1. Introduction
The Pacific white shrimp (
Litopenaeus vannamei) has a pivotal role in the global shrimp aquaculture industry, contributing to over 70% of the total global shrimp production [
1]. However, in recent years, the emergence of
Enterocytozoon hepatopenaei (EHP) and its associated hepatopancreatic microsporidiosis (HPM) has led to significant economic losses in the Asian shrimp farming sector [
2,
3]. EHP is a single-celled eukaryotic microsporidian parasite belonging to the phylum microsporidia. It was initially identified in black tiger shrimp in Thailand in 2004 and was officially named based on histological, morphological, and phylogenetic data in 2009 [
4,
5,
6]. Approximately half of the known microsporidia species can infect aquatic organisms [
7]. Currently, three cultivated shrimp species are recognized as hosts for EHP:
P. monodon,
L. vannamei, and
P. stylirostris [
8]. While EHP infection does not cause lethal outcomes, it significantly suppresses shrimp growth rates and may induce immune dysfunction and complications such as White Feces Syndrome (WFS) [
9,
10]. Previous studies have indicated that EHP possesses a highly simplified genome and organelles, lacking metabolic pathways for glycolysis or oxidative phosphorylation to generate ATP [
11]. Its genome is enriched with ATP/ADP transporters, indicating its reliance on continuously siphoning energy from the host [
12]. Compared to other microsporidian parasites within the Enterocytozoonidae family, EHP exhibits unique patterns during host-parasite interactions [
13].
The shrimp hepatopancreas possesses remarkable regenerative and self-repair mechanisms, mitigating some of the damage caused by EHP infection [
14]. Consequently, EHP often establishes a long-term symbiotic relationship with shrimp [
9]. Previous research has shown that shrimp with severe EHP infection exhibit down-regulated lipid metabolism compared to those with mild infection. Analysis of high (H) and low (L) infection severity groups revealed a positive correlation between infection severity and metabolic changes [
13]. Additionally, as EHP infection intensifies, the abundance of certain microbial groups in shrimp intestines increases, including
Pseudomonas,
Bradyrhizobium,
Bacteroides, and
Vibrio [
15]. These findings suggest that the effects on shrimp vary with the duration of EHP infection. Nevertheless, significant gaps remain in our understanding of the long-term effects of EHP infection in shrimp.
Transcriptome sequencing (RNA-Seq) enables the study of gene function under specific conditions through genomic structure analysis and functional annotation [
16]. Metabolomics reveals changes in metabolic pathways through systematic analysis of small molecule metabolite spectra [
17]. These omics techniques offer significant advantages, making them ideal for in-depth investigations into pathways and mechanisms of life events [
18]. In recent years, multi-omics analyses have provided a comprehensive understanding of the molecular mechanisms of EHP infection. For instance, combined analysis of transcriptomics and the microbiome has revealed a positive correlation between
Bacillus spp. in shrimp intestines and immune genes associated with antibacterial processes following EHP infection. Furthermore, integrated proteomic and metabolomic research has uncovered changes in growth-related proteins such as ecdysteroid-regulated protein and juvenile hormone esterase-like carboxylesterase 1, along with inhibited energy metabolism, contributing to the slow growth of shrimp following EHP infection [
19,
20].
In this study, through sampling of infected shrimp at two distinct life stages, we gained valuable insight into the EHP-shrimp interaction during different phases of the infection process. Comparative to single time-point analyses, analyzing juveniles versus adults offers a higher-resolution perspective into EHP pathogenesis during shrimp development. Our primary goal was to comprehensively investigate the interaction between Pacific white shrimp and EHP through a combined transcriptomics and metabolomics approach. By examining EHP infections in juvenile versus adult shrimp hepatopancreas, we explore the relationship between shrimp life stage and gene expression as well as metabolic pathways in diseased shrimp. Our research provides a rich dataset of the interaction between Pacific white shrimp and EHP, potentially yielding a better understanding of the changing impacts on shrimp during development and EHP progression, and offering deeper insights for future studies.
