1 INTRODUCTION

As crucial live feed for marine fish and crustacean larvae, brine shrimp Artemia has been widely applied in hatchery production. With the expansion of aquaculture industry, the demand for Artemia cysts has continued to increase. The annual consumption is now estimated at 3 500–4 000 t, supporting the production of over 900 billion crustacean post-larvae and fish fry valued at more than USD 2 billion. Approximately 90% of current Artemia cysts yield relies on the harvest from inland salt lakes; therefore, the future of hatchery industry could be at risk due to unpredictable cyst production under pressure of global climate changes (Patrick Sorgeloos, personal communication). The controlled Artemia farming in hypersaline water or in saltponds integrated with salt production have been practiced for decades, in order to guarantee the sustainable provision of Artemia cysts as well as Artemia biomass for shrimp nursery and maturation (Van Stappen et al., 2020). Moreover, indoor Artemia tank culture is also considered an alternative Artemia source, providing Artemia with tailor-made size, controlled nutritional quality and biosecurity.

The non-selective filter-feeder Artemia can take up a wide range of food particles with size ranging from 4–8 μm (Makridis and Vadstein, 1999) to 50 μm (Lavens and Sorgeloos, 1996) from nauplii to adult. Moderately halophilic Proteobacteria Halomonas are widely presented in hypersaline water body, where Artemia are mostly dominant microzooplankton in the common environment. And Halomonas also have been identified as one of the major Artemiaassociate genera in Artemia gut (Tkavc et al., 2011; Bellisario et al., 2013; Quiroz et al., 2015). Several Halomonas species can synthesize massive amount of poly- β -hydroxybutyrate (PHB), a biodegradable and biocompatible material that are applied in environment protection and medical industry (Yin et al., 2015). For a decade, PHB has been considered a potential biocontrol agent in aquaculture as it can be partially degraded into β-hydroxybutyrate monomer in the gut of aquatic animals, and hence improve the growth of aquatic animal as well as enhance their disease resistance (Defoirdt et al., 2011). The studies in gnotobiotic Artemia have revealed that providing bacterial PHB accumulated in Brachymonas denitrificans (Halet et al., 2007) and Bacillus (Laranja et al., 2018) improved the resistance of Artemia against Vibrio campbellii. However, no study has been conducted with Artemia culture, regarding to the Artemia associated bacteria Halomonas and its storage compound PHB.

In our previous study, a super PHB-accumulating strain was obtained from local saltponds, in which the PHB content reached up to 65% cell dry weight (Sui et al., 2015). In this study, the effect of Halomonas-PHB on survival, pathogen resistance and gut microflora of Artemia were studied in gnotobiotic and xenic culture conditions. The aim of the study is to investigate the probiotic effect of Halomonas-PHB on Artemia, which can be further applied in the intensive Artemia culture to guarantee the bio-secured Artemia production as well as sustainable aquaculture.

2 MATERIAL AND METHOD 2.1 Bacteria culture

Halomonas 100-16-2 strain (CGMCC No. 13730) were fermented in a 5-Lfermenter at 37 ℃. The culture medium was prepared by dissolving 10-g/L yeast extract and 7.5-g/L acid hydrolyzed casein into the diluted brine (salinity 30) and adjusting to pH 7.5. To improve the PHB accumulation in Halomonas, 30-g/L glucose was added to the culture medium at start and 18 h in progress of 36-h fermentation. To obtain Halomonas without PHB accumulation, no glucose was supplemented in the medium. The cultures were collected at plateau growth phase by centrifugation at 8 000 r/min, 10 min. The cell pellets were rinsed and re-suspended with diluted brine (salinity 30), and stored at -4 ℃, for PHB content analysis and feeding experiments.

