1 Introduction
Viruses are an important part of marine ecosystems. With the development of genetic technology, more and more new marine RNA and DNA viruses have been discovered (1, 2). Despite their tiny size, viruses play a large role in marine ecosystems and food webs. On the one hand, marine viruses can infect a variety of oceans organisms, lysing their cells and releasing carbon and other nutrients that impact the food web (3). On the other hand, marine viruses often contain host-derived metabolic genes (i.e., auxiliary metabolic genes; AMGs), which are hypothesized to increase viral replication and alter ecosystem-level productivity through reprogramming host metabolism (4). To date, several crustacean viruses have been found to induce metabolic reprogramming to promote survival and replication, including white spot syndrome virus (WSSV) (5–8), Taura syndrome virus (TSV) (9) and IHHNV (10). In recent years, with global warming and increasing marine pollution, some novel marine viruses have become widespread in the aquaculture industry, causing huge losses to the aquaculture industry (11). Decapod iridescent virus 1 (DIV1) is one of the newly discovered marine viruses. Since China initiated the DIV1 surveillance in 2017, DIV1 has been detected in shrimp culture ponds in several provinces (12). In addition to farmed shrimp, in 2020, Srisala et al. also detected DIV1 in wild populations of Penaeus monodon in the Indian Ocean (13). Up to now, DIV1 has been known as a highly lethal virus with global risk of transmission, capable of infecting freshwater and marine crustaceans, including Marsupenaeus japonicus (14).
Kuruma shrimp M. japonicus is widely distributed in the Indo-Western Pacific region and the East and South China seas (15). Due to its high economic value, strong environmental adaptability and suitability for long-distance transportation, it has now become one of the most farmed prawns in China (16, 17). Our previous study demonstrated that M. japonicus showed obvious clinical symptoms after DIV1 challenge, including empty stomach and intestine, atrophy of the hepatopancreas with yellowing, red body and soft shell. The shrimp mortality rate increased as the virus dose increased, and reached 100% mortality after at 76 hpi injected with 3.95 × 109 copies/μg DNA DIV1 inoculum (14). Through mRNA-seq and miRNA-seq analyses and their association analysis, we preliminarily revealed the hemocyte and intestinal immune response of M. japonicus to DIV1 infection (11, 14), and proposed that DIV1 can promote the Warburg effect by regulating host miRNA and mRNA expression. The Warburg effect was also observed in DIV1-infected Litopenaeus vannamei and Penaeus monodon. However, only the miRNA and mRNA expression profiles under DIV1 infection were revealed, the deep mechanism of the immune response needs further analysis, especially the intestinal immunity.
As an important immune and digestive organ of shrimp, the intestine and symbiotic microorganisms together constitute a complex ecosystem, which plays an important role in maintaining the function of the shrimp immune system. The intestinal immune system of shrimp is mainly composed of three parts, including the mechanical barrier formed by the tight junction and adhesion junction of intestinal mucosal cells, the innate immune barrier formed by intestinal hemocytes and their secreted immune factors, and the biological barrier formed by the intestinal microbiota and their secretions (18). The potential cooperation of bacteria and viruses in promoting disease development has received extensive attention from researchers in recent years. Numerous studies have shown that viral infection can alter the composition and function of the shrimp intestinal microbiota, induce secondary bacterial infection, and impair host immunity (19–21). However, similar to other invertebrates, shrimp lack specific immunity and cannot acquire antibodies through vaccination. Therefore, regulating host metabolic reprogramming by exogenous metabolites to enhance host immunity and ability to withstand environmental has become one of the best strategies for shrimp to resist viral infection (22–24). It will be interesting to explore the effect of metabolic reprogramming on host intestinal metabolome and microbiota and the consequence on immune response. However, there are no reports on the effects of DIV1 infection on the intestinal metabolome and microbiome of shrimp.
To gain a more comprehensive understanding of the interaction between DIV1 and shrimp, this study is the first to investigate the changes of intestinal microbiota and metabolites of M. japonicus under DIV1 infection through the integrated analysis of intestinal microbiome and metabolomics, demonstrating that DIV1 can induce secondary bacterial infection and metabolic reprogramming, and several highly related bacteria and metabolites highly associated with DIV1 infection were screened. The results are beneficial to provide a theoretical basis for virus control technology.
