Introduction

Various kinds of healthy and high value-added nutrition have been discovered in microalgae, such as proteins, carbohydrates, pigments (e.g., phycobillins and carotenoids), and polyunsaturated fatty acids (PUFAs) (e.g., eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)). Due to their potential nutraceutical and pharmaceutical properties, many microalgae species can be utilized as feed and food additives in the aquaculture industry (Dhandayuthapani et al. 2021; Yaakob et al. 2014). Along with the increasing production of aquatic animals, the demand for suitable microalgae in the aquaculture industry is increasing. The interplay between nutrition and the immune system is well recognized (Martin and Krol 2017). It is reported that microalgae can be an excellent immunostimulant that plays a role in enhancing fish immunity and improving the capacity of anti-infection (Ma et al. 2020). Microalgae and their derived bioactive ingredients have antibacterial, antiviral, and immunomodulation capabilities to modulate aquatic animals’ immune status and to protect them from pathogens.

Several studies have evaluated the beneficial effects of microalgae (such as Tetraselmis sp., Phaeodactylum tricornutum, Chlorella sp., Nannochloropsis oculata, Nannochloropsis gaditana, Lobosphaera incisa, and Schizochytrium sp.) on farmed fish, and explored the effects of microalgae on intestinal health of aquatic animals, including intestine immune status and microbial ecology (Bravo-Tello et al. 2017; Cerezuela et al. 2012, 2013;et al. Lazado et al. 2019; Nayak et al. 2018; Shi et al. 2021). The intestine is regarded as an important part of the immune system in fish. Gut-associated lymphoid tissue (GALT) is one of the mucosa-associated lymphoid tissues (MALT) of teleost (Salinas and Parra 2015; Yu et al. 2020), consisting of the mucus layer and a variety of immune cells, which are responsible for the complicated immune responses to diseases. Besides, the microbial community inside the digestive tract is crucial to the health of fish (Bates et al. 2006). Interestingly, shortly after hatching, a variety of microorganisms that colonize the teleost intestine directly or indirectly interact with the host immune system and affect the host immune status (Kelly and Salinas 2017; Pérez et al. 2010). Nowadays, intensive aquaculture increases the severity and frequency of fish diseases. The high morbidity and mortality in commercial aquaculture are mainly caused by fish diseases, leading to the decrease of fish production and associated economic loss. Given the functional importance of the intestinal microbiome in host physiology, the regulation of the microbiome may be one of the feasible strategies to mitigate fish diseases in aquaculture. Changes in the microbiome brought by the diet may affect the susceptibility and potential resistance of fish to diseases (Xiong et al. 2019). Hence, a clear understanding of how animals’ physiological response and intestinal microbiota change after receiving microalgae feed is required. However, the mechanisms by which feed influences host-microbial immune interactions and its impact on fish health remain largely unexplored (Brestoff and Artis 2013; Clements et al. 2014; López Nadal et al. 2020). Since zebrafish have a short growth cycle and play an important role in the evolution of fish, they are excellent models to examine the effects of diet on the complex interactions between the microbiome and gut immune function (Galindo-Villegas 2016; Volff 2005). Very recently, a few studies investigated the effect of dietary microalgae L. incisa on gut health of model animal zebrafish (Lazado et al. 2019; Nayak et al. 2020).

In this work, we systemically evaluated the effects of dietary Schizochytrium sp., Spirulina platensis, Chlorella sorokiniana, Chromochloris zofingiensis, and Dunaliella salina on the growth, immune status, and microbiota of zebrafish (Fig. 1). We examined the indices of these microalgae dietary supplementation on growth performance, and expression of immune and inflammatory genes in zebrafish. Also, we performed next-generation sequencing of 16S ribosomal ribonucleic acids (rRNA) amplicons, which were carried out on the total deoxyribonucleic acids (DNA) samples from zebrafish gut microbiota after feeding the diets supplemented with different microalgae. The results showed that these five different microalgae diets containing characteristic nutrition had positive effects on the expression of immune genes and the composition of gut microbiota.

Fig. 1
figure 1

Schematic diagram of experimental design

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Materials and methods

Animal experiment design

Diet preparation

Lyophilized microalgae powder, including S. platensis, C. sorokiniana, C. zofingiensis, and D. salina, was kindly supplied by SDIC Microalgae Biotechnology Center, SDIC Biotechnology Investment Co., Ltd (https://biotech.sdic.com.cn/gtsw/index.htm, Xizhimen Nanxiao Street, Xicheng District, Beijing, China). Schizochytrium sp. (CCTCC AF 2,010,001) was obtained from China Center for Type Culture Collection. The strain was first cultured in 500-mL flask at 28 °C, and after 4 days, cells were harvested and lyophilized. The commercial diet, dried bloodworm, was grounded into a powder with a mixer. Experimental feeds were prepared by adding 15% (w/w) lyophilized microalgae powder from Schizochytrium sp., S. platensis, C. sorokiniana, C. zofingiensis, and D. salina to a commercial diet bloodworm (named as the Schi15%, Spla15%, Csor15%, Czof15%, and Dsal15% groups respectively). The mixtures which were doped with microalgae powder and bloodworm powder were added an appropriate amount of double distilled water, and made into a doughy shape. Then the mixture was made into wet pellets by passing through a 0.5 mm sieve. After that, the pellets were freeze-dried for 24 h, and then stored at − 20 °C in vacuumed plastic bags until used. Commercial feed without adding microalgae was processed with a similar procedure, as a control feed (the bloodworm group).