The PHB content of Halomonas was determined by HPLC after acid-hydrolyzing PHB into crotonic acid (Gao et al., 2019). Briefly, dry cell pellet was digested with 98% H₂SO₄ at 100 ℃ for 1 h. The mixture was diluted 50-fold with 5-mmol/L H₂SO₄ and PHB content was determined by HPLC (1260 Infinity, Agilent Technologies, USA) equipped with an Aminex HPX-87H ion exclusion organic acid column (Bio-Red, USA). Elute was 5-mmol/L H₂SO₄ at a flow rate of 0.7 mL/min. PHB content was determined at 210 nm and calculated according to the standard curve (PHB concentration of 0, 1, 2, 3, 4, and 5 mg/L), of which PHB powder (Good Fellow, UK) was treated with the same procedure as for the cell pellet. The PHB content in PHBaccumulating Halomonas was 48.69% on cell dry weight, while PHB could not be detected in Halomonas cultured without glucose supplementation.

Vibrio anguillarum (strain MCCC 1A07299) was cultured in LB broth at 28 ℃, 150 r/min for 7 h. The cells were collected by centrifugation at 3 000 r/min, 10 min. The cell pellets were rinsed and re-suspended with diluted brine (salinity 30), and applied immediately to the challenge test.

2.2 Gnotobiotic Artemia culture

All procedures were manipulated under sterile conditions. Two hundred milligram of parthenogenetic Artemia cysts originated from Aibi Lake, China (Boaifeng Biotech, Co., Ltd., China) were hydrated for 1 h in a falcon tube containing 18-mL distilled water. The chorion of the cysts was removed by adding 10-mL NaOCl solution (containing 10% active chlorine) and 660-μL 40% NaOH solution. When the chorine color turned into orange, 14-mL 10-g/L Na₂S₂O₃ solution was added to terminate the reaction. After rinsing thoroughly over a 100-μm sieve with sterile diluted brine (salinity 30) and transferring to a falcon tube containing 30-mL sterile diluted brine, the decapsulated cysts were hatched on a rotator at 28 ℃, 10 r/min. 24 h later, newly hatched nauplii were pipetted into the sterile glass tubes. Each tube contained 20 Artemia and 20-mL sterile diluted brine.

Artemia were fed Halomonas-PHB (HM.PHB) and Halomonas without PHB (HM) for 72 h, respectively. Feeding was conducted immediately after transferring Artemia into the tubes and given once per day with a daily feeding ration of 10⁷-CFU/mL medium. The cell density was determined by spectrophotometer, whereby OD₆₀₀ of 1.000 corresponded to 1.2×10⁹ cells/mL according to the McFarland standard (BioMerieux, Marcy L'Etoile, France). The starved Artemia was considered as control (Starvation). Separately, V. anguillarum (10⁷-CFU/mL medium) were daily added to Artemia culture, which referred as HM. PHB+VB, HM+VB, and VB group. Each treatment had four replicates. The gnotobiotic culture was terminated when the starved Artemia in Ctrl died completely (72 h). The axenity of Artemia culture were verified at the end of trial following the procedure described by Marques et al. (2006a).

2.3 Artemia culture in xenic condition

Artemia cysts were hatched at 28 ℃ in diluted brine (salinity of 30) for 24 h. The newly hatched Artemia nauplii were collected and transferred into a 50-L cylinder tank at a stocking density of 1 ind./mL. Artemia were reared at 25 ℃ for 7 days, and fed microalgae Chlorella in a daily feeding ration of 106 cells/mL. The on-grown Artemia were then collected and transferred into 10-L cylinder tanks containing 8-L diluted brine. In the following 14- day culture, Artemia were fed Halomonas (HM), Halomonas-PHB (HM.PHB) and microalgae Isochrysis galbana powder (ISO, SDIC Microalgae Biotechnology Center, China), respectively, with daily feeding ration of CFU/mL. Each treatment contained five replicates.

2.4 Parameter determination 2.4.1 Gnotobiotic Artemia culture

At the end of the gnotobiotic culture, the survival percentage of Artemia were determined. The Artemia body length (10 Artemia per treatment) were measured under stereoscopic microscope (Olympus S261, Japan).

