Next Article in Journal
Transcriptome Sequencing Reveals That Intact Expression of the Chicken Endogenous Retrovirus chERV3 In Vitro Can Possibly Block the Key Innate Immune Pathway
Next Article in Special Issue
Mitigating the Environmental Impacts from Pig and Broiler Chicken Productions: Case Study on a Citrus Extract Feed Additive
Previous Article in Journal
The Effects of Sodium Acetate on the Immune Functions of Peripheral Mononuclear Cells and Polymorphonuclear Granulocytes in Postpartum Dairy Cows
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Animals
Volume 13
Issue 17
10.3390/ani13172722
animals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effects of Microorganisms on Growth Performance, Body Composition, Digestive Enzyme Activity, Intestinal Bacteria Flora and Antimicrobial Peptide (AMP) Content of Black Soldier Fly Larvae (Hermetia illucens)

by
Yongqi Meng
1,2,
Xiuxia Zhang
1,3,
Zelong Zhang
1,3,
Jiajun Li
1,3,
Peihua Zheng
1,3,
Juntao Li
1,3,
Jiarui Xu
1,
Jianan Xian
1,2,3,* and
Yaopeng Lu
1,3,*
1
Hainan Provincial Key Laboratory for Functional Components Research and Utilization of Marine Bio-Resources, Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
2
Ocean College, Hainan University, Haikou 570228, China
3
Key Laboratory for Biology and Genetic Resources of Tropical Crops of Hainan Province, Hainan Institute of Tropical Agricultural Resources, Haikou 571101, China
*
Authors to whom correspondence should be addressed.
Animals 2023, 13(17), 2722; https://doi.org/10.3390/ani13172722
Submission received: 1 July 2023 / Revised: 22 August 2023 / Accepted: 24 August 2023 / Published: 26 August 2023
(This article belongs to the Special Issue Adapting Novel Nutritional Practices to Enhance Product Quality and Environmental Sustainability in Ruminant Livestock Sector)
Browse Figures
Versions Notes

Abstract

:

Simple Summary

Antimicrobial peptides (AMPs) are a class of small-molecule active polypeptides. Because of their good antibacterial activity, stability and safety, AMPs are considered one of the potential alternatives to antibiotics. Black soldier fly (Hermetia illucens) is an excellent material for the study of AMPs. This study aims to screen optimum microbial strains and concentration to support the growth performance and AMP induction of H. illucens. Six microorganisms were selected as feed additives for H. illucens by tracking the growth performance, proximate composition, digestive ability and AMP content in the first trial. Microorganism efficiency screening results showed that Rhodopseudomonas palustris (RP) could improve growth performance, digestive ability and AMP content of H. illucens. Therefore, RP was selected to prepare the diets and was incorporated into diets for H. illucens at different levels. The results showed that RP is superior to the other strains as a feed additive for the H. illucens larvae, and we recommend the addition of 1.22 × 109–1.22 × 1010 CFU/g RP to promote the growth and AMP content of H. illucens. This study provides new ideas for large-scale production of AMPs and lays the foundation for the development of AMPs as feed additives to substitute antibiotics.

Abstract

Escherichia coli (EC), Staphylococcus aureus (SA), Bacillus subtilis (BS), Rhodopseudomonas palustris (RP), Saccharomyces cerevisiae (SC) and Lactobacillus plantarum (LP) were selected as feed additives for black soldier fly (Hermetia illucens) by tracking the growth performance, proximate composition, digestive ability and antibacterial peptides (AMPs) content in the first trial. Microorganism efficiency screening results showed that RP could improve growth performance, digestive ability and AMP content of H. illucens. Therefore, RP was selected to prepare the diets and was incorporated into diets for H. illucens at levels of 0 (R0), 1.22 × 106 (R1), 1.22 × 107 (R2), 1.22 × 108 (R3), 1.22 × 109 (R4) and 1.22 × 1010 (R5) CFU/g. After 5 d of feeding, larvae fed the R2-R5 diets had higher weight gain and specific growth rates. Different concentrations of RP had no significant effect on larval body composition. R4–R5 could improve the digestibility and expression of AMPs in larvae. Moreover, RP could significantly increase the abundance of Lactobacillus and Rhodopseudomonas and decrease the abundance of Proteus and Corynebacterium. Therefore, RP is superior to the other strains as a feed additive for H. illucens larvae, and we recommend the addition of 1.22 × 109–1.22 × 1010 CFU/g RP to promote the growth and AMP content of H. illucens.