Zebrafish husbandry

Wild-type zebrafish (mean weight 0.21 ± 0.03 g) were obtained from local supplier and allocated to the breeding system with one group set per one tank (10 L) per dietary group type, about 110 tails per group. The experiment lasted 4 weeks and the temperature was maintained at 25 °C with a light/dark cycle of 14/10 h. During the culture period, the water quality parameters were maintained at a normal level, and the water was changed according to the water quality. Fish were hand-fed to visual satiety twice daily. The total fish weight of each group was recorded at the beginning and end of the experiment, and the feed consumption during the experiment was also collected. After gathering the final weights, the fish were randomly selected for pathogen challenge and sample collection.

Pathogen challenge

The wild-type Vibro anguillarum strain MVM425 was previously isolated and identified in our laboratory (Xiao et al. 2011). Strain V. anguillarum MVM425 was grown in LB20 medium at 28 °C for 16 h. Bacteria harvested by centrifugation were rinsed twice in sterile physiological seawater (PSW, NaCl, 20 g/L; NaHCO3, 0.11 g/L; KCl, 0.7 g/L; MgCl2⋅6H2O, 4.8 g/L; MgSO4⋅7H2O, 3.5 g/L; and CaCl2⋅2H2O, 1.6 g/L; pH 7.2) and diluted to the appropriate concentration in PSW for infection. Zebrafish were bath-challenged in the aerated bacterial PSW suspension of 2 × 108 CFU/mL (CFU, colony-forming units) V. anguillarum MVM425 for 10 min, then transferred to clean water for 5 min, and finally removed back to the rearing water.

Sample collection

At the end of the feeding trial, the fish in each tank were fasted for 24 h. Zebrafish were randomly selected from each group and placed on ice to be anesthetized and then dissected. The entire zebrafish gut was carefully removed, avoiding contamination from other smaller organs and visceral fat. Intestinal content samples for microbial community analysis were collected in sterile 1.5-mL tubes on dry ice, immediately snap-frozen in liquid nitrogen, and stored at − 85 °C until analysis. After this, the middle and the posterior intestine were separated and immediately placed in RNAstore Reagent (Tiangen, Beijing, China) for gene-expression analysis. From each group, thirty fish were randomly selected to collect the intestinal tissues and the gut microbial samples for gut associated gene expression and gut microbial community analysis, respectively. These thirty fish were assigned into three biological replicates (ten fish per pool). Another forty-five fish were randomly selected for challenge with V. anguillarum MVM425, and then thirty of them were collected the intestinal tissues (ten fish per pool) for gut associated gene expression analyses.

RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)

The intestines of 10 fish were pooled (three pools per group) and frozen at − 85 °C. TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) was used for total RNA extraction according to the manufacturer’s protocol. The quality and quantity of RNA were checked by Nanodrop oneC (Thermo Scientific, Waltham, MA, USA). Then, 800 ng total RNA was used to synthesize first-strand complementary DNA (cDNA) using the PrimeScript™ RT Master Mix (TaKaRa, Shiga, Japan) for real-time qPCR following the manufacturer’s instructions. The resulting cDNA was used as a template for qPCR in a Bio-Rad CFX96™ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR Green mix (TOYOBO, Osaka, Japan). Reaction mixture (20 μL) contained 10 μL SYBR Select Master Mix, 1 μL of cDNA sample, 1 μL each of forward and reverse primers (500 nM), and 7 μL PCR-grade water. Primers used in this study are shown in Table S1 (Online Resource 1). The running program of real-time qPCR was composed of an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, and 60 °C for 30 s.

Relative expressions of target genes before challenge were calculated by the 2−ΔΔCT method (Livak and Schmittgen 2001), and β-actin was selected as the reference gene.

Biomolecule components analysis

Protein content analysis

The Kjeldahl method was used to determine the protein content of freeze-dried bloodworm and microalgae materials with some modifications (Nielsen 2017). Samples were precisely weighed at 500 mg. Ammonium sulfate as the positive standard and saccharose as the negative standard were also weighed. Briefly, the materials were digested in sulfuric acid with potassium sulfate and copper sulfate pentahydrate as catalysts for 1 h to convert the amine nitrogen into ammonium ions. Then, the ammonium ions in the samples were converted into ammonia and separated from the digestion mixture with a distiller (Xianjian Shanghai KDN-102A, Shanghai, China). Finally, the ammonia was determined by titration with a standard solution of hydrochloric acid. The percentage of nitrogen was converted to the content of crude protein with a factor of 6.25. Each sample was analyzed in duplicate.