2.4.2 Xenic Artemia culture

At the end of the culture, Artemia biomass were collected with sieve and rinsed with distilled water. The enzyme activity of trypsin, pepsin, lipase, and catalase (CAT), total superoxide dismutase (TSOD), and total antioxidant capacity (T-AOC) of Artemia were detected with analytical kits (Nanjing Jiancheng Bioengineering Institute, China).

For gut microbial diversity analysis, thirty Artemia were randomly collected from each group. The food residue in the gut was evacuated in duration of 24 h, by adding 1.5-g/L sodium cellulose solution into sterile diluted brine, which were renewed every 6 h. Artemia were then rinsed with PBS and cleaned with 75% ethanol. The dissection was conducted entire intestines of Artemia were dissected under stereoscopic microscope using sterile tweezers. The entire Artemia gut (including foregut, midgut and hindgut) was obtained by gently pressing the head and stretching the trunk, and removing the external chitin layer. Each of 10 guts from the same replicate were pooled under sterile conditions, and preserved immediately into liquid nitrogen, and stored at -80 ℃. Total genomic DNA were extracted using bacterial DNA extraction kit (TIANAMP, China), and were sent to Novogene Bioinformatics Institute (Tianjin, China) for high throughput sequencing analysis. The V3–V4 fragment of 16S rRNA gene was amplified with universal bacterial primer 341F/ 806R. Shannon index was calculated based on the OTUs to estimate the microbial diversity. The relative bacterial abundances were determined at phylum and genus level, to compare the gut microbiota structure in different groups.

Twenty Artemia were randomly collected from each tank and transferred to conical glass tubes containing 200-mL diluted brine (salinity 30). V. anguillarum was added at a final density of 5× 10⁸ CFU/mL. The survival percentage of Artemia was determined after 144 h. No feed was given during challenge.

2.5 Statistical analysis

Data were presented as mean±SD. Significant difference among the treatments was determined using one-way ANOVA followed by Duncan's test at P < 0.05 (SPSS 20.0 software).

3 RESULT 3.1 Survival and growth of Artemia in gnotobiotic culture

Under gnotobiotic condition, the starved Artemia all died three days post hatching (Fig. 1), and there was no significant difference in survival percentage between HM and HM.PHB groups (P>0.05). When challenged by V. anguillarum, HM.PHB+VB group had similar survival percentage comparing to the unchallenged HM and HM. PHB groups (P>0.05), but significantly higher than HM+VB group (P < 0.05), hile the survival percentage in VB group remained the lowest (P < 0.05).

Artemia in HM group had significantly better growth than HM-PHB group (P < 0.05). When challenged by V. anguillarum, Artemia in HM+VB group had significantly better growth than HMPHB+VB group (P < 0.05), while Artemia body length in VB group remained the lowest (P < 0.05) (Fig. 2).

3.2 Survival percentage of Artemia in xenic culture

Under xenic condition, the survival percentage of Artemia in HM, HM-PHB, and ISO groups ranged from 40% to 50% (data were not shown). When challenged by V. anguillarum for 144 h, the survival percentage of Artemia in ISO+VB group was significantly lower than that in HM-PHB+VB group, while HM+VB group was in between (P < 0.05) (Fig. 3).

3.3 Enzyme activity analysis

Under xenic condition, there was no significant difference in trypsin and lipase activity among the groups (P>0.05), while HM and HM-PHB feeding resulted in significantly higher pepsin activity than ISO (P < 0.05) (Table 1).

T-AOC and CAT in HM group was significantly higher than that in ISO group, and HM was in between (P < 0.05) (Table 2). T-SOD in HM.PHB was significantly higher than that in HM and ISO group (P < 0.05).

3.4 Gut bacterial composition

Value of Goods-coverage in different groups ranged of 0.998–0.999, suggesting that 16S rRNA gene in each library represented most bacteria in Artemia gut (Table 3). The Shannon index of ISO group was significantly higher than HM. PHB (P < 0.05) (Table 3), while HM group was in between.

