Aquaculture is one of the fastest-growing and largest food production sectors that play an important role in providing sustainable livelihood opportunities and food security for the world population (Dawood et al., 2019; Stentiford et al., 2020). The development of practice models that contributes to the outstanding growth of the aquaculture industry and promotes the increase in terms of production has been considered. Pacific white shrimp (Penaeus vannamei) is one of the most important marine aquaculture species globally, accounting for approximately 80% of the total annual production (Zheng et al., 2021b). Similar to the general situation of large-scale intensive aquaculture systems, the shrimp aquaculture industry faces to deteriorating farming environment and frequent disease outbreaks, which cause great economic loss (Deng et al., 2013; Zheng et al., 2021a). The developed methods of treatment that overcome growth performance and increase disease resistance of shrimps are needed (Deng et al., 2013). Several reports suggested that aquatic animals fed with a diet supplemented with prebiotics, probiotics, synbiotics, or postbiotics revealed an enhancement in growth performance, immune response, and disease resistance when compared to their non-added counterparts (Hai, 2015; Hoseinifar et al., 2016; Ringø, 2020; Tran et al., 2020; Tran and Li, 2022). In crustaceans, probiotics bacteria, such as the members of Bacillus, Enterococcus, Lactobacillus, and Pediococcus, that showed improving growth and digestive enzyme activity, modulating gut microbiota, and enhancing immune response and hematological parameters have been widely investigated (Ringø, 2020; Tran and Li, 2022). In Pacific white shrimp, for example, the mixed-species probiotics (Bacillus subtilis, Bacillus licheniformis, and Lactobacillus sp.) have shown benefits to their hosts, including enhancing growth and non-specific immunity, changing intestinal morphology, and modulating gut microbiota (Xie et al., 2019). Furthermore, the application of yeasts, yeast extract, and other fungal-origin compounds (such as β-glucan) showed beneficial effects on growth, antioxidant ability, immunity, disease resistance, and intestinal microbiota of Pacific white shrimp (Murthy et al., 2009; Deng et al., 2013; Boonanuntanasarn et al., 2016; Li et al., 2019; Ma et al., 2019; Ayiku et al., 2020; Zheng et al., 2021a).

The fungus Aspergillus niger has been reported as one of the predominant species of the genus Aspergillus harboring the body of Pacific white shrimp (Cruz da Silva et al., 2011). Previous studies have shown that A. niger used for the fermentation of groundnut oil cake, soybean meal, sunflower oil cake, and plant protein mix can enhance growth, feed efficiency, and hemolymph indices in Pacific white shrimp (Jannathulla et al., 2018a; Jannathulla et al., 2018b; Dayal et al., 2020; Jannathulla and Dayal, 2022). Recently, in common carp (Cyprinus carpio), supplementing A. niger in the diet increased survival rate, growth performance, feed utilization, immune responses, hematological indexes, digestive enzyme activity, and the load of the fungi in the intestine (Jasim et al., 2022). Therefore, results of previous studies have proven the potential use of A. niger as a probiotic organism conferring health benefits to aquatic animals. However, the influence of A. niger as the dietary probiotic supplementation in Pacific white shrimps has not been investigated. This study aimed to evaluate the influences of A. niger (Insighter Co. Ltd., Guangzhou, Guangdong, China) on growth, oxidative activity, immune response, and gut microbiota of juvenile Pacific white shrimps under farming conditions. The results of this study provided basic information on the potential use of A. niger as a probiotic organism in shrimp aquaculture.

