Pacific white shrimp Litopenaeus vannamei has become an important species in global aquaculture due to the advantages of its rapid growth and strong stress resistance (FAO, 2020). However, some gastrointestinal diseases, such as white feces syndrome (WFS) (Hou et al., 2018) and early mortality syndrome (EMS) (Zorriehzahra and Banaederakhshan, 2015) have caused significant losses to shrimp farming in recent years. As a vital organ for nutrient uptake and immune defense, the intestinal structure and biochemical barrier are closely related to the health status of the shrimp (Suo et al., 2017).

The intestinal structure and the biochemical barrier refer to the integrity of the histological structure of the intestine. The midgut of shrimp consists of the outer epidermis, muscular layers, protruding mucosal folds, and the inner epidermis, and an intact intestinal structure forms the basis for the functioning of the intestine (Xie et al., 2020). In addition, the immune function of the gut should be considered, as pathogens in the external environment can pass through the intestine (Pilotto et al., 2020). The antimicrobial peptides such as alf (anti-lipopolysaccharide factor), cru (crustins), and pen3 (penaeidins 3) are regulated by secreted cytokines and immune signaling pathways, such as the Toll and IMD (immune deficiency). When Toll and IMD pathways are activated in early response to pathogens, the expression of downstream factors dorsal and relish can regulate pathways activities (Qiu et al., 2014). Intestinal microbiota are an important part of the intestinal biochemical barrier (Qi et al., 2017). The intestinal microbiota have been extensively studied and shown to benefit aquatic animals with its functions in immune response, nutrient uptake, and homeostasis maintenance (Li et al., 2018), which is mainly influenced by environmental and dietary factors (Egerton et al., 2018). Although close contact with the aquatic environment may influence the early gut colonization (Roeselers et al., 2011), feed causes a continuous effect on the intestinal microbiota composition over a longer culture period (Ringø et al., 2016).

In shrimp diets, fish meal (FM) is the most commonly used protein source, usually accounting for 25–35% of the formulation (Xie et al., 2016). The depletion and shortage of fishery resources and the high cost of FM have encouraged researchers to look for alternatives (Cai et al., 2020). In the last decade, experimental studies on feeding of insect meal–based diets have been carried out in L. vannamei, which has been conducted on mealworm (Tenebrio molitor), silkworm pupae (Bombyx mori L.), as well as fly maggots (Cao et al., 2012; Choi et al., 2018; Rahimnejad et al., 2019). Besides, the black soldier fly is a saprophytic insect (Hermetia illucens) commonly used for the sustainable recycling of animal waste and other organic wastes. Its larvae are rich in proteins and lipids, and also it is considered as a potential protein source for aquatic animals (Barragan-Fonseca et al., 2017). Several studies have been conducted to evaluate the effectiveness of BSF as an FM replacement in the diets of L. vannamei. In general, most growth responses could be obtained if the amount of BSF replacement FM is limited to 25% of the ratio without changing the nutrition levels of the diet (Cummins et al., 2017). Another study showed that BSF substitution of <30% FM in diets did not negatively affect the growth performance of L. vannamei (Hu et al., 2019). Also, dietary BSF has been shown to affect the intestinal health status; however, it causes different effects on different animals. In previous studies, a total replacement of the FM with BSF did not impair the intestinal health of Atlantic salmon (Salmo salar L.) (Li et al., 2020). However, replacement of 75% FM with defatted BSF led to significant pathological changes in the intestinal wall of Jian carp (Cyprinus carpio var. Jian) (Li et al., 2017). It is still unclear how dietary BSF affects the intestinal health of L. vannamei and our experiments were conducted to study the effect of BSF on the growth performance, intestinal structure, and immunity as well as the intestinal microbiota communities of L. vannamei.