2 Materials and methods
2.1 Shrimp and rearing conditions
Healthy M. japonicus with an average body weight of 10.5 ± 1.6 g were randomly obtained from a local farming pond in East Island Marine Biological Research Base, Guangdong Ocean University (Zhanjiang, China). The shrimp were randomly sampled and tested by PCR to ensure that they were free from WSSV, IHHNV, and DIV1. The detection method was consistent with the previous study (25). Every 30 M. japonicus were acclimatized in 0.3-m3 tanks with aerated and filtered seawater (salinity 30‰, pH 7.5, temperature 28°C) for 7 days before the DIV1 challenge experiment. Commercial feed was provided to the shrimp three times per day at a feeding rate of 5% of their body weight, and nearly 90% of the seawater was exchanged per day.
2.2 DIV1 challenge and sample collection
After 7 days of acclimation, the healthy shrimp were divided into two groups: the PBS-injected group and the DIV1-infected group. Each group included three replicate tanks, and each tank containing 30 individuals. Base on LC50 test results from previous studies (14), we set the DIV1 injection concentration of this study as 3.95 × 109 copies/μg DNA. Each M. japonicus from the DIV1-infected group was intramuscularly injected with 50 μL of DIV1 inoculum, while each M. japonicus from the PBS-injected group was intramuscularly injected with 50 μL of phosphate-buffered saline (PBS; pH 7.4). The methods of viral inoculum preparation and quantification can be found in previous studies (26).
Based on previous research (14), the M. japonicus at twenty-four hour post-injection (hpi) were collected as samples under an aseptic condition. The intestines and their contents from 3 random individuals in the same group were combined as one sample and immediately frozen in liquid nitrogen before storing at -80°C until experimental analysis. In detail, each group had 6 samples were used for enzyme activity analysis, 6 samples were used for intestine microbiome analysis and 3 samples were used for intestinal metabolomics analysis. For histological analysis, the intestines of 6 shrimp per group were used to assess intestine tissue damage. Intestines of these shrimp were fixed in 10% formalin.
2.3 Histological analysis
After fixed in 10% formalin at 4°C for 24 h, the fixed intestine were dehydrated in gradient ethanol, hyalinized in xylene, and embedded in paraffin wax. Next, the paraffin blocks were sectioned at 5-μm thickness. The sections were collected on glass slides and stained with hematoxylin and eosin (H&E). The intestinal sections were examined by a microscope (Olympus, Nikon, Tokyo, Japan). The electronic images were further analyzed using ImageJ software to assess the dimensions of intestinal villus height and muscle thickness.
2.4 Enzyme activity analysis
Enzymatic biomarkers of functional responses in the intestine were measured using commercial detection kits (Jianglai Bioengineering Institute, Shanghai, China) according to protocols of the manufacturer. Prior to analysis, 6 samples in each group were homogenized in pre-chilled PBS (1:9 dilution) and then centrifuged for 10 min (4°C and 5,000 × g) to obtain the supernatant for further use. Superoxide dismutase (SOD), catalase (CAT) and lysozyme (LYZ) activities were used to reflect the nonspecific immunity in the shrimp intestine. α-Amylase (α-AMS), lipase (LPS) and trypsin (TPS) activities were used to reflect the digestive function of shrimp.
2.5 Intestinal microbiome analysis
Total bacterial DNA was extracted by the EasyPure
® Marine Animal Genomic DNA Kit (TransGen Biotech, China) following the manufacturer’s directions. The concentration and purity of total DNA were determined by SimpliNano (GE Healthcare, United States) and 1% agarose gels. The primers pair 515F (5′-GTGCCAGCMGCCGCGG-3′) and 806R (5-′GGACTACNNGGGTATCTAAT-3′) were used to amplify the V4 hypervariable region of 16S rRNA gene, which was modified with a barcode tag with a random 6-base oligos. The PCR amplification reaction was performed with a 25 μL reaction mixture containing 12.5 μL rTaq mix (Takara Biotechnology (Dalian) Co., Ltd.), 1 μL of each primer, 1μL template and 9.5 μL ultrapure water. The reaction cycle parameters were as follows: an initial denaturation step of 95°C for 3 min; 30 serial cycles of a denaturation step of 95°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 45 s; and a final extension step of 72°C for 10 min.
PCR amplification reaction system and parameters can refer to previous studies (27). After the PCR products were purified and mixed in equidensity ratios, sequencing libraries were constructed, and then sequenced by BGI (Shenzhen, China) with the Illumina Genome Analyzer technology. All raw sequencing data of intestinal microbiota was submitted to the Sequence Read Archive (SRA) (accession: PRJNA720257).
