spores upregulated genes related to ribosome, proteasome, peroxisome, and metabolic processes as a survival response to drug exposure. These findings highlight chlorogenic acid's potential as a therapeutic agent against \u003cem\u003eA. portunus.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Chlorogenic acid on the in vitro germination, invasion and intracellular proliferation of Ameson portunus (Microsporidia)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 03:18:00","doi":"10.21203/rs.3.rs-6226867/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de32e0ad-873d-4750-b98e-b0daee1aa71f","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-21T14:23:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-07 03:18:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6226867","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6226867","identity":"rs-6226867","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}