1. Introduction

The large-scale and intensive development of aquaculture has caused many farming diseases, and antibiotics have been widely used to prevent and control bacterial diseases [1,2,3]. However, the misuse of antibiotics can result in many problems in aquatic animals, such as the emergence of drug-resistant bacteria, drug residues, microecological imbalances and immune system disruption [4,5]. Multidrug resistant bacterial infections caused by antibiotics threaten people’s life and health safety [4]. Many countries around the world have issued laws banning the use of antibiotics [6]. Therefore, finding new efficient and environmentally friendly feed additives to replace antibiotics has become an urgent task for research and development.
Antimicrobial peptides (AMPs) are a class of small-molecule active polypeptides. Because of their good antibacterial activity, stability and safety, AMPs are considered one of the potential alternatives to antibiotics [7]. AMPs have broad-spectrum resistance to bacteria, fungi, viruses and parasites [8,9]. In the immune system, AMPs are important effectors and modulators that perform a variety of physiological functions, including the recruitment of immune cells, release of chemokines and regulation of pro-inflammatory responses [10,11]. Antimicrobial peptides have the advantages of good stability, natural source and no pollution, which is a good alternative to antimicrobials [12]. However, there are still many problems in the application of AMPs in aquaculture. There are many kinds of AMPs, but the expression of the antimicrobial mechanism is not clear. To apply AMPs to aquatic animal feed, we should understand the relationship between the function and structure of AMPs, and a suitable one that should be selected for different species. In addition, the AMO production cost is high. The extraction process is complex. The yield is limited and cannot be used on a large scale in animal breeding. The promotion of AMPs depends on their efficient production and cost reduction.
Black soldier fly (Hermetia illucens) can transform organic waste into usable biological resources. Its nutritional value is high. The content of protein, lipid and trace elements in the body accounts for 35–64% of the dry weight of the larvae and has good application in feed protein replacement [13,14]. Hermetia illucens contains more than 50 AMP genes, and AMPs produced in its body have a highly effective antibacterial ability [15]. Hermetia illucens is an excellent material for the study of AMPs [16]. There are several methods for the production of AMPs from H. illucens, such as the molecular technology method [17,18], the hemolymph collection method [19] and the grinding and extraction method [20]. The AMP content in the body of H. illucens is low under normal conditions. Microbial needling infection is a relatively common method to induce the expression of AMPs in H. illucens. Park et al. reported that using Staphylococcus aureus needling could induce the production of AMPs in H. illucens [21]. Lee et al. reported that using Lactobacillus casei with acupuncture could induce the production of AMPs in H. illucens [22]. Similarly, the expression of AMPs can be induced by Escherichia coli needling [19]. Bacterial injection and other methods are commonly used to stimulate the production of AMPs in H. illucens [21,22]. However, these methods are not applicable to the production of AMPs and are not realistic in production practice.
Therefore, this study aimed to screen optimum microbial strains and concentration to support the growth performance and AMP induction of H. illucens. The microbial strain efficiencies of growth enhancement and AMP induction improvement for H. illucens of six microbial strains were evaluated. The optimal microbial strains of feed at different concentrations were prepared, and their mechanism of influence was evaluated by tracking the growth performance, intestinal tract bacteria and AMPs induction.

2. Materials and Methods

2.1. Microbial Culture

Six microorganisms, including E. coli (EC), S. aureus (SA), Bacillus subtilis (BS), Rhodopseudomonas palustris (RP), Saccharomyces cerevisiae (SC) and Lactobacillus plantarum (LP) were used in this study. EC, SA and BS were cultured in LB (Luria–Bertani) (Huankai Biology, Guangzhou, China) media at 37 °C for 12 h. RP was incubated in media at 30 °C for over 96 h under 3000 lx light intensity. SC was cultured in YPD (yeast extract peptone dextrose) (Huankai Biology, Guangzhou, China) media at 30 °C for 12 h. LP was incubated in a stationary state in MRS (deMan, Rogosa and Sharpe) (Huankai Biology, Guangzhou, China) media at 37 °C for 12 h. After cultivation, bacteria were collected by centrifugation at 3000× g for 10 min and resuspended in phosphate buffer solution (PBS) to prepare experimental diets.