Soluble sugar content analysis

The content of polysaccharides was measured by the phenol–sulfuric acid method with some modifications (Dubois et al. 1956). Samples (10 mg freeze-dried materials) were resuspended with 1 mL deionized water and broken up by ultrasonication (Scientz ultrasonic homogenizer JY92-IIN, Ningbo, China) at 150 W (working for 2 s, intermittent for 2 s) for 15 min. The mixture was centrifuged at 2000 g (Eppendorf 5810R, Hamburg, Germany) for 10 min at 4 °C. Briefly, the diluted samples were mixed with 0.5 mL 6% phenol solution and 3.5 mL concentrated sulfuric acid. After keeping still for 10 min, the mixture was vortexed and kept at 40 °C for 10 min. The absorbance at 490 nm was recorded. Later, the concentration was calculated according to the absorbance and the standard curve determined by dextrose solution.

Chlorophylls and total carotenoids analysis

The chlorophylls a and b, and total carotenoids content were determined by using the UV–VIS spectroscopy method (Lichtenthaler and Buschmann 2001). Samples were precisely weighed at 20 mg and the pigment content was extracted in 0.8 mL acetone by grinding for 2 min and ultrasonication (Scientz ultrasonic homogenizer JY92-IIN, Ningbo, China) at 150 W (working for 2 s, intermittent for 2 s) for 5 min on the ice. The mixture was centrifuged at 2000 g (Eppendorf 5810R, Hamburg, Germany) for 10 min at 4 °C. The pigment-containing phase in acetone was collected and transferred to a clean vessel. A further 0.8 mL of acetone was added to the residue, and the pigments were extracted and centrifuged again. After the extracted solution was mixed, filtrated, and diluted, an aliquot of this solution was added to a glass cuvette and exposed to the selected wavelengths (661.6 nm, 644.8 nm, and 470 nm). The absorbance was recorded, using acetone as the blank, and calculated according to the Lichtenthaler and Buschmann equations (Lichtenthaler and Buschmann 2001).

Lipid extraction

Lipids were extracted from the freeze-dried materials using a modified Folch method (Folch et al. 1957). Samples (60 mg freeze-dried materials) were dissolved with 3 mL chloroform and methanol solution (w/w = 2:1), vortexed for 2 min and broken up by ultrasonication for 3 min. Then the mixture was centrifuged at 2000 g (Eppendorf 5810R, Hamburg, Germany) for 10 min at 4 °C. The phase containing chloroform lipid was collected and transferred to a clean vessel. A further 2 mL of chloroform and methanol solution was added to the residue, vortexed, and centrifuged again. The combined lipid extracts were then evaporated under nitrogen gas flow.

Fatty acids composition analysis

Samples were transmethylated with 1% H2SO4 in dry methanol (v/v) at 70 °C for 0.5 h. Fatty acid methyl esters (FAMEs) were extracted with cyclohexane containing 0.133 g/L methyl heptadecanoate (Aladdine, Shanghai, China) as the internal standard. FAME samples (1 µL) were then separated by gas chromatography (Agilent GC 6890, USA) with HP-5 Column (30 m × 0.25 mm × 0.25 µm) (Agilent Technologies, Santa Clara, CA, USA), during which the temperature gradient is from 120 (hold time 1 min) to 170 (linear increase of 10 °C min−1; hold time 2 min) and finally to 260 °C (linear increase of 3 °C min−1; hold time 3 min). Then the samples were detected by mass spectrometry (Agilent Technologies MS 5975I, Santa Clara, CA, USA). Compound identification was achieved by matching with mass spectra database (NIST/EPA/NIH Mass Spectral Library, Version 2.0d, USA). FAMEs were quantified by using the ratio of the peak area of each target compound to that of the internal standard.

Next-generation sequencing of 16S rRNA amplicons

Extraction of genomic DNA

The DNA of the intestinal content was extracted by FastDNA SPIN Kit for soil (MP Biomedicals, Solon, OH, USA). The purity and concentration of DNA were tested by Nanodrop oneC (Thermo Scientific, Waltham, MA, USA). DNA solutions were stored at − 85 °C.

Denaturing gradient gel electrophoresis (DGGE) analysis

Primer F357 (5′-CCTACGGGAGGCAGCAG-3′ with GC clamp in the 5’end) and R518 (5′-ATTACCGCGGCTGCTGG-3′) were chosen for variable V3 region of the bacterial 16S rRNA gene amplification, which had been proved to be suitable for detecting zebrafish gut microbiota diversities in soil using DGGE analysis(Li et al. 2012). A GC-clamp was added to the terminal primer to improve electrophoretic separation amplicons by DGGE. The PCR reactions were conducted with Taq DNA polymerase (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol. The touchdown PCR was performed, and a two-step cycle was applied in the touchdown PCR; i.e., a denaturing step and an annealing step. The denaturing temperature was 95 °C, and the annealing temperature (68 °C) was gradually lowered (1 °C lower after each set of cycle) to 59 °C. A thirty-cycle amplification was then performed with an annealing temperature of 58 °C. Products were checked by electrophoresis in 2% (w/v) agarose gels and ethidium bromide staining. PCR products from each sample were separated by DGGE system (BioRad, Hercules, CA, USA) as follows: 9% polyacrylamide gels and a denaturing gradient from 35 to 65% were used; gels were electrophoresed in 1 × TAE buffer at 60 °C and 130 V for 6 h.