3. Discussion
Microsporidian infections often impact the host’s behavior and interact with various regulatory mechanisms within the host’s body [
21]. In this study, for the first time, we revealed distinct patterns of host response through infection samples taken at different time points, observing significant differences between early and late infection stages. We identified 2444 DEGs and 374 SDMs during the early infection, and 1769 DEGs and 323 SDMs during the late infection. PCA analysis demonstrated substantial differences in transcription and metabolic levels between juveniles and adults in the absence of EHP infection. However, these differences diminished after EHP infection, with partial similarity observed between the two groups. These findings suggest the activation of immune defense-related genes in both shrimp groups. The early immune response appeared to be broad spectrum, yet a substantial portion of immune-related genes remained suppressed, possibly due to EHP invasion. In the late infection stage, most immune responses gradually recovered, and some immune-related genes exhibited more pronounced activation, likely indicating the shrimp’s adaptation to EHP infection. Moreover, both shrimp groups experienced profound impacts on metabolic pathways. Energy metabolism pathways were significantly enriched in both early and late stages, highlighting the potential importance of energy metabolism pathways for EHP at both time points, consistent with EHP’s absolute dependence on host energy [
22]. Additionally, we identified certain energy metabolism-related genes as potential pathways for EHP to acquire energy. Notably, immune response-related DEGs were significantly affected during the early infection stage, with a large number of up-regulated DEGs indicating EHP activation of host defense mechanisms. However, down-regulated expression of certain DEGs may signify key pathways for EHP invasion into host cells and interaction with the host. Importantly, we also observed disruptions in growth-related metabolic pathways, likely contributing to the slow growth of juveniles following EHP infection. In the late infection stage, EHP primarily disrupted lipid, energy, and carbohydrate metabolism pathways, likely due to the extensive reproduction of spores within shrimp, which divert host energy and resources to sustain their proliferation.
Table S4 details the expression profiles of select DEGs across different developmental stages, corroborating our findings.
Under the stress of EHP infection, there are significant differences in the immune responses of shrimp during the early and late stages. Shrimp identified more immune-related DEGs during the early infection compared to the late infection, suggesting that EHP may activate a broad spectrum of immune defenses in the early stages. In the late infection stage, the expression of these DEGs returned to normal, possibly indicating a strategy employed by EHP to establish a long-term symbiotic relationship with the shrimp while reducing the host stress response. However, during the early infection, we observed that certain immune-related genes might be regulated by EHP to facilitate invasion. Previous research indicates that microsporidia can suppress host immunity during invasion [
23].
We observed a significant impact on the transcriptional expression of two genes, Ftz-F1 and SEPs, during the early infection stage. Ftz-F1 overexpression can inhibit phagocytosis of blood cells. This suggests that in the early stages of EHP infection, the parasite might manipulate the host’s Ftz-F1 to evade phagocytosis and promote its infection [
24,
25]. Similarly, it is known that small open reading frame (sORF)-encoded peptides (SEPs) can inhibit the proliferation of white spot syndrome virus (WSSV) [
26]. The down-regulation of SEPs-related genes during the early stages of EHP infection might lead to a decrease in antimicrobial peptide (AMP) levels, promoting EHP invasion. Additionally, in the Asp vs. Jsp comparison, we identified a unique significant enrichment of the PI3K-Akt signaling pathway. The PI3K-Akt pathway is involved in shrimp’s immune response and plays a crucial role in shrimp’s defense against bacteria and viruses [
27,
28]. This suggests that PI3K-Akt may also play an important role in EHP invasion. In summary, EHP significantly impacts the host’s immune system during the early stages of infection.
In contrast, the reduced number of DEGs during the late infection stage suggests that the effects of EHP are gradually diminishing. This includes most immune-related DEGs that exhibited significant differences in the early stages, such as the expression of SEPs which returned to normal in the late infection stage. Interestingly, some immune activities become more pronounced. For example, the expression of Ftz-F1 is significantly down-regulated in the late infection stage, leading to a significant increase in the cell’s phagocytic ability against pathogens [
24]. Additionally, we observed that antioxidant and detoxification-related pathways, such as oxidative phosphorylation and cytochrome P450 xenobiotic metabolism pathways, seem to play an important role in shrimp during the late infection stage. This includes the significant activation of pathways related to ascorbic acid, aldehyde metabolism, and detoxification. Ascorbic acid (vitamin C) is generally considered a potent antioxidant capable of scavenging free radicals [
29]. However, recent studies have shown that high doses of vitamin C can also have pro-oxidant effects, protecting larvae of Daphnia magna from
Vibrio harveyi infection [
30]. We observed that the expression of ascorbic acid is significantly down-regulated during the early infection stage but significantly up-regulated during the late infection stage. Taken together, these results suggest that in the late infection stage, the high parasite load of EHP might generate harmful substances to the host. To counteract the toxic effects of these substances, shrimp may protect themselves by continuously up-regulating detoxification and antioxidant systems. Additionally, to cope with prolonged infection, the shrimp’s immune system gradually recovers and remains in a continuously activated state. In conclusion, these strategies may explain why shrimp can establish a long-term symbiotic relationship with EHP during the later stages of infection.