Although differences in the metamorphic stage of insects (larvae and pupae) (Huyben et al., 2019), nutrient composition caused by different food sources (Barroso et al., 2017), and processing techniques (Larouche et al., 2019) may lead to distinguishing experimental results, many studies have demonstrated the potential of BSF for application in aquatic animal feeds (Li et al., 2017; Renna et al., 2017; Wang et al., 2019). Similar to the previous studies, the present study showed that better growth performance was obtained when 20% of FM was replaced with BSF, but replacing 30% of FM significantly reduced the final body weight, weight gain, and specific growth rate of the shrimp (Cummins et al., 2017; Hu et al., 2019). Due to the high lipid content (10–40%), BSF is often processed by oil separation methods into protein-rich components to minimize the risk of fat oxidation and compositional changes. The use of defatted BSF in shrimp feed has also been studied, and defatted BSF can replace 60% of FM without negatively affecting the growth performance and antioxidant and immune capacity of shrimp (Wang et al., 2021). This suggests that the inhibition of growth performance by the addition of high levels of BSF to shrimp diets may be due to the fatty acid composition in BSF. Under the present experimental conditions, the fatty acid composition of BSF was rich in lauric acids, but very low in EPA and DHA, compared to FM. Lipids are not only a major source of energy and carriers of fat-soluble vitamins but also serve as components of cell membranes and precursors for many important metabolites (Zhou et al., 2019; Xie et al., 2020). Replacement of FM with BSF in the diet reduced the highly unsaturated fatty acid contents in the diet, and the hepatocyte disruption was observed in L. vannamei (Wang et al., 2021) and Cyprinus carpio var. Jian (Li et al., 2017). In addition to BSF, other insect proteins have been also studied in shrimp diet, such as silkworms (Bombyx mori L.). Replacing 75% of FM with defatted silkworm meal did not affect the growth performance of L. vannamei but higher levels of silkworm meal would lead to hepatopancreas tubular damage (Rahimnejad et al., 2019). However, the effect of dietary BSF on the intestinal health remains unknown. Excessive replacement of FM may lead to impaired nutritional sensitivity and metabolism (Xu et al., 2016; Shao et al., 2020). Besides, intestinal histology is directly related to the digestion and absorption of nutrients by the shrimp, thus affecting growth.

In addition to the growth status of shrimp, issues related to animal welfare need to be considered, which includes the effects of immune status, oxidative status, and diet on the intestinal structure, directly affecting the digestive function and immune response of shrimp (Bruni et al., 2018). The intestinal structural barrier separates the internal from the external environment and is the first line of defense against invasion by pathogens (Rahimnejad et al., 2018). In the results of HE-staining sections, it was clear that the width and height of intestinal mucosal folds decreased with increasing BSF replacement. The shrinkage of intestinal mucosal folds will reduce the contact area between the intestine and the food of shrimp, which is not conducive to food digestion and nutrient absorption. Besides, results of TEM showed that shrimp fed with BSF30 showed early signs of apoptosis: chromatin consolidation, separation and distribution along the nuclear membrane, swelling of the endoplasmic reticulum vesicles, and abnormal mitochondrial structures, demonstrating that BSF replacement for 30% FM can damage the intestinal epithelial cell structure of shrimp and may explain the reduction of growth performance (Xie et al., 2018). In a previous study, the reduction of FM in diets upregulated the expression of endoplasmic reticulum stress-related genes, resulting in severe endoplasmic reticulum stress in the intestine (Xie et al., 2020). The content of highly unsaturated fatty acids in the diet decreases with the proportion of FM and fish oil in the diet, which negatively affects the biophysical properties of cell membranes and may lead to apoptosis (O'shea et al., 2009).

The non-specific immune system of the shrimp plays an important role in the resistance to pathogen invasion (Gui et al., 2019). To evaluate the effect of BSF on resistance to V. Parahaemolyticus infection, a challenge trial was conducted to compare the disease resistance of shrimp fed diets containing different levels of BSF. Colonizing in the intestine, V. parahaemolyticus would result in EMS, also known as acute hepatopancreatic necrosis disease (Zorriehzahra and Banaederakhshan, 2015; Qi et al., 2017). In this study, the survival rate of shrimp fed with BSF10 was significantly higher than those fed with FM after infection, indicating that BSF replacement of 10% FM could improve the resistance of L. vannamei to V. Parahaemolyticus.

Furthermore, the effect of BSF on the non-specific immune gene expression in the intestine was investigated. dorsal and relish are Rel/NF-κB transcription factors, the former factor activating antifungal and antimicrobial responses in the Toll pathway through a cascade amplification effect, and the latter one activating antimicrobial peptide gene expression in the IMD pathway (Li and Xiang, 2013). The Toll and IMD pathways induce immune-related gene expression during pathogen invasion, thereby regulating the early stages of the immune response (Jin et al., 2018). The results showed that intestinal dorsal and relish mRNA levels were maintained at the control level in the shrimp fed with BSF10 but downregulated in those fed with BSF20 and BSF30. Furthermore, antimicrobial peptides are products of the immune response and are capable of directly killing or removing pathogens (Bachère, 2003; Wang et al., 2007). Anti-lipopolysaccharide factor has been reported in L. vannamei (De La Vega et al., 2008), Fenneropenaeus chinensis (Li et al., 2013), and Penaeus monodon (Supungul et al., 2004), which showed a broad-spectrum of antimicrobial activity against Gram-negative bacteria and endotoxins (Silveira et al., 2018). Crustins are antimicrobial peptides prevalent in crustaceans with antibacterial activity against Gram-positive bacteria, as well as inhibition of fungi and white spot syndrome virus (Shockey et al., 2009; Chen et al., 2019). Penaeidins 3 is an antimicrobial peptide isolated from L. vannamei (Destoumieux et al., 1997) with antibacterial activity against Gram-positive bacteria and fungi (Takuji, 2007). In the present study, mRNA level of alf upregulated in shrimp fed with BSF10, while downregulated in those fed with BSF30, which was consistent with the results of the challenge trial, indicating that dietary BSF replacing 10% FM can improve intestinal immunity in shrimp.