2.2. Experimental Diet Preparation

The basic feed of black soldier fly larvae were made based on a previous study [23]. The diet consists of 50% wheat bran, 30% soya bean meal and 20% corn meal. Distilled water was added to make the water content of the feed 55%. The proximate composition of the experimental diet is shown in Table 1. Two batches of the experimental diet were prepared in this study. For the first batch of diet preparation, EC, SA, BS, RP, SC and LP were individually added to the experimental diets at the level of 1.22 × 108 colony forming unit (CFU)/g. The diet supplemented with PBS was used as the control diet (control) for the first trial. For the second experimental diet preparation, RP was incorporated into experimental diets at levels of 0 (R0), 1.22 × 106 (R1), 1.22 × 107 (R2), 1.22 × 108 (R3), 1.22 × 109 (R4) and 1.22 × 1010 (R5) CFU/g. The diet supplemented with PBS was used as the control diet (R0). In the experiment, the strains were resuspended in equal doses of PBS added to the feeds. To ensure the safety of the experiment, pathogenic bacteria (EC, SA and BS) were added to the feed only after being inactivated.

2.3. Hermetia illucens Acclimation

Hermetia illucens eggs were bought from a private farm in Guangzhou, Guangdong, China. Eggs in plastic bottles with corn flour were transported to the laboratory at the Institute of Biology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China. During acclimation, eggs were reared for 3 d in the storage box (60 cm × 45 cm × 30 cm) at 28 °C and 55% relative humidity. When hatching, eggs were put in a cool place to avoid direct sunlight and rapid water evaporation.

2.4. Experimental Design and H. illucens Rearing

Two experiments were conducted in this study. In the first experiment, optimal microorganisms were screened for growth and induction of AMPs expression in H. illucens. Seven experimental diets containing a single bacterium (Control (supplement with PBS), EC, SA, BS, RP, SC and LP) were offered to H. illucens larvae (initial weight of 0.01 ± 0.002 g) for 5 d. After 5 d of feeding, growth performance and AMP concentration were determined. Because the RP improved the growth performance and AMP concentration of the H. illucens larvae in the first trial, RP was used to prepare the diet, and the optimal concentration of the RP was evaluated in the second trial. In the second trial, larvae with an initial mean weight of 0.01 ± 0.002 g were assayed for growth performance, proximate composition, including ash, crude protein and crude lipid, and digestive ability, including amylase, lipase and trypsin activities in the gut and intestinal microbiota, and AMP concentration after 5 d of feeding with the RP diets (R0 (supplement with PBS), R1, R2, R3, R4 and R5).
During the period of experiments, healthy pretreated larvae of the same size were put into feeding boxes (ladder PP plastic boxes with top bottom 21.5 cm × 14.5 cm, bottom 17.5 cm × 10.5 cm and height 6.9 cm). Feed (45 g) and bacteria solution (55 mL) are added to each box of the experimental group and mixed well. For the first experiment, the concentration of each bacterial solution was 1 × 108 CFU/mL. For the second experiment, the concentration of the bacterial solution in each RP group was 1 × 106 (R1), 1 × 107 (R2), 1 × 108 (R3), 1 × 109 (R4) and 1 × 1010 (R5) CFU/mL. Each treatment group was set up with 6 replicates, and 100 larvae were in each feeding box. The pretreated feed was added to the feeding box and placed in a biochemical incubator (temperature 28 °C, relative humidity 55%) for feeding. The experimental feed was spread evenly on the bottom of the box, and the black soldier fly larvae were kept on the feed. The feed was given sufficiently during the experiment, and the water content was relatively stable.