In the context of metabolic pathways, our data indicate a strong reliance of EHP on the host’s energy metabolism. During the early infection stage, ATP synthase exhibits significant up-regulation. ATP synthase is essential for electron transport and ATP synthesis [
31]. Moreover, other studies have shown that white spot syndrome virus (WSSV) up-regulates ATP synthase in infected blood cells to enhance its own replication [
32]. Considering that EHP is nearly incapable of ATP production and relies entirely on obtaining ATP from host cells [
13], we speculate that EHP might similarly up-regulate the expression of ATP synthase in host cells and heavily exploit the host’s ATP to support its proliferation during the early infection stages. Interestingly, within the purine metabolism pathway, we also observe significant up-regulation of genes involved in the degradation of ATP and GuaR (
Figure 9). ATP and GuaR are vital components of the purine metabolism pathway, playing crucial roles in energy transfer and nucleic acid synthesis. Previous research indicates that microsporidia lack many of the genes required for nucleotide synthesis. However, they retain the core enzyme toolkit for nucleotide salvage, suggesting that microsporidia manipulate host cell processes to promote nucleotide synthesis and hijack the host’s ATP and nucleotides [
33]. Additionally, radiolabeled purine nucleotides have been utilized by
Trachipleistophora hominis for the critical purine building blocks of its DNA and RNA [
34]. Our study suggests that EHP might manipulate host nucleotide-regulating genes to facilitate its replication. The up-regulation of genes involved in the degradation of ATP and GuaR further supports this notion. Additionally, we observe a significant enrichment of cytochrome c oxidase (COX) during the early infection stage. Previous research suggests that microsporidia can gain energy by interacting with the host mitochondria through spore surface protein (rEhSSP1) [
35]. COX is mainly found in the inner mitochondrial membrane, catalyzing redox reactions [
36]. Mitochondria, as the main energy-producing organelles, may be a prime target for EHP. Therefore, we speculate that COX might be an essential pathway for EHP to acquire mitochondrial energy. However, further research is required to confirm this hypothesis and elucidate the exact mechanism through which EHP exploits host nucleotide resources.
In contrast, during the late infection stage, EHP seems to become more reliant on the host’s energy metabolism. Several fundamental ATP-generating pathways, including glycolysis and oxidative phosphorylation, are significantly affected. Glycolysis is a key carbohydrate metabolism pathway. Although genes related to glycolysis are missing in the Enterocytozoon family of microsporidia, each lineage retains genes from different parts of the glycolytic pathway [
13]. This suggests that glycolysis remains an important energy-generating pathway in these microsporidia. Phosphoglucomutase (PGM) catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. This allows microsporidia to use glucose 1-phosphate in glycolysis and glucose 6-phosphate (G6P) in trehalose synthesis [
37]. Our data indicate a significant up-regulation of PGM during the late infection stage. Hexokinase, which is important for glycolysis, is significantly down-regulated in the Asn and Asp groups. EHP possesses only one copy of hexokinase and lacks a complete glycolytic pathway. Yet, it utilizes secreted hexokinase to disrupt host metabolism and support its development, similar to other Enterocytozoon microsporidia [
38,
39,
40,
41]. Notably, hexokinase, as the enzyme responsible for the first step of glycolysis, also catalyzes the conversion of G6P. Furthermore, G6P might promote increased ATP production for microsporidian growth in the host. A significant up-regulation of G6P has also been observed after
Nosema bombycis infection, another microsporidian [
42,
43]. These findings suggest that PGM, hexokinase, and G6P play a crucial role in EHP’s energy acquisition pathway. EHP may target the host’s PGM to acquire the necessary G6P, while the down-regulation of hexokinase might indicate the parasite’s manipulation of the host’s enzymes to complete its glycolysis. In conclusion, these enzymes and G6P likely play key roles in obtaining ATP, regulating the host, and promoting EHP’s lifecycle, especially during the progression of infection.