At phylum level, Proteobacteria were dominant in ISO (94.7%±1.5%) and HM.PHB (81.6%±11.3%) groups, while Actinobacteriota dominated in HM group (54.2%±9.05%) (Fig. 4). The average percentage of Firmicutes and Bacteroidetes in all groups were 0.75%–0.93% and 0.67%–0.71%, respectively. Phylogenic analysis showed that the bacterial composition within the replicates clustered together, and HM.PHB group were closer to ISO group. The relative abundances of genus in Artemia gut are shown in Table 4. The top three bacteria genus in HM.PHB group consisted of Halomonas (69.4%), Brachybacterium (19.4%), and Vibrio (6.5%); while Brachybacterium (69.9%), Halomonas (18.2%), and Vibrio (2.3%) were in HM group. On the other hand, Halomonas (51.7%), Vibrio (34.6%), and Brachybacterium (1.5%) were in ISO group.

4 DISCUSSION

The gnotobiotic Artemia system provides a unique opportunity to clarify how the target microbes or compounds influence the growth and survival in an organism, because no interference of other microorganism presents in the system (Marques et al., 2006b). In this study, gnotobiotic Artemia culture system were used to clarify if Halomonas-PHB and Halomonas can the only food for Artemia and its role in protecting Artemia from Vibrio challenge. Meanwhile, Artemia were also cultured in xenic mass culture to obtain more Artemia biomass for enzymatic and gut microbial diversity analysis, which was relevant to the controlled Artemia production practice.

It has been reported that certain bacteria species have beneficial effect on the growth and survival of Artemia (Intriago and Jones, 1993). Bacteria also contribute to the nutritional value of Artemia as a major source of proteins and amino acids (Gorospe and Nakamura, 1996). Our study showed that under gnotobiotic condition, 50% Artemia survived on day 3 when receiving only HM or HM-PHB, while all starved Artemia died. This revealed that HM and HM-PHB could be the sole food for Artemia nauplii. Moreover, HM feeding resulted in a longer body length than HM-PHB, indicating the better nutritional value of Halomonas. PHB is accumulated in condition of limited nitrogen and phosphorus, but excess carbon sources (Quillaguamán et al., 2006). Actually, PHB accumulation is at the expense of other cell compounds. The PHB content of Halomonas (HMPHB) reached 48.69% cell dry weight in this study, thus major nutritional components (e.g., proteins and lipids) should be less than the Halomonas without PHB accumulation (HM). On the other hand, 10% survival percentage was obtained when exposing Artemia to V. anguillarum only, suggesting that V. anguillarum did not only provide pathogenic elements, but also contributed microbial (nutritional) compounds to Artemia.

For decades, probiotics and prebiotics have received much more attention in order to replace antibiotics in aquaculture (Sapkota et al., 2008). The beneficial effects of dietary PHB supplement on the growth, survival, immune status, and gut microbial condition of marine fish and shrimps have been investigated (De Schryver et al., 2010, 2011; Duan et al., 2017; Franke et al., 2017a, b). And it has been claimed that bacterial-storage amorphous PHB granules are more effective than solvent-extracted crystalline PHB, as the former is easier to be degraded in the gut of aquatic animals (Thai et al., 2014; Gao et al., 2019, 2020). For Artemia, the bacterial PHB accumulated in Brachymonas denitrificans (Halet et al., 2007) and Bacillus (Laranja et al., 2018) also enhanced the resistance of gnotobiotic Artemia against Vibrio campbellii. Our results showed that under both gnotobiotic and xenic culture conditions, Halomonas-PHB (HMPHB) feeding significantly improved the Artemia survival upon challenged by V. anguillarum, which confirmed the beneficial effect of Halomonas-PHB on the robustness of Artemia.