Sequences from raw data were analyzed and filtered by QIIME (v1.8.0). Sequence analysis was performed with UPARSE software (v7.0.1090), and the operational taxonomic units (OTUs) were defined with ≥ 97% similarity. Chimeric sequences were identified with UCHIME (v4.2.40). Alpha diversity was calculated using mothur software with five metrics, including the observed OTUs, Chao1, ACE, Shannon and Simpson indexes. Rarefaction curves were generated based on these metrics. The shared and unique OTUs between two groups were figured out by a Venn diagram. Beta diversity index based on the phylogenetic relationship between OTUs was used to calculate the Unifrac distance (weighted and unweighted Unifrac) and the results of Beta diversity were showed through PCoA and UPGMA Phylogenetic Dendrogram. A bar plot of the microbial community was constructed at the phylum, family and genus level respectively. To study the functional characteristics of bacterial communities, Kyoto Encyclopedia of Genes and Genomes (KEGG) functions were predicted using the PICRUSt software.
2.6 Intestinal metabolomics analysis
Six intestinal samples replicates of shrimp from each group were used for metabolomic analysis. All the samples were taken from the refrigerator at -80°C and thawed in the refrigerator at 4°C, and metabolite extraction was performed using methanol and 2-chlorobenzalanine. Twenty microlitres of each sample was taken for quality control (QC), and the rest was used for LC-MS detection. Liquid chromatography was accomplished in a Thermo Ultimate 3000 system equipped with an ACQUITY UPLC® BEH C18 (1.7 μm 2.1 × 100 mm, Waters, USA) column. Mass spectrometry was executed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, USA). Data-dependent acquisition (DDA) MS/MS experiments were performed with HCD scans. Dynamic exclusion was implemented to remove some unnecessary information in the MS/MS spectra.
Peak-identification, peak-alignment and compound identification was conducted using Compound Discoverer software (v3.1). All the data were determined using quality control (QC) and quality assurance (QA). Partial least squares-discriminant analysis (PLS-DA) of the metabolomics data was performed using the R language ropls package. All the metabolites were classified according to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Human Metabolome Database (HMDB). The PLS-DA model was used to determine the differential metabolites (DMs) between the PBS-injected group and the DIV1-infected group with the first principal component of variable importance in projection (VIP) values (VIP ≥ 1) combined with a q-value ≤ 0.05. The DMs were annotated with KEGG pathway analysis to further identified the change characteristics of the functional metabolites related to the immunity of the shrimp.
2.7 Correlation analysis of intestinal bacteria and DMs
Canonical correlation analysis and spearman correlation analysis were employed to reveal the correlation between significantly altered intestinal bacteria and intestinal DMs, and the results were shown with scatterplots and heat maps. The thresholds for correlation coefficients and p-values were not set. p < 0.05 was regarded as statistically significant, p < 0.01 was regarded as very significant, and p < 0.001 was regarded as extremely significant.
4 Discussion
Growing evidences suggested that viral infection or environmental stress can lead to changes in the structure and function of the host’s intestinal microbiota, and further affect the normal metabolism of the host and caused various adverse reactions (28–31). To date, there are no reports on the effects of DIV1 infection on the intestinal microbiota and metabolites of M. japonicus. The present study provides insights into the interaction of M. japonicus and DIV1 through the histological analysis, enzyme activity analysis and the integrated analysis of intestinal microbiome and metabolomics.