Sharma et al. (2022) indicated that A. niger is a potential probiotic, which can increase egg production and quality, and decrease the cecal microbial load of pathogenic bacteria (such as Clostridium perfringens, Salmonella spp., and Escherichia coli) in laying hens. In aquaculture, A. niger has been demonstrated to be a potential probiotic that confers health benefits on common carp (including the enhancement of growth performance, immunity, hematology, the load of the gut fungi, and digestive enzymes) (Jasim et al., 2022). However, the probiotic potential of A. niger on shrimp has not been evaluated yet. In this study, the effects of A. niger on growth performance, antioxidative activity, immune response, and changes in the gut microbiota of juvenile Pacific white shrimp under farming conditions were investigated for the first time. Results showed that the inclusion of A. niger can enhance the weight gain, specific growth rate, and length gain of shrimps, especially in the A1.5, indicating the growth promotion of A. niger (at 1.5 g/kg diet) shrimps under culture conditions. This similar to the previous findings in common carp fed a diet supplemented with A. niger (at 1×10³ or 1×10⁶ CFU/g diet) (Jasim et al., 2022). Jasim et al. (2022) showed an improvement in the final length and weight, weight gain, thermal growth coefficient, protein efficiency ratio, lipid efficiency ratio, feed conversion ratio, immune response, and activity of digestive enzymes of fish fed with diets supplemented with A. niger. Also, in another study, supplementation of Aspergillus oryzae singly or in combination with β-glucan can increase the growth, feed efficiency, antioxidant status, and immunomodulation of Nile tilapia (Dawood et al., 2020). The improvement in the growth of fish by A. niger supplementation has been discussed to relate to the positive benefits on feed digestibility, activity of digestive enzymes (protease, amylase, lipase, alkaline phosphatase, and trypsin), and feed absorbance efficiency with an increase in the absorptive area, microvilli density and height (Dawood et al., 2020; Jasim et al., 2022). Our study observed that A. niger added to the diet of shrimps (at 1.5 g/kg diet, A1.5) showed the best effectiveness in the growth performance of shrimps; however, the mechanisms of action were lacking, which merits further investigations. Collectively, the outcomes of this current study indicate that the low dose of A. niger (1.5 g/kg diet) is more suitable for use in shrimp aquaculture.

The antioxidant system plays an important role in the physiological response of hosts in responding to effects of noninfectious or infectious agents. SOD and catalase are the main components in the antioxidant system that protects hosts against oxidative stress, specific to the overproduction and residuals of reactive oxygen species during inflammatory responses (Parrilla-Taylor et al., 2013). Our results revealed that the activities of SOD and catalase of shrimps were increased in A1.5 and A3.0 as compared to the control, indicating that A. niger-containing diets can enhance the antioxidant capacity in juvenile Pacific white shrimp. Moreover, NOS, which is one of the oxidation-derived compounds, has been proven to have antimicrobial effects on the innate immunity of crustaceans (Li et al., 2012b). The result herein showed the increased expression of the NOS gene in shrimps fed the A. niger-containing diet (A3.0), suggesting the stimulation of the probiotic to the induction of NOS. A similar observation was found in mouse macrophagocytes that showed the expression of inducible NOS after being treated with β-glucan derived from Aureobasidum pullans SM-2001 (Choi et al., 2016). Thus, it is speculated that A. niger can activate the non-specific defense of shrimps by stimulating NOS production.

Lysozyme activity is important in innate immunity, which is responsible for attacking and causing damage to the cell structures of invading bacteria (Saurabh and Sahoo, 2008). The lysozyme activity was found to increase in Nile tilapia (Oreochromis niloticus) fed with probiotic-or synbiotic-supplemented diets (Sîrbu et al., 2022), Chinese drum (Miichthys miiuy) fed with live or dead cells of Clostridium butyrium CB2-supplemented diets (Pan et al., 2008), and red sea bream (Pagrus major) fed with β-glucan-supplemented diets (Dawood et al., 2017) but to remain unchanged in grouper (Epinephelus coioides) fed with Bacillus pumilus or B. clausii (Sun et al., 2010). However, our findings found that dietary A. niger supplement significantly decreased the lysozyme activity in shrimps, referring to that the probiotic showed negative effects on the activity of lysozyme in the innate immunity of shrimps. Differences in results among studies may be due to differences in probiotic organisms, dosage, host species, experimental designs, and nutritional and environmental factors. Furthermore, acid phosphatase (ACP) and alkaline phosphatase (AKP) are important in the immune system of crustaceans, where the former is responsible for digesting invading pathogens and the latter is for detoxifying during the normal living and phagolysis and digesting and absorbing nutrients (Xie et al., 2019). The findings herein showed an increase in activities of ACP and AKP in the shrimps fed A. niger-containing diets. Similar results were observed in the Pacific white shrimp fed with a probiotic mixture (Xie et al., 2019) and ridgetail white prawn (Exopalaemon carinicauda) fed with Psychrobacter sp. B6 (Lai et al., 2022).