Lipids, as a principal component of hepatopancreas, play a vital role in energy storage, among other functions. During the late infection stage, the high load of EHP leads to the disruption of hepatopancreas function, resulting in the disturbance of lipid metabolism pathways. In this study, we observed a significant enrichment in glycerophospholipid and sphingolipid metabolism pathways. This aligns with Ding et al.’s research, which showed that microsporidian infection disrupts glycerophospholipid and sphingolipid metabolism in
Eriocheir sinensis [
44]. Our temporal analysis further corroborated this transition, indicating a sustained increase in glycerophospholipid-related metabolism from the non-infected group to the long-term infected group. Glycerophospholipids, also known as phospholipids, are implicated in the synthesis of membrane phospholipids and possess crucial roles in various microsporidia for membrane synthesis and other cellular functions [
45]. The significant accumulation of phospholipids suggests that EHP may have specific requirements for phospholipids during its growth and development. Furthermore, we observed a significant down-regulation of phosphoethanolamine N-methyltransferase. El Alaoui et al. suggested the presence of phospholipid synthesis pathways in microsporidia and observed elevated activities of phospholipid acylserine decarboxylase and phospholipid ethanolamine N-methyltransferase following microsporidian infection. The combined action of these enzymes can convert phosphatidylethanolamine and phosphatidylserine into phosphatidylcholine [
46]. Additionally, another study demonstrated that phosphatidic acid is a limiting host metabolite for
Tubulinosema ratisbonensis proliferation [
47]. These findings indicate that the enrichment of phospholipids might signify EHP’s regulation of PE and PS-related genes, with phospholipids potentially playing a significant role in EHP’s growth and development.
Sphingolipids, as another important class of lipids, are integral components of membrane lipids and play vital roles in signal transduction. In this study, we observed a continuous decrease in sphingolipids as the infection cycle progressed. Existing research suggests that microsporidia might form invasion vesicles through interactions with the host cell membrane during invasion [
44,
48]. This implies that EHP might utilize potential interactions with the host cell membrane to facilitate invasion. Sphingomyelin can be transformed into sphingosine through the action of serine palmitoyltransferase long chain (SPTL). Sphingosine can further react to form ceramide. The work of JH Jeon et al. discovered that sphingolipid levels might impact microsporidian proliferation. This is because microsporidia might have diverse enzymes generating C20-ceramides, and the study indicated that only the infected group exhibited an enrichment of ceramide species, suggesting that microsporidia could hijack host enzymes to generate ceramides. This indicates that sphingolipids, especially sphingosine, might play a significant role in microsporidian infection, potentially promoting their growth [
49]. Our data display a notable reduction in sphingosine and a significant up-regulation of ceramide phosphate ethanolamine lyase, which might result in an overall decrease in sphingosine levels. This suggests that EHP might similarly intervene in host sphingolipid metabolism and promote its proliferation. In conclusion, these lipid changes pose intriguing questions about their impact on the EHP-host interaction and the mechanisms through which EHP manipulates host lipid metabolism, warranting further investigation.
Our study also revealed a significant down-regulation of genes involved in glutathione metabolism during the early EHP infection period. The glutathione system plays a crucial role in antioxidant defense and maintaining redox balance, which are essential during the infection process. A study focusing on
Nosema ceranae highlighted the importance of the glutathione system in microsporidian invasion. Knocking down the expression of γ-glutamyl-cysteine synthetase and thioredoxin reductase genes resulted in a significant reduction in spore load within honeybees [
50]. Interestingly, our data indicate a substantial inhibition of glutathione metabolism during the early infection phase, which becomes significantly activated in the later stages of infection. This suggests that the glutathione system may serve as a crucial defense mechanism against EHP invasion in the early stage, but it is significantly suppressed in the context of EHP’s invasion strategy. In the later stages, the substantial spore load might prompt the activation of shrimp’s antioxidant defense to cope with prolonged microsporidian infection. However, the exact role of the glutathione system in the EHP-host interaction, as well as how EHP suppresses the host’s glutathione system to facilitate invasion in the early phase, remains to be elucidated.