2.5. Sample Collection

Each group of H. illucens was rinsed with water and sterilized with 75% alcohol, after which, H. illucens were collected and frozen in liquid nitrogen after the breeding experiment. The intact black soldier fly larvae were stored at −80 °C for further analysis.

2.6. Growth Performance

After a 5 d feeding trial, 20 larvae were chosen randomly from every replication to calculate weight gain rate (WGR) and specific growth rate (SGR). The following methods by Zokaeifar et al. [24] and Yeh et al. [25] were used:
WGR (%) = [(Final weight − Initial weight)/Initial weight] × 100
SGR (% day−1) = [(ln Final weight − ln Initial weight)/Days] × 100.

2.7. Proximate Composition

The crude protein, crude lipid and ash of whole larvae were determined according to the Association of Official Analytical Chemists (AOAC) (1995) [26]. Crude protein, crude lipid and ash were determined using K8400 Kjeltec Analyzer (Fossana Lyticab, Shanghai, China), Soxtec System HT (SE-A6, Alvah, Shanghai, China) and muffle furnace, respectively.

2.8. Digestive Ability

Whole larvae were used to determine the digestive ability of H. illucens. Amylase, lipase and trypsin activities were determined using their respective kits (catalog no. C016-1-1, A054-2-1 and A080-2) following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.9. Intestinal Microbiota

Three mixed samples per group were used in performing the intestinal flora analysis. Total bacterial DNA was extracted from the peeled larvae using Tguide S96 DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The quality and concentration of DNA were measured using a NanoDrop One spectrophotometer (Thermo, Shanghai, China). The V4 region of the bacterial 16S rRNA gene was amplified by PCR using the primers, including 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′). PCR was performed in a 10 μL reaction system containing 5 μL PCR buffer (KOD FX Neo buffer, Toyobo, Osaka, Japan), 1 μL DNA template, 2 μL dNTP, 0.2 μL KOD FX Neo, 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer (10 μM) and 1.2 μL ddH2O. The PCR conditions were as follows: 95 °C for 5 min, followed by 25 cycles of 95 °C for 30 s, 50 °C for 30 s and 72 °C for 40 s and finally 72 °C for 7 min. The purified PCR products were sequenced on the Illumina novaseq6000 platform (Illumina, San Diego, CA, USA) to generate paired-end sequences. According to quality of single nucleotide, raw data were primarily filtered by Trimmomatic [27] (version 0.33). Identification and removal of primer sequences were performed using Cutadapt [28] (version 1.9.1). PE reads obtained from previous steps were assembled by USEARCH [29] (version 10) and followed by chimera removal using UCHIME [30] (version 8.1). The high-quality reads generated from the above steps were used in the following analysis. OUT, diversity, rarefaction curve and abundance were all performed by Beijing Biomarker Technologies (Beijing, China).

2.10. Extraction and Determination of AMPs

The lyophilized larvae were well-ground and extracted with acidified methanol (methanol/water/acetic acid; 90/9/1) [31]. Extracts were centrifuged at 1600× g for 10 min at 4 °C and dried in a rotary evaporator under reduced pressure. Proteins and lipids were removed from the samples by sequential extraction with chloroform and ethyl acetate. All fractions were lyophilized and stored at −20 °C for further use. The concentration of AMPs was determined using kits following the manufacturer’s instructions [32] (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.11. Antibacterial Effect

Using the filter paper diffusion method [32], the extract was set as the negative control (NC) and 120 μg/mL of florfenicol was set as the positive control (PC). In addition, 20 μL of E. coli at 1 × 108 CFU/mL was evenly coated in the plates containing LB medium. Then, filter paper of 6 mm in diameter was sterilized and soaked in the crude extract of AMPs in the control group and each experimental group for 2–5 min and taken out with sterile forceps for 5 min at room temperature into drug-sensitive paper sheets. The distance between each paper sheet on the culture medium should be greater than 24 mm. The distance between the paper sheet and the inner edge of the Petri dish should be greater than 15 mm.

2.12. Statistical Analysis

All experimental data are presented as means ± standard deviation (SD). The data were analyzed for significance by one-way analysis of variance (ANOVA). When differences were found among groups by one-way ANOVA, Tukey’s multiple-comparison test (SAS Institute, Cary, NC, USA) was conducted to examine differences among the controls and treatments. Statistical analyses were performed with Statistical Package for the Social Science version 18 (SPSS 18.0) software with p < 0.05 as statistically significant.