Antimicrobial peptides (AMPs) contribute to the innate immune system as the first line of defense against invading pathogens. The current study found that crustin, which showed antibacterial activity against Gram-positive and Gram-negative bacteria, and virus (Du et al., 2019), was down-regulated in the shrimps fed with A. niger. This is consistent with findings of the giant tiger shrimp crustin-like, which was down-regulated in the Candida haemulonii S27-fed shrimps, and then up-regulated in these animals after challenge with WSSV (Antony et al., 2011). Furthermore, ALFs and PENs are important in the immune defense of shrimps, showing broad-spectrum antimicrobial activities against bacteria, fungi, parasites, and viruses (Gu et al., 2018; Wu et al., 2019). The increase of gene expression of ALF1, ALF2, PEN2, PEN3, and PEN4 in shrimps fed with A. niger-containing diets suggested that the probiotic can promote the production of these AMPs and confer health benefits on shrimps. Therefore, our results indicated the role of A. niger in improving the immune response of shrimps.

In crustaceans, the proPO system, which is activated by the pattern recognition receptor family (e.g., β-1, 3-glucanase related protein), is vital in the humoral immune response. The binding of β-1, 3-glucan to these proteins activates the immune signaling pathway and enhances the PO activity (Li et al., 2019). Herein, the increase of proPO mRNA expression and PO activity in shrimp fed with A. niger-containing diets suggests interactions between A. niger and the proPO system regulation in promoting shrimp humoral immune response. A similar finding was previously reported in black tiger shrimp where β-glucan participates in the activation of the proPO system and increase of PO activity, resulting in the protection of hosts against WSSV (Chang et al., 2003; Thitamadee et al., 2014). Collectively, dietary A. niger supplement shows an effectiveness in stimulating immune responses in shrimps, which may help hosts in resisting invading pathogens during the culture period.