16S rDNA Illumina sequence analysis

The V3-V4 regions of the 16S rRNA genes were targeted for 16S rDNA Illumina sequence analysis and following taxonomy analysis. A linker with index was added to the end of the PCR product of 16S rDNA by PCR for next-generation sequencing (NGS), and the library was purified with magnetic beads. Then the library concentration was quantified to 10 nM, and PE250/FE300 paired-end sequencing was performed according to the Illumina MiSeq/Novaseq (Illumina, San Diego, CA, USA) instrument manual. The sequence information was read by the MiSeq Control Software (MCS)/Novaseq Control Software (NCS) (https://support.illumina.com/sequencing/sequencing_instruments/miseq). The sequencing raw data was deposited in the NCBI Sequence Read Archive database (accession: PRJNA759359).

Following quality control, the sequences were aligned, checked for chimeric sequences, and clustered into different operational taxonomic units (OTUs) on the basis of 97% sequence similarity using VSEARCH (1.9.6) software (Rognes et al. 2016). The sequences were then classified according to the Silva 138 database (Yilmaz et al. 2014). Then, the RDP (Ribosomal Database Program) classifier Bayesian algorithm (Wang et al. 2007) was used to analyze the representative sequence of OTU species taxonomy, and count the community composition of each sample under different species classification levels.

Statistics and data analysis

Data are presented as mean ± SEM. t-tests were performed with GraphPad Prism software (Version 8.0.1, GraphPad Software Inc., San Diego, CA, USA) to determine statistical significance. p-values less than 0.05 were considered statistically significant.

Results

Growth performance

The growth performance of the fish fed with the different experimental diets is shown in Table 1. The growth of all microalgae additive groups was better than that of the bloodworm-fed group. Regarding weight gain (WG) and weight gain rate (WGR), increases were found in the fish fed with different microalgae diets compared to the values of the fish in the bloodworm-fed group. Diet supplementary of S. platensis, C. sorokiniana, and C. zofingiensis showed a similar WGR of zebrafish, which were 2, 1.68, and 1.69 times to the WGR of the bloodworm group, respectively. The fish in the Schizochytrium sp. and D. salina additives groups displayed a significant bodyweight increase with above threefold of WGR compared to the bloodworm group. As for specific growth rate (SGR), the values in all the microalgae additives groups were observed to be higher than that in the bloodworm group, especially in the Schizochytrium sp. and D. salina additives groups. The best feed conversion ratio (FCR) was obtained from the group with a diet containing 15% D. salina, which means the fish in this group had the best growth. The other microalgae additive groups also showed better FCR.

Table 1 Zebrafish growth performance fed with different microalgae diets for 4 weeks
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Innate immunity response

To evaluate the impact of algal dietary supplementation on the expression of genes related to immune function, a real-time qPCR was performed using RNA isolated from the intestine of zebrafish fed with the different experimental diets. The expression of these genes is shown in Fig. 2.

Fig. 2
figure 2

Innate immune related gene expression after 4 weeks rearing. Real-time PCR quantification of expression of genes involved in immune responses in zebrafish intestine. Data are presented as 2-ΔΔCT (expression normalized to housekeeping gene β-actin). Significant differences between microalgae diet group and the bloodworm group are indicated by asterisks and corresponding p-value was determined by t-test. (n = 3 biological replicates, a pool of 10 fish each). Differences were considered significant at p < 0.05. Schi Schizochytrium sp.; Spla S. platensis; Csor C. sorokiniana; Czof C. zofingiensis; Dsal D. salina. The percent 15% (w/w) means the proportion of microalgae biomass added to commercial fish feed bloodworm

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For antigen recognition and signal transduction molecules, gene expression of toll-like 2 receptor (TLR2) and of TLR4 had a few changes in microalgae additive groups, compared to the bloodworm group. The gene TLR2 was downregulated in the Schi15% additive group, but not significant, and the TLR4 gene showed a similar downward tendency. The expression of myeloid differentiation primary response gene 88 (MyD88) and of the gene for nuclear factor kappa B (NF-κB) were significantly downregulated in the Schi15% additive group, and there was no difference between the bloodworm diet group and the others.

The gene expression of the inflammation related cytokines was examined, including the genes for pro-inflammatory cytokines tumor necrosis factor alpha (TNFα), for interleukin beta (IL1β) and IL8, and for anti-inflammatory cytokine IL10. In the groups fed with Schi15%, Spla15% and Csor15% supplemented diets, the gene expression of pro-inflammatory cytokines TNFα and IL1β was significantly downregulated, but a downregulation of the IL8 was not apparent, as compared to the control. In the Dsal15% additive group, the gene expression level of IL1β was significantly downregulated, while the levels of TNFα and IL8 were also downregulated but not apparent. In addition, in the Czof15% group, these four cytokines displayed an upregulated expression trend compared with the bloodworm diet group.

Gene expression of antibacterial proteins with microalgae supplemented diet was also detected. Comparing the expression level to the control, the Csor15% supplemented diets led to an increasing trend in the gene expression of lysozyme; however, the other microalgae additive groups had no change.

Disease resistance

To further investigate the immunomodulatory properties of microalgae diets, the expression of immune-related genes in the intestine of surviving fish was measured. To assess the impact of microalgae diets on bacterially-challenged zebrafish immunity, the transcriptions of immune related genes were detected at 12 h post challenge with V. anguillarum MVM425, as shown in Fig. 3.