It is worth noting that polyamine metabolism, as a part of the glutathione metabolic pathway, is significantly affected both in the early and later infection phases. Polyamines include putrescine, spermidine, and spermine. Notably, spermidine levels show significant down-regulation in both early and late infection phases, while the expression of spermine synthase is significantly up-regulated in both periods. Interestingly, the reduction in spermidine levels is more pronounced in the later phase, while the transcriptional level of spermine synthase is even higher in the later phase. Polyamine metabolism is considered important for parasite survival [
51]. Related studies have indicated that
Enterocytozoon cuniculi exhibit polyamine synthesis and transformation activities before germination, shown by the conversion of spermine to spermidine. Furthermore, the use of polyamine analogs as inhibitors effectively interferes with spore uptake and conversion of polyamines before germination [
52]. This suggests that polyamines may play a crucial role in microsporidian survival, particularly in relation to germination activity. However, further research suggests that microsporidia have adapted to rely more on polyamine uptake and interconversion rather than de novo synthesis [
53]. Some microsporidia, like
Enterocytozoon cuniculi, demonstrate significant potential for polyamine transformation [
52]. These findings indicate that polyamines are vital for microsporidian survival and proliferation, with uptake being more critical than synthesis. This seems to explain the continuous up-regulation of spermine synthase expression and the persistent down-regulation of spermidine levels observed in our study. The role of polyamines in EHP infection and how EHP regulates host polyamine metabolism merits further investigation. Additionally, polyamine analogs could hold potential research value in EHP control strategies.
Finally, in the infection cycle of EHP, we observed that shrimp seem to experience more pronounced growth inhibition during the early infection phase. In the early stages of EHP infection, we observed significant disruption in amino acid metabolism, particularly for growing essential amino acids such as tryptophan and histidine. Our KEGG topology analysis of SDMs suggests that this disruption could impair shrimp growth and development, as these amino acids are crucial for protein synthesis and various metabolic processes [
54] Furthermore, the enrichment of pathways related to purine, pyrimidine, α-linolenic acid, taurine, and hypotaurine metabolism, as well as glycosaminoglycan biosynthesis, indicates that EHP might interfere with host nucleotide metabolism, fatty acid metabolism, osmotic regulation, and extracellular matrix composition during the early infection phase. Our temporal analysis also revealed sustained down-regulation of taurine metabolism. Another microsporidian,
Paranosema locustae, significantly reduces taurine levels in infected
Locusta migratoria, leading to growth inhibition in the infected locusts [
55] This suggests that the persistent down-regulation of taurine could be another important factor contributing to the growth inhibition and molting impairment in shrimp after EHP infection. Moreover, Li et al.’s study showed that when taurine was supplemented to infected locusts through injection, the delayed development phenomenon was suppressed [
55] However, it is worth noting that taurine metabolism mechanisms in shrimp and locusts could be different, and the specific impact of EHP infection on shrimp taurine metabolism requires further investigation.
In our study, we also found that certain metabolic pathways, such as tryptophan, phenylalanine metabolism pathways, and lysine degradation pathways, were significantly enriched in both early and late infection stages, albeit in different host groups (Asn vs. Jsn and Asp vs. Jsp). Among them, lysine degradation seems to be more affected, as it was significantly enriched in Clusters 3, 6, and 7 in the temporal analysis. Lysine-rich proteins, such as extreme-heat pipe protein 2 (EHPPTP2), were identified in EHP’s infective polar tube [
56] This suggests that lysine could be an important amino acid for EHP growth and host interaction, possibly contributing to the structure, function of EHPPTP2, and the mechanisms of EHP infection. In conclusion, the early growth inhibition observed in shrimp might be due to the widespread effects caused by EHP, with inhibition of amino acids and steroid metabolism closely associated with growth possibly leading to stunted growth in shrimp.
In summary, our study provides new insights into the metabolic adaptations of shrimp during EHP infection. However, our understanding of the complex interactions between EHP and its host is still in its early stages. Further research is needed to validate these findings and uncover the molecular mechanisms behind these metabolic changes. This study not only enhances our understanding of the pathogenesis of EHP but also identifies potential targets for therapeutic intervention.