In addition to the immune defense of shrimp, the gut microbiota has been found to have a remarkable effect on animal health, growth, and survival and is of increasing interest to researchers and breeders (He et al., 2017; Li et al., 2018). To date, the effect of BSF on the intestinal microbiota has been studied on aquatic animals, such as rainbow trout (Terova et al., 2019), zebrafish (Danio rerio) (Osimani et al., 2019), rice field eel (Monopterus albus) (Hu et al., 2020), marron (Cherax cainii) (Foysal et al., 2019), and other animals. Since there was no significant difference in the growth performance of shrimp fed with BSF10 and BSF20, we explored the intestinal microbiota of shrimp fed with FM, BSF20, and BSF30 diets. Results showed that there were no significant differences in Anosim tests of beta diversity analysis, indicating that the intestinal microbiota community was not separated in shrimp fed with FM, BSF20, and BSF30 diets. At the phylum level, Proteobacteria was the most common bacteria in the shrimp gut, followed by Bacteroidetes, Firmicutes, and Actinobacteria, which was in line with the results of a previous study (Amoah et al., 2020). Proteobacteria do not change in abundance with environment or diet and are the most stable bacteria in L. vannamei, while the abundance of the other major bacterial phyla (Firmicutes, Bacteroidetes, Actinobacteria) may vary with developmental stage, diet, and environmental factors (Li et al., 2018). At the genus level, Vibrio and Photobacterium are known as opportunistic pathogens and have been reported in shrimp diseases (Avendaño-Herrera et al., 2006; Liu and Qiu, 2007; Wang et al., 2020). Tenacibaculum and Acinetobacter have also been reported as bacterial pathogens, while the former was identified in marine fish, which causes an ulcerative disease (Avendaño-Herrera et al., 2006) as well as the latter was identified as a potential pathogen of red leg disease in the Pacific white shrimp (Huang et al., 2020). Moreover, Candidatus_Bacilloplasma may contribute to WFS in L. vannamei (Hou et al., 2018). In the present study, the reduction of some harmful bacteria in shrimp fed with BSF20 may help improve intestinal health. On the other hand, some beneficial bacteria also differed among FM, BSF20, and BSF30. Haloferula has been reported in sea cucumbers (Apostichopus japonicus) and was reduced by the high levels of selenium and vitamin C supplementation, indicating that Haloferula is related to the antioxidant capacity of the gut (Zeng et al., 2020). Moreover, Pseudoalteromonas has been shown to produce extracellular antibacterial compounds, which could enhance the resistance of shrimp to V. parahaemolyticus (Wang et al., 2018). Bacillus is a non-pathogenic Gram-positive bacterium and is beneficial to the growth performance and immunity of the Pacific white shrimp (Wen et al., 2011; Zokaeifar et al., 2012; Tang et al., 2015). The increase of Pseudoalteromonas in BSF20-fed group and Bacillus in BSF30-fed group may enhance the intestinal health. According to the present study, the increase of beneficial bacteria and the reduction of harmful bacteria suggest that BSF20 diet may improve the intestinal bacterial communities of L. vannamei.

The present study evaluated the effects of BSF on growth, intestinal health, and susceptibility to V. parahaemolyticus of L. vannamei. Results showed that BSF replacement of 10% FM did not negatively affect the growth performance and intestinal histology of the shrimp L. vannamei, but improved the survival rate after infection of the bacteria V. parahaemolyticus. When the replacement of FM was increased to 20%, there was no negative effect on the intestinal integrity and promoted a positive modulation in the intestinal microbiota. However, FM replacement up to 30% promoted intestinal cell apoptosis and degeneration and was deleterious to the shrimp.

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 at: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA729583.

This animal study was reviewed and approved by the Animal Ethical and Welfare Committee of Guangdong Ocean University.

YC and SX designed the experiments. YC carried out the experiments and drafted the manuscript. SC, SZ, XD, QY, and HL: accountable for some aspects (such as ingredients and sites) of the study in ensuring that the experiment can be carried out properly. SX and BT reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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