Fig. 3
figure 3

Immune related gene expression after challenging Vibrio anguillarum 425. Real-time PCR quantification of expression of genes involved in immune responses in zebrafish intestine. Data are presented as 2-ΔΔCT (expression normalized to housekeeping gene β-actin). Significant differences between microalgae diet group and the bloodworm group are indicated by asterisks, corresponding p-value was determined by t-test. (n = 3 biological replicates, a pool of 10 fish each). Differences were considered significant at p < 0.05. Schi Schizochytrium sp.; Spla S. platensis; Csor C. sorokiniana; Czof C. zofingiensis; Dsal D. salina. The percent 15% (w/w) means the proportion of microalgae biomass added to commercial fish feed bloodworm

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The gene expression of pathogen recognition receptors and downstream signaling cascades was examined at 12 h post challenge with V. anguillarm, including TLR2, TLR4, TLR5, MyD88, and NF-κB. For TLR2, the expression in all the microalgal supplementary groups had a higher level than that in the bloodworm diet group. In contrast, the gene expression of TLR4 and TLR5 in the microalgae additive groups was apparently downregulated. The expression of MyD88 in these algal supplementary groups attenuated, positively associated with the downregulation of TLR4 or TLR5, except that in the Csor15% additive group, the expression of MyD88 did not change. However, the expression of NF-κB in the Csor15% additive group appeared to rise as compared to that in the bloodworm diet group.

The transcriptional changes of cytokines related to inflammation in the mucosal tissues of zebrafish were further investigated after the V. anguillarum MVM425 challenge. The gene expression of IL1β, IL6, IL8, TNFα, and the gene for C–C Motif Chemokine Ligand 20 (CCL20) in the Schi15% additive group was apparently lower than in the bloodworm diet group. Similarly, downregulation by other microalgae supplementation was observed on the expression of IL6. The Csor15% diet group exhibited higher expression of the genes IL8, TNFα, and CCL20 compared to the bloodworm diet group.

The biomarker genes, mpeg1 and b7r, representing macrophages and neutrophils, as well as mpx, the marker gene of zebrafish mononuclear phagocytes (Wittamer et al. 2011), were also selected to investigate the changes in intestine of zebrafish after infection. The qPCR data indicated the expression of b7r in the Csor15% supplementary group was higher than that in the bloodworm diet group; while, the expression level of b7r in other microalgae additive groups was lower than that in the bloodworm group. As for mpx, the expression level was higher in the Spla15% additive group than that in the bloodworm group, and the other microalgal supplementary groups exhibited a downward trend. However, there was no significant change regarding the expression of mpeg1 among the groups.

Gut microbiota

DGGE analysis and 16S rDNA Illumina sequence analysis were performed to investigate the gut microbiota composition as shown in the Fig. S1 (Online Resource 1). In order to obtain species OTUs corresponding to classification information, we selected a representative sequence for each of the OTUs, and the RDP classifier was used for classification of species on the representative sequence annotation to obtain each sample’s community composition (Online Resource 2).

To estimate the number of OTUs in a community, abundance-based coverage estimator (Ace) and Chao1 indices are commonly used to measure community richness (Chao 1984, Chao and Lee 1992). Shannon and Simpson indices are commonly used to characterize community diversity (Hill 1973; Izsák and Papp 2000). Compared with the bloodworm group, the intestinal microbiome abundance of zebrafish in the Spla15% and Czof15% groups decreased (shown in Table 2). Conversely, the community abundance in the Csor15% group increased. In general, alpha-diversity decreased when fish were fed with microalgae diets.

Table 2 Richness and diversity of the samples
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The 30 most abundant OTUs were selected for clustering to show the similarity and difference between the data (Fig. 4a). In order to further investigate the diversity difference among the groups, principal coordinate analysis (PCoA) analysis was performed (Fig. 4b), and the clustered samples were made up of similar microbial communities. Comparing the relative abundances of phylum in each group, it is found that the two most abundant species were Fusobacteriota and Proteobacteria (Fig. 4c). Interestingly, the proportion of Fusobacteriota in each algae meal diet group increased compared to the bloodworm diet group. Firmicutes accounted for 20.4% only in the bloodworm diet group, and none of the proportion in the other groups exceeded 2.5%. According to the community abundance data of different groups, differences of community structure between groups were detected, and the significant differences of species composition between groups were analyzed at the genus level. The genus with significant differences in abundance among the groups were Cetobacterium, Mycoplasma, Chitinibacter, Vibrio, and Shewanella (Fig. 4d). Comparing the relative distribution of at the genus level of each group, Cetobacterium was the most abundant genus among all groups. Moreover, the proportion of Mycoplasma in the bloodworm feed group was higher than that in the other groups. Compared with the ordinary diet group, the abundances of Chitinibacter in the Dsal15%, Schi15%, and Czof15% groups significantly decreased (p < 0.05), and Shewanella also significantly reduced in the Dsal15% diet group (p < 0.05).