3. Results

3.1. First Trial: Screening Possible Microbial Strain Candidates

3.1.1. Growth Performance and Survival Rate

During 5 d of the growth trial, the larvae did not die. Growth parameters of H. illucens larvae are presented in Table 2. RP, SC and SA groups exhibited significantly higher WGR and SGR than the control group (p < 0.05).

3.1.2. Proximate Composition

The effect of dietary microbiota on the proximate composition (Crude protein, crude lipid and ash) of H. illucens is presented in Table 3. All treatments showed no significant differences in crude protein content. SC group exhibited a significant increase (p < 0.05) in crude lipid and ash content compared to the other groups.

3.1.3. Digestive Ability

Microbial strain supplementation of H. illucens larvae affected the activity of α-amylase, lipase and trypsin in the gastrointestinal tract of H. illucens larvae (Table 4). The H. illucens larvae fed RP had the highest α-amylase, lipase and trypsin activities. The α-amylase activity of RP and SC groups was significantly enhanced (p < 0.05) compared to that of the other groups. H. illucens larvae fed with the RP, SC and SA had increased lipase activity significantly higher than that of the other groups (p < 0.05). Trypsin activity was significantly higher in the RP, BS, SA and SC groups when compared to that of the other groups (p < 0.05).

3.1.4. AMPs Content

The AMP content of each group of H. illucens larvae was measured after feeding diets were mixed with EC, SA, BS, RP, SC and LP (Table 5). The AMP content of H. illucens in EC, SA, RP and SC groups was significantly higher than that in the control group, with the RP group having the most significant AMP content (p < 0.05).

3.1.5. Antibacterial Effect

The diameter of the inhibition circles of each feed group, NC and PC, are shown in Table 6. The corresponding inhibition pictures are shown in Figure S1. The diameter of the inhibition circle of the NC group was 0. The diameter of the inhibition circle of the PC group was greater than that of all experimental groups of the AMPs in H. illucens (p ˂ 0.05). The diameter of the inhibition circle of the RP group was significantly greater than that of the BS, LP and control groups (p ˂ 0.05); the diameter of the EC, SA, RP and SC groups was not significantly different (p > 0.05).

3.2. Second Trial: The Evaluation of Efficiencies of Different Concentrations of the Microbial Strain

3.2.1. Growth Performance and Survival Rate

To evaluate the effect of RP on the growth performance and survival rate of H. illucens, we prepared diets containing five different concentrations of RP, R1 (1.22 × 106 CFU/g), R2 (1.22 × 107 CFU/g), R3 (1.22 × 108 CFU/g), R4 (1.22 × 109 CFU/g) and R5 (1.22 × 1010 CFU/g), to feed H. illucens. After a 5 d feeding period, the larvae did not die and WGR and SGR were measured. Supplementation with the RP tested improved growth performance compared to the control group (Table 7). In addition, R4 and R5 groups displayed significantly higher WGR and SGR than the R0 group (p < 0.05).

3.2.2. Proximate Composition

The whole-body compositions are shown in Table 8. R4 and R5 promoted the crude lipid content of the body compositions of H. illucens larvae compared to groups R0, R1, R2 and R3. There were no significant differences in ash content between the five treatments and the control group (p > 0.05).

3.2.3. Digestive Ability

The activities of α-amylase, lipase and trypsin in larvae fed with RP are presented in Table 9. The activities of α-amylase, lipase and trypsin in the intestinal tract of H. illucens after feeding with a diet containing RP were significantly increased (p < 0.05).

3.2.4. Antibacterial Peptide Content

After feeding diets mixed with different concentrations of RP, the AMP content of each group of H. illucens larvae was measured (Table 10). As the concentration of RP increased, the AMP content of H. illucens increased significantly (p ˂ 0.05). The AMP content of H. illucens in R1, R2, R3, R4 and R5 groups was significantly higher than that in R0, with the most significant AMP content in groups R4 and R5 (p ˂ 0.05).