The results of histological analysis showed that severe intestinal mucosal damage was observed in the intestine of DIV1-infected shrimp, some intestinal epithelial cells were detached from the basement membrane and the intestinal villus height was significantly reduced. Similarly, Xue et al. found that WSSV-infected crayfish Procambarus clarkii exhibited worse intestinal histomorphology, with thinner intestinal walls and shorter intestinal villi compared to healthy crayfish (32). Liang et al. found that the intestinal epithelial cells of L. vannamei infected with Vibrio parahaemolyticus were completely detached from the basement membrane (33). Except for the intestinal mucosal damage, the disturbance of intestinal immunity and digestive function was also observed in the DIV1-infected shrimp. Enzyme activity analysis showed that all digestive enzymes and immune enzymes were significantly altered (p < 0.001), indicating that DIV1 infection severely affected the normal immune and digestive functions of the M. japonicus intestine. Studies have found that shrimp hemocytes can produce a large amount of free radicals and reactive oxygen species (ROS) during phagocytosis, thereby effectively killing the invading pathogens (34, 35). However, the mass accumulation of ROS in animals will cause serious cell damage, resulting in various diseases (36). SOD and CAT are two kinds of antioxidant enzymes, which can effectively remove free radicals and ROS, thereby protecting cells from oxidative stress (37–39). Under the action of SOD, ROS react with hydrogen ions to generate less toxic hydrogen peroxide, and then react with hydrogen ions under the action of CAT to finally produce harmless water (40). Therefore, only when the activities of SOD and CAT are coordinated, the free radicals and ROS in the organism can be maintained at a low level. However, the trends in the activity of SOD and CAT were not always consistent. In L. vannamei, intramuscular injection of 0.1 μg (g shrimp)−1 fluorescent red polyethylene microspheres (41) and experimental infection with Enterocytozoon hepatopenaei (42) both resulted in a significantly increase in SOD activity and a significant decrease in CAT activity. In Strongylocentrotus intermedius, the activity of SOD increased significantly and the activity of CAT decreased significantly with increasing temperature (43). In our study, the activities of SOD was significantly increased in the intestine of the M. japonicus under DIV1 infection, while the activities of CAT was significantly decreased. These results are supported by the previous findings, and suggested that DIV1 infection may cause a large amount of intestinal reactive oxygen species to be produced and inhibit the antioxidant capacity of the intestine to some extent. The specific mechanism needs to be further studied. LYZ has been shown to play an important role in shrimp resistance to viral and bacterial infections (44–46). The activity of LYZ can directly reflect the strength of shrimp immune function. After infection with DIV1, the activity of LYZ in the intestine of M. japonicus was significantly decreased, which indicated that the intestinal immune function was suppressed to some extent. α-AMS, LPS and TPS are three important digestive enzymes that also serve as markers to help diagnose diseases. Several studies had reported that the significantly increased α-AMS and LPS activities can be used as the basis for the diagnosis of mammalian enteritis and pancreatitis (47–51), while trypsin deficiency is closely related to enteritis (52–54). In this study, the intestinal activities of α-AMS and LPS were significantly increased after infection with DIV1, while the activities of TPS was significantly decreased. This suggested that DIV1 infection may cause damage to the shrimp intestine, causing enteritis and digestive dysfunction. In addition, this may also be one of the reasons for the decreased feeding rate of DIV1-infected shrimp and caused the symptoms of empty stomach and intestine (14, 25, 26, 55, 56).
In intestinal microbiome analysis, after DIV1 infection at 24 h, the intestine microbiome was significantly changed in the composition and diversity at the phylum, family and genus levels. In this study, the intestinal microbiota in M. japonicus were dominated by five phyla of Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cyanobacteria in both the PBS-injected group and DIV1-infected group, which is consistent with the previous microbiome studies in L. vannamei. At the family level, the relative abundance of Vibrionaceae significantly increased in the intestine of DIV1-infected M. japonicus compared with healthy M. japonicus, which mainly reflected in the significant increase in the relative abundance of Photobacterium and Vibrio at the genus level. In addition, both the LEfSe cladogram and LDA score of LEfSe-PICRUSt showed that Photobacterium and Vibrio dominated in the DIV1-infected group. Several previous studies had shown that WSSV infection can cause an increase in the relative abundance of opportunistic pathogens in the intestine of shrimp, such as Aeromonas, Arcobacter, Vibrio and Trichococcus (19, 21, 32). Photobacterium had been shown to cause various diseases in fish and shrimp (57–61) causing huge losses in aquaculture. Vibrio, which is generally considered to be an opportunistic pathogen of shrimp and widely exists in the intestine of shrimp, can cause shrimp disease when the environment changes drastically (62–65). These results suggested that DIV1 infection can cause an increased abundance of opportunistic pathogens in the intestine, which may probably due to DIV1 infection disrupting the mechanical barrier of the intestine and impairing the ability of intestine to select microorganisms. The results of PICRUSt functional prediction showed that, in the KEGG level 2, the relative abundance of “carbohydrate metabolism”, “metabolism of cofactors and vitamins” and “amino acid metabolism” were the top 3 in the two groups. It meant that intestinal microbiota played an important role in regulating host vitamin metabolism, carbohydrate metabolism and amino acid metabolism. Notably, the relative abundance of “infectious diseases: bacterial” was significantly increased under DIV1 infection, which further suggested that DIV1 infection could cause secondary bacterial infection. In-depth analysis of the top 3 KEGG level 2 terms showed that the abundance value of amino acid metabolism, vitamin metabolism, glycolysis/gluconeogenesis and pyruvate metabolism were significantly increased after DIV1 infection. It meant that DIV1 can promote the Warburg effect by affecting the function of the host intestinal microbiota.