Fig. 4
figure 4

Heatmap of hierarchy cluster results for the relative abundance of top 30 OTUs (a). PCoA of all samples based on Bray–Curtis distance matrix (b). Species relative abundance at phylum level (c) and genus level (d) in the zebrafish intestine. (n = 3 biological replicates, a pool of 10 fish each). Schi Schizochytrium sp.; Spla S. platensis; Csor C. sorokiniana; Czof C. zofingiensis; Dsal D. salina. The percent 15% (w/w) means the proportion of microalgae biomass added to commercial fish feed bloodworm

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Discussion

Microalgae diets enhanced the growth performance and immune status of zebrafish

Microalgae is an important food source for many aquatic animals (Shah et al. 2018; Yarnold et al. 2019). The non-toxic and nutritious characteristics of some microalgae meet the nutritional needs of fish. We used zebrafish as a model and added 15% of Schizochytrium sp., S. platensis, C. sorokiniana, C. zofingiensis, and D. salina to the feed. The data showed that dietary addition of these microalgae could improve the growth performance of zebrafish. Similar to our conclusion, Furbeyre et al. (2017) studied the effects of a microalgae diet on growth performance of mammalian piglets, and the results showed that pigs receiving diets supplemented with the Spirulina sp. or Chlorella sp. had higher digestibility in the jejunum compared with the control group.

Besides, the microalgae cells contain naturally active ingredients as shown in the Table 3. Because of their antioxidant, anti-inflammatory or anti-cancer activity, they have aroused interest in the food and pharmaceutical industry (Ávila-Román et al. 2018; Bito et al. 2020; Christaki et al. 2013; Ferrazzano et al. 2020; Talero et al. 2015). It is worth noting that, every microalga has its characteristic nutrients. For example, S. platensis has the highest protein content, C. sorokiniana contains the highest soluble polysaccharide, and C. zofingiensis has the highest total fatty acids. In our study, although the nutritional components of Schizochytrium sp. were similar to the ones of bloodworm (the control group), the zebrafish in the Schi15% additive group showed better growth performance than the fish in the control group. The analysis result of fatty acids composition indicated that DHA was solely detected in Schizochytrium sp., as shown is Table S2 (Online Resource 1), which means DHA might be the key factor of the fish’s rapid development in the Schizochytrium sp. group. Moreover, a certain amount of pigments were found in all the other microalgae we used except Schizochytrium sp. With the fact that every group of zebrafish grew well and were in healthy immune status, pigments could have a function of promoting the health of fish. In general, the nutritional components in every group had huge differences, and the growth performances of the fish in the microalgae diet groups were all better than those in the control group.

Table 3 TFAs, proteins, soluble polysaccharides, and pigments content of the feed material
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We explored the intestinal inflammatory status of zebrafish after the four-week microalgae dietary supplement, and most inflammatory cytokines in the Schi15%, Spla15%, Csor15%, and Dsal15% group declined with respect to the ordinary bloodworm feed group. These four microalgae diets attenuated the intestinal inflammatory responses at the normal physiological state of zebrafish. Similarly, Lazado et al. (2019) observed that the expression of IL1β was decreased in the zebrafish intestine with the increase of microalgae L. incisa feed. Another work showed that the expression of NF-κB was significantly increased in the kidneys of fish fed with the wild type and mutant L. incisa supplements; while, the gene for pro-inflammatory cytokine TNFα was significantly downregulated in the fish fed with both of the supplemented diets, as compared to the control (Nayak et al. 2018). However, in piglets, oral administration of S. platensis elevated the expression of IL8 in the ileum and oral administrated Chlorella vulgaris elevated IL1β expression in the jejunum (Furbeyre et al. 2018). Cerezuela et al. (2013) showed that the addition of microalgae (Tetraselmis chuii and P. tricornutum) alone or in combination with Bacillus subtilis increased the expression of the gene for pro-inflammatory cytokine IL8 in the gut of gilthead seabream (Sparus aurata L.). Reyes-Becerri et al. (2013) demonstrated that dietary lyophilized Navicula sp. caused a higher expression of IL8 and IL1β than a silage diet in gilthead seabream (Sparus aurata) intestine. Nevertheless, the details in these studies about how the microalgae diets elevate pro-inflammatory factors are undefined. Similarly in our study, IL8 and IL1β possessed a higher expression in the Czof15% group than in the bloodworm diet group. Strangely, the gene for the anti-inflammatory cytokine IL10, whose product shows an opposite effect to the pro-inflammatory factor, exhibited higher expression in the Czof15% group, which needs to be clarified in future research. These results suggest that different bioactive molecules contained in microalgae exert anti-inflammatory function through a variety of mechanisms (Tabarzad et al. 2020).

Microalgae dietary supplementation improved the resistance to infection by V. anguillarum

When the pathogen associated molecular patterns (PAMP) are recognized by TLRs, cellular signaling cascades can be activated through MyD88-dependent pathways that lead to the production of inflammatory cytokines (Rauta et al. 2014). TLRs can activate MyD88, and the downstream cascade is triggered and activated by related kinases. Finally, the signal is transmitted to the nucleus through NF-κB nuclear translocation to initiate an inflammatory response. The transcription factor NF-κB exists in almost all the animal cell types and plays a vital role in inflammatory signaling pathways, regulating the immune response to infection. It controls the expression of various cytokines, such as IL1β, IL6, IL8, and TNFα (Kong et al. 2021).