3.2.5. Intestinal Microbiota

Using 16S rDNA high-throughput sequencing, we measured the microbiota composition in the H. illucens intestine after feeding with a diet containing R0 (Control), R1 (RP-A) and R5 (RP-B). Raw sequencing information is shown in Table S1. Sangon Biotech sequencing of intestinal microbes produced 446,670 sequences from nine samples. Using cluster analysis of valid sequences according to 97% similarity, a total of 141 operational taxonomic units (OTUs) were obtained. The dilution curve tended to approach the saturation plateau, which indicates that most of the microorganisms have been detected in this study, and the results are representative (Figure 1).
Venn diagram showed that 105 OTUs were shared among the three groups, and 117, 119 and 137 core OTUs were identified in the control, RP-A and RP-B groups, respectively. The DNA sequences from samples of the RP-B group had the most abundant unique OTUs (14) (Figure 2).
The composition of intestinal microbes from the three groups at the phylum level was mainly Proteobacteria, Firmicutes and Actinobacteria (Figure 3). However, the relative proportion in each group differed. Proteobacteria was the dominant phylum in the three groups, accounting for 76.60%, 70.59% and 56.85%, respectively. Interestingly, the relative abundance of Firmicutes in the RP-B group (37.39%) increased compared with that in the control group (11.16%). The relative abundance of Actinobacteria in the RP-B group (5.03%) decreased compared with that in the control group (11.91%).
At the level of genus classification (Figure 4), Proteus was dominant in the three groups, and the average relative abundance of Proteus in the RP-B group was 34.41%. Still, the relative abundance was significantly different between the control (57.64%) and RP-A groups (56.26%) (p < 0.05). The genera of Corynebacterium (11.84%) and Providencia (5.16%) were dominant in the control group. Corynebacterium (9.84%) and Lactobacillus (8.39%) were dominant in the RP-A group. Lactobacillus (19.42%) and Rhodopseudomonas (8.90%) were dominant in the RP-B group.
Moreover, the Shannon and Ace indexes of the intestinal microorganism community of the RP-B group were significantly higher than those of the control and RP-A groups. Regarding the Chao index, the RP-B group had the same index as the control and RP-A groups; no significant differences were observed. The Simpson index was significantly lower than that of the control and RP-A groups (Table 11).
The microbial communities of each group appeared as one major cluster along PCA2 and along PCA1 (Figure 5). The principal coordinate (PCoA) cluster analysis indicated a separation between the control and RP groups. NDMS analyses revealed a clear separation of the three groups.
Welch’s t-test was performed to evaluate the differentially abundant genera. RP-A and RP-B groups showed a significantly higher abundance of Lactobacillus and Pediococcus than the control group (Figure 6A). The RP-B group presented a significantly higher abundance of Rhodopseudomonas and a lower abundance of Corynebacterium and Proteus (Figure 6B,C). The RP-B group displayed a significantly higher abundance of Rhodopseudomonas, Lactobacillus and Clostridium and a lower abundance of Corynebacterium and Proteus than the RP-A group.

4. Discussion

Insect meal is a good substitute for fish meal in aquatic feed because of its high protein content and rich nutrition [33]. Insect AMPs have good antibacterial and immunomodulatory activities and are a good substitute for antibiotics [17]. Insects can turn waste into biological resources. They can reproduce on a large scale, in line with the requirements of modern health, environmental protection and sustainable development [34]. The production of AMPs using H. illucens has the advantages of environmental protection, high yield and low cost.