A recent study found that the “linoleic acid metabolism” and “arachidonic acid metabolism” were also the most disturbed pathways by HCoV-299E infection (66). Interestingly, exogenous supplement of linoleic acid or arachidonic acid in HCoV-229E-infected cells significantly suppressed HCoV-229E virus replication (66). Another study showed that linoleic acid could directly bound to WSSV to inhibited the viral replication and indirectly participate in the immune response against WSSV by activating the ERK-NF-κB signaling pathway to promote the expression of antimicrobial peptides and IFN-like gene Vago5 (67). Consistently, the intestinal metabolomics analysis in this study showed that lipid metabolism in the intestine of M. japonicus was remodeled after DIV1 infection, and a total of seven types of lipid fatty acids were significantly changed, including arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, 9-oxo-10(e), 12(e) -octadecadienoic acid, gamma-linolenic acid, 8(s)-hydroxy-(5z,9e,11z,14z) eicosatetraenoic acid and 16-hydroxyhexadecanoic acid. In addition, two Warburg effect marker metabolites were significantly up-regulated, including L-(+)-lactic acid and pyruvic acid. KEGG enrichment analysis indicated that the “Linoleic acid metabolism” and “arachidonic acid metabolism” pathways were the most affected by DIV1 infection. These results suggested that the lipid metabolic reprogramming of M. japonicus was significantly associated with DIV1 infection and replication. After infected with DIV1, two marker metabolites of the Warburg effect were significantly up-regulated in the shrimp intestine, including L-(+)-lactic acid and pyruvic acid, and significantly enriched in the “pyruvate metabolism” and “glycolysis/gluconeogenesis” pathways. In addition, some amino acid metabolism-related pathways were also significantly enriched under DIV1 infection, including “tyrosine metabolism”, “phenylalanine metabolism” and “alanine, aspartate and glutamate metabolism”. These phenomena also appeared in previous studies (11, 25, 26), which may be attributed to the material and energy requirements of DIV1 replication and suggested that DIV1 may contain AMGs for regulating host amino acid metabolism, carbohydrate metabolism and energy metabolism. It was worth noting that some pathways related to vitamin metabolism were significantly enriched, such as “retinol metabolism”, “vitamin B6 metabolism” and “pantothenate and CoA biosynthesis”. Retinol, also known as vitamin A (VA), plays an important and positive role in intestinal mucosal immunity and repair (68). The B vitamins can regulate the energy metabolism, nutrient accumulation and immune defense function of aquatic animals (69). Our previous study showed that DIV1 infection can regulate the vitamin metabolism of shrimp by affecting the expression of intestinal miRNA and mRNA (11, 26). In this study, from the metabolome level, it was further demonstrated that DIV1 infection can regulate vitamin metabolism in the intestine, thereby affecting the normal physiological function of M. japonicus.
Intestinal microbiota variation caused by viral infection or environmental stress can affect the metabolism and immunity of their host, and increase disease susceptibility (70, 71). To further explore the relationship between intestine microbiota variation and metabolites, the correlation analysis of dominant intestinal microbial and marker DMs was carried out. In this study, the increased levels of Photobacterium and Vibrio were positively correlated with changes in Warburg effect marker metabolites L-(+)-lactic acid and pyruvic acid. This indicated that Photobacterium and Vibrio may synergize with DIV1 to jointly promoted the Warburg effect, providing material and energy for the successful replication of DIV1. In addition, the abundances of Photobacterium and Vibrio were positively associated with arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and 8(s)-hydroxy-(5z,9e,11z,14z) eicosatetraenoic acid and negatively associated with 9-oxo-10(e),12(e)-octadecadienoic acid, gamma-linolenic acid and 16-hydroxyhexadecanoic acid. However, Corynebacterium, which dominated the PBS-injected group, was just the opposite of the above. It suggested that Photobacterium and Vibrio may be involved in the metabolic reprogramming of the shrimp intestine after DIV1 infection.