The probiotic bacteria offered protection against Vibrio anguillarum challenge in Artemia franciscana nauplii through modulating Artemia defense system (Giarma et al., 2017). Our study showed that feeding Artemia with HM-PHB and HM in xenic culture enhanced the T-AOC, T-SOD, and CAT activities, comparing to the microalgae Isochrysis feeding. The better antioxidant and immune status of Artemia fed with HM-PHB and HM should be in favor of the protection towards V. anguillarum challenge (Duan et al., 2015; Qiao et al., 2019, 2020). However, even though microalgae are prevailing live feed for zooplanktonic Artemia, the inferior performance was observed for Artemia fed with Isochrysis dry products. This might be caused by the less digestibility of the dehydrated Isochrysis cells.

It has been reported that dietary PHB increased bacterial richness and changed bacterial community in the intestine of European seabass Dicentrarchus labrax juvenile (De Schryver et al., 2010, 2011), white-leg shrimp Litopenaeus vannamei (Gao et al., 2019), and half-tongue sole Cynoglossus semilaevis (Gao et al., 2020). Artemia has a hooked and tubular gut structure, and the epithelium lining of the entire gut consisted of a single cell layer (Gunasekara et al., 2011). In this study, the entire gut of Artemia was dissected after 24-h evacuation. Therefore, the microbiota remained in the gut are the microorganisms that mostly adhered and/or resident to the gut epithelium. The significantly reduced gut microbial diversity in HM-PHB and HM should be causes by daily supplementing of HM-PHB and HM to the water column.

In this study, Proteobacteria were dominant in ISO and HM.PHB groups, while Actinobacteriota dominated in HM group. Although Firmicutes and Bacteroidetes are beneficial to the food digestion and absorption (Turnbaugh et al., 2006), the two phyla remained minor in all groups (less than 1% of total OTUs). On the other hand, high proportion of Halomonas presented in Artemia gut fed with Isochrysis maybe due to the universal bacterial primer used for high throughout sequencing, by which the eukaryotic microalgae could not be detected.

Some Vibrio species are common aquatic pathogens threat the sustainable development of aquaculture. Live feed Artemia has been argued of being a possible pathogen carrier causing the outbreak of Vibriosis (Quiroz-Guzmán et al., 2013). Although not all Vibrio species in the gut are harmful or potentially harmful to the host (Yévenes et al., 2021), it is worth noting that the proportion of genus Vibrio was largely suppressed in HM-PHB group. This should relate to the PHB degradation in the gut (Halet et al., 2007), though the gut pH could not be measured for the tiny Artemia gut. It has been reported that β-hydroxybutyrate monomer could lower the gut pH and consequently inhibited the growth of Gram-negative bacteria (such as Vibrio, Escherichia. coli, and Salmonella) (Cherrington et al., 1991), and provide energy to the gut epithelium cell through diffusion (Topping and Clifton, 2001; Pryde et al., 2002). The interactive and/or negative effect between resilience to Vibrio anguillarum and the reduction of gut diversity may impact other metabolic features of the live feed, and so its value/quality.

It has been reported that bioencapsulation of probiotic bacteria such as Lactobacillus latics, L. plantarumis, and Bacillus subtilis is an effective biological approach for the disease control in larviculture, through suppressing Vibrio growth or improving the digestive enzyme activities which in turn lead to further stimulation of endogenous enzymes in the fish and shrimp larvae (Touraki et al., 2012, 2013). Therefore, we suppose the administration of Halomonas-PHB in Artemia culture could not only result in the resistance to a Vibrio challenge and carrying fewer Vibrio in the gut, but also more important lead to the production of biosecure live feed Artemia.

5 CONCLUSION

Halomonas could be the sole food of Artemia, and PHB containing Halomonas conferred protection to gnotobiotic Artemia towards V. anguillarum infection. The higher resistance against Vibrio challenge, better antioxidative status, and suppression of Vibrio in Artemia gut resulted by HalomonasPHB and Halomonas make it a promising feed or feed supplement for a bio-secured intensive Artemia culture.

6 DATA AVAILABILITY STATEMENT

The data used to support this study are available from the corresponding author upon request.