Gut microbiota is important in dietary polysaccharide digestion, nutrient absorption, physiological homeostasis, and immune stimulation in their hosts. The gut microbiota in aquatic animals has been known to be affected by several factors, such as host species, nutrient supplementation, aquatic environment, and geolocation (Tran et al., 2017). In this study, the supplementation of A. niger significantly increased the alpha diversity and distinctly separated the microbial community structure in shrimps, referring to the great contribution of probiotics in shaping the gut microbiota of shrimps. This is in agreement with the previous findings in Pacific white shrimps fed with the probiotic mixture of B. subtilis, B. licheniformis, and Lactobacillus sp. (Xie et al., 2019). However, different results were observed in another study which found a decrease in the richness and diversity, but unchanged relative abundance (at the phylum level) of gut microbiota in Pacific white shrimps fed with β-glucan (Li et al., 2019). The difference herein may be explained by the difference in water salinity used for the culture of experimental animals between the studies. Our results found that the bacterial phyla Proteobacteria, Firmicutes, Bacteroidota, Verrucomicrobiota, Actinobacteriota, and Cyanobacteria are the core bacteria in the gut of shrimp, which is similar to the previous findings (Duan et al., 2018; Li et al., 2019). Bacteroidetes and Firmicutes are responsible for the metabolism of undigested food remnants (Tran et al., 2018a). The members of Bacteroidetes are known as producers of short-chain fatty acids (especially propionate) during the anaerobic fermentation, which has been demonstrated to confer health benefits on their hosts (Rios-Covian et al., 2017; Tran et al., 2020). The abundance of Firmicutes gives a good parameter concerning the health status of the gut, of which many members, such as Lachnospiraceae, Clostridium, Bacillus, Lachmoclostridium, and Lactobacillus, are beneficial bacteria (Duan et al., 2018). Among them, the members of the family Lachnospiraceae are capable of producing butyrate via the butyryl-CoA or acetate CoA transferase pathways or the butyrate kinase pathway by using lactate and acetate (Zhang et al., 2019a). Our findings showed that the abundance of members of Lachnospiraceae was increased in both A1.5 and A3.0 as compared to that in the control (data not shown), indicating the positive effects of the A. niger supplementation on the gut health of shrimps. Proteobacteria is related to energy disequilibrium and unstable gut microbiota (Shin et al., 2015), which was enriched in grass carp (Ctenopharyngodon idellus) with intestinal disease (Tran et al., 2018b) and humans with Crohn’s disease and inflammatory bowel disease (Li et al., 2012a; Zhu et al., 2014). Our results showed that the relative abundance of Firmicutes and Bacteroidota was increased, but such of Proteobacteria was decreased in the shrimps fed A. niger-supplemented diets. Several bacterial genera, such as Vibrio, Marivita, ZOR0006, Roseobacter, Ruegeria, Psychromonadaceae, Edwardsiella, Clostridium sensu stricto 1, Halioglobus, and Shimia, were predominated in the gut of shrimps. Of these genera, Vibrio, Marivita, and Roseobacter were found to be increased in the A. niger-fed shrimps, which is almost consistent with the previous findings (Li et al., 2019; Liao et al., 2022). Roseobacter 27-4 is a probiotic bacterium for protecting turbot (Scophthalmus maximus) larvae against Vibrio anguillarum (Planas et al., 2006). Vibrio and Edwardsiella are known as opportunistic pathogens causing diseases in both cultured and wild aquatic animals (Park et al., 2012; Li et al., 2018). Vibrio spp. may cause an outbreak of vibriosis in cultured shrimps under the deterioration of the environment, but the pathogenic effect of Vibrio spp. is species-specific (Intaraprasong et al., 2009; Li et al., 2019). Intaraprasong et al. (2009) found that the extracellular products of Vibrio harveyi cause high toxicity to black tiger shrimp (P. monodon) but not Pacific white shrimp. Also, the previous study has demonstrated that the susceptibility of Pacific white shrimp is depended on the Vibrio species and doses (Aguirre-Guzmán et al., 2001). The study showed that V. alginolyticus showed a high survival of shrimp larvae, while V. harveyi and V. parahaemolyticus induced high mortality only at high doses (10⁵ and 10⁷ cfu/mL), and V. penaeicida caused high mortality and only at low dose (10³ cfu/mL) in postlarvae 1 (Aguirre-Guzmán et al., 2001). An increase in the abundance of Vibrio in shrimps fed A. niger (in the A1.5 and A3.0) may speculate that these microorganisms are non-pathogenic species causing infections in shrimps. Also, the overgrowth of Vibrio spp. in A. niger-fed shrimps may indicate their beneficial role to the host. This is supported by the findings that Vibrio alginolyticus UTM 102 can enhance feed utilization and disease resistance against V. parahaemolyticus in Pacific white shrimp (Balcázar et al., 2007). Similarly, Edwardsiella sp. 34HN showed an effectiveness in promoting growth, immunity, and expression of myostatin and pituitary adenylate cyclase-activating polypeptide in African catfish (Clarias gariepinus) (Selim et al., 2019). Thus, A. niger can promote beneficial bacteria and inhibit opportunistic pathogens colonizing the gut of shrimps. Similar findings were observed in Pacific white shrimps fed with beer yeast and yeast extract (Zheng et al., 2021a).

This study proved the beneficial effects of A. niger in promoting weight gain, specific growth rate, and length gain, as well as, improving antioxidant activity and immune response of juvenile Pacific white shrimps. Also, the addition of A. niger can promote the selective growth of gut microbiota (with an increase in the abundance of beneficial bacteria and a decrease in that of opportunistic pathogens). Therefore, A. niger can be used as a probiotic in shrimp aquaculture. Based on growth promotion, the recommended supplementation of A. niger is a 1.5 g/kg diet. However, the limitation of this study is lacking an evaluation of the germination ability of fungal conidia in the gut of shrimps. Also, whether the positive impact of A. niger on shrimp health status is through the composition of fungal conidia or through the mycelium after germination and colonization of shrimp gut need to be further evaluated in the future.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA900677.

The animals were processed according to “the Regulations for the Administration of Affairs Concerning Experimental Animals” established by Guangdong Provincial Department of Science and Technology on the Use and Care of Animals, and the experiments were approved by the Institutional Animal Care and Use Committee of Shantou University.

MZ and HL: Investigation, Data curation. YL, YZ, ZT and WC: Data curation. SL and XP: Conceptualization, Project administration, Funding acquisition, Writing- review and editing. NT: Conceptualization, Investigation, Writing- original draft, review and editing. All authors contributed to the article and approved the submitted version.