Our previous study has reported that up-regulation of TLR5 triggers the MyD88-dependent signaling pathway after bath-vaccination of a live attenuated V. anguilarum vaccine (Liu et al. 2014). In this study, the expression discrepancy trend of TLR5 and MyD88 was consistent, verifying that V. anguillarum MVM425 stimulated the immune response through the signal transmission pathway mediated by the coupling of TLR5 and MyD88. TLR5 binds to bacterial flagellin, activates signal transduction through NF-κB, and triggers the innate immune response to invading pathogens (Yoon et al. 2012). Meanwhile, it was reported that the gene expressions of IL1β and IL8 were increased significantly within 12 h after V. anguillarum MVAV6203 vaccination (Liu et al. 2014). As is known, IL1β is a major initiator of inflammatory and immune responses, and IL8 facilitates migration of neutrophils in pathogen resistance (Oehlers et al. 2010). In this work, we detected the expressions of these cytokines at 12 h post infection with V. anguillarum MVM425, and the expressions of these cytokines showed varying degrees of decline in most microalgae diet groups compared to the bloodworm diet group.

Bravo-Tello et al. (2017) evaluated the effects of five microalgae on zebrafish, including Tetraselmis sp., P. tricornutum, Chlorella sp., N. oculata, and N. gaditana, as additives in a soybean meal-based diet, to be specific, on intestinal inflammation and survival after Edwardsiella tarda infection. The study showed that all microalgae reduced intestinal inflammation triggered by the soybean meal, and only Tetraselmis sp. and Chlorella sp. diets decreased the mortality of larval fish against bacterial infection. This appears to be supported by an increase in neutrophils. Nayak et al. (2018) reported that dietary supplementation with the ω6 LC-PUFA-rich microalga L. incisa improved the zebrafish’s resistance to Streptococcus iniae infection, due to the balance between n-3 and n-6 LC-PUFA in sustaining the inflammatory homeostasis. DHA and EPA have been found to inhibit NF-κB from reducing the gene expression of cytokines in macrophages (Gutiérrez et al. 2019; Mendivil 2021). It is demonstrated that the antibacterial efficacy of DHA is the highest among long-chain PUFAs (Desbois and Lawlor 2013). Schizochytrium sp. is a fast-growing thraustochytrid microalga rich in DHA (Sprague et al. 2015). In our study, DHA was detected in the Schi15% group but not in the other groups. The lower gene expression of inflammatory factors in this group compared to the bloodworm group might be attributed to DHA. Similarly, it has been reported that when tilapia (Oreochromis niloticus) was intraperitoneally injected with DHA, the gene expressions of IL1β, IL6, IL8, and TNFα were significantly down-regulated 12 h after the fish was infected by Vibrio vulnificus (Pan et al. 2017).

In rats, it was found that S. platensis had anti-inflammatory effects on acetic acid-induced experimental colitis (Tabarzad et al. 2020). Similar results were shown in our experiments. The cytokines and chemokines of the Spla15% and Dsal15% groups as well as the Schi15% and Czof15% groups maintained similar levels or lower levels with those of the ordinary feed group. Mucosal immune cells such as macrophages, lymphocytes, and granulocytes are distributed in the epithelium and lamina propria, and play a crucial role in the defense system of the intestinal mucosal surface (Lazado and Caipang 2014). Neutrophils are the first type of leukocytes to arrive at infection sites and display potent microbicidal functions. Therefore, they have classically been viewed as the first line of defense (Rosales 2020). In our study, compared with the bloodworm feed group, the gene expression of neutrophil marker b7r in the Csor15% group was increased, which indicates that it was activated after infection. Subsequently, it promoted the expression of TNFα and CCL20, improving the disease resistance of zebrafish.

We compared the relative expression of intestinal immune genes before and after V. anguillarum infection by the 2−ΔCT method as shown in the Fig. S2 (Online Resource 1). It was interestingly found that, before the infection, IL8 and IL1β expressions were at a lower level in the remaining microalgae diet groups compared to the red worm dry diet group. In contrast, after bacterial infection, NF-κB, IL1β, IL8, TNFα, etc. were all obviously promoted in expression. Physiologically, this is explained by the fact that these pro-inflammatory factors are expressed at low levels in the normal physiological state of the body to avoid inflammation and this mechanism appears to be more beneficial to health. Once infection occurs, the host rapidly produces inflammatory factors that activate the immune response, suppressing the exacerbation of infection. This conclusion can be imprinted by our experiment that the microalgae diets modulated the immune function of zebrafish and improved the resistance to infection by V. anguillarum.

Microalgae diets modulate gut health through alternating gut microbiota

The composition of the gut microbiota is closely associated with host immune health and plays a crucial role in maintaining host innate immune balance (Gómez and Balcázar 2008). Microalgae diet could regulate the abundance and homeostasis of probiotic and harmful bacteria, and plays a positive effect on the health of zebrafish. Cetobacterium is the most common bacterium found in the gut of freshwater fish (Ramirez et al. 2018; Sugita et al. 1991). Since Cetobacterium can produce vitamin B12 with high efficiency, it can supply vitamin B12, which is necessary for the physiological activities in freshwater fish (Tsuchiya et al. 2008). In our study, all of the microalgae diets increased the abundance of Cetobacterium compared with the bloodworm diet, especially in the Spla15% group (p < 0.05). Thus, the microalgae diets might exhibit probiotic properties, modifying the microbiota composition and improving fish health.

The metabolites of the intestinal bacteria, which were derived from dietary lipids, could be utilized as bioactive modulators for the host health and disease. For example, Stenotrophomonas could metabolize linoleic acid into hydroxy fatty acids (i.e., 10-hydroxy-cis-12-octadecenoic acid (HYA))—decreasing the expression of surface receptor associated with intestinal epithelial cells, and thereby preventing the receptor-mediated inflammation and protecting the epithelium (Nagatake and Kunisawa 2019; Saika et al. 2019). Our experiment showed that the abundance of this genus was higher in the Schi15% group than in the other groups (p < 0.05). A low inflammatory level consistently exhibited in this group, which might have a certain correlation with this genus.

The abundance of Chitinibacter was reduced in the microalgae diet groups. However, there are few studies on the association between Chitinibacter and host intestinal health. Chitinibacter could secrete chitinase-a key enzyme to digest chitin in crustaceans and is closely related to the growth and immune function of crustaceans (Niu et al. 2018). Perhaps the lack of chitin in our diets has reduced the abundance of this probiotic.

Mycoplasma was considered as a member of the phylum Firmicutes (Davis et al. 2013). It is a common inhabitant in rainbow trout (Oncorhynchus mykiss) intestines with insect-based diets. As a result of a long-established symbiosis, this microbe benefits from gut fermentable substrates and the host benefits from the lactic and acetic acid produced by bacterial fermentations (Rimoldi et al. 2019). However, there has been reported that Mycoplasma is potentially pathogenic and can lead to intestinal lesions and even tumorigenesis in zebrafish (Burns et al. 2018; Huyben et al. 2020; Kent et al. 2021). Shi et al. (2021) reported that diet supplemented with Schizochytrium sp. could reduce the abundance of harmful pathogen Mycoplasma in the zebrafish intestine. Interestingly, our experiments showed that the bloodworm diet accounted for the highest abundance of Mycoplasma in the intestinal tract. Meanwhile, the abundance of Mycoplasma decreased in the Schi15% group, as well as in the other microalgae diet groups, indicating that all tested microalgae diets improve zebrafish health.

Vibriosis is one of the most prevalent bacterial diseases affecting diverse aquatic animals (Ina-Salwany et al. 2019). It was reported that intestinal Vibrio spp. could increase the expression level of MyD88 and TLR signaling pathway genes in the conventionally reared zebrafish (Xin et al. 2020). Furthermore, the abundance of Vibrio in the Schi15%, Spla15%, and Csor15% groups decreased. Coincidentally, the gene expressions of TLR2, TLR4, and MyD88 decreased or remained unchanged in these groups. Therefore, Vibrio might be associated with these gene expressions. Similarly, Zhang et al. (2020b) indicated that the Vibrio community was positively correlated with TNFα and IL1β of the koi carp (Cyprinus carpio) intestine, and dietary supplementation with oregano essential oil decreased the Vibrio community. Fish might have a mutualistic relationship with V. cholera (Halpern and Izhaki 2017), but the stakes between this pathogen and fish need to be carefully discussed.

Shewanella is an opportunistic pathogen for fish that is widespread in the aquatic environment (Jung-Schroers et al. 2018). Species of Shewanella collected from freshwater fish were genetically diverse (Pazdzior et al. 2019). Commensalism of several Shewanella species played a beneficial role in improving the host gut health (Camara-Ruiz et al. 2020; Cordero et al. 2015; Hao et al. 2017; Wei et al. 2021; Zhang et al. 2020a). However, the number of pathogenic bacteria in this genus is unknown. The abundance of Shewanella in our study was decreased in all microalgae diet groups. It might be explained that the microalgae diets could reduce the abundance of opportunistic pathogenic Shewanella sp.

Anyway, further elucidation of the microalgae diet-microbiota-host health interactive link is still needed in future. In addition, with the development of genetic engineering technology of many algal species, algal cells are modified to produce new bioactive substances, such as vaccines, antimicrobial peptides, and growth hormone (Charoonnart et al. 2018; Fajardo et al. 2019), providing the feasibility of functional algae feed for the aquatic industry.

In this study, we found that dietary supplemented with Schizochytrium sp., S. platensis, C. sorokiniana, C. zofingiensisi, and D. salina could enhance zebrafish growth performance and immune status. Microalgae diets possessed a positive effect on resistance to V. anguillarium infection, suggesting that such a nutritional replacement can be an efficient approach to improve the immune status and resistance to bacterial infection in fish. In addition, dietary supplementation with microalgae could modulate the intestinal community, forming a new homeostasis of probiotics and harmful bacteria. In a nut shell, these microalgae can be used as high-quality aquatic feed additives and have broad application prospects in the aquaculture industry. Also, these microalgae could be appropriate candidates for development of an oral vaccine vector in the future.