4.1. Effect of Feed Supplementation with Different Strains on H. illucens Larvae

4.1.1. Effect of Feed Supplementation with Different Strains on the Growth and Development of H. illucens Larvae

The impact of microbiota on host development, metabolism and immunology has made microbial research an integral part of organismal biology. The addition of microorganisms to feed may affect the growth and development of animals [35]. This study determined the growth performance and body composition of H. illucens-fed diets containing different strains. Adding microorganisms to feeds can promote animal growth and development [35,36,37]. Lee et al. [38] reported that adding B. subtilis to feed improved growth performance and nutrient digestibility in weaned piglets. Merrifield et al. [39] reported that feed supplementation with probiotics could improve feed conversion, specific growth rate and protein efficiency in rainbow trout (Oncorhynchus mykiss). Kannan et al. reported that S. cerevisiae contributes to altered metabolic pathways in the H. illucens larvae and enhances larval body weight [40]. This study showed that feed addition of inactivated SA, RP and SC significantly improved the growth performance of H. illucens larvae. However, the addition of EC, BS and LP did not significantly affect the growth performance of H. illucens larvae. The large variation in the effect of different strains on the growth of H. illucens may be due to the different modes of action between the strains and the microorganisms in the H. illucens or because of the direct effect the development of the host. Studies reported that feed supplementation with microorganisms failed to promote the growth of the host. Hidalgo et al. [41] reported that feed supplementation with probiotics could not improve the growth performance of juvenile dentex (Dentex dentex), which indicates that the growth promoting effect of different strains on animals varies [42]. In the present study, the growth performance of H. illucens was significantly improved by the addition of SA, RP and SC to the feed, which implies a greater advantage of the application of these microorganisms in the feed of H. illucens. The H. illucens with feed supplementation of SA, RP and SC groups also had higher digestive enzyme activity, which can be explained by the fact that higher digestive enzyme activity implies higher digestive capacity and growth rate. The effect of probiotics on growth improvement was positively correlated with digestive enzyme activity and has been confirmed in many animal studies, such as in gilthead bream (Sparus aurata) [43], tilapia (Oreochromis niloticus) [44] and pacific white shrimp (Litopenaeus vannamei) [45], which may be attributed to the production of extracellular enzymes by the microflora after successful adhesion and colonization of the organism, which improves digestion [46]. This promotes the growth of the host.

4.1.2. Effect of Feed Supplementation with Different Strains on the Body Composition of H. illucens

Feeding SC significantly increased the crude lipid and ash content of the H. illucens but had no significant effect on crude protein. Yu et al. [47] showed that feed supplementation with probiotics did not affect the body composition of L. vannamei. Seenivasan et al. reported that feed supplementation with yeast, Bacillus and Lactobacillus significantly increased the crude protein, crude lipid and ash content of freshwater prawn (Macrobrachium rosenbergii) [48]. Microbial effects on host components vary across species studies. The mechanisms of microbial effects on host components are unclear. In this study, only SC increased the crude lipid and ash content of the H. illucens, which could be due to SC affecting the metabolism of lipids in H. illucens and increasing lipid deposition. In addition, the changes in crude lipid and ash may be due to altered digestion and absorption of the feed, which may be a function of bioactive components (vitamins and amino acids) in the SC that facilitate the absorption of nutrients in the digestive tract. Ovie et al. reported that dietary yeast increased the crude lipid content and decreased the ash content of fingerlings (Heterobranchus longifilis) [49]. However, Darafsh et al. reported no significant effect of dietary yeast on crude lipid and ash content of Persian sturgeon (Acipenser persicus) [50]. These studies differed from the results of the present study, suggesting that there are differences in the mechanisms by which strains affect the body composition of different species. The effects of different microbe on the host vary and the exact mechanism remains to be explored.

4.1.3. Induction of AMPs by Feed Supplementation with Different Strains

Insects have adapted to different habitats because their innate immune system provides them with a range of cellular and humoral responses against microorganisms [51]. In recent decades, significant advances have been made in the theory of immune defense due to the wide diversity of insects. They are an excellent source of biologically active molecules [52]. Insect AMPs are acquired immune factors, which can be induced, and have antibacterial, antidisease and antitumor properties [53]. Bacteria can induce the expression of host defense peptides in the organism, which improves the immune function of the organism [54].
Feeding diets containing different strains can induce the production of AMPs in yellow mealworm (Tenebrio molitor) larvae [55]. In this study, the addition of EC, SA, RP and SC to the diet-induced the production of AMPs in H. illucens, with RP, in particular, having the best inhibition effect. Choi et al. reported that E. coli could induce AMPs in H. illucens. AMPs had strong inhibition activity against Gram-negative bacteria, demonstrating the feasibility of feeding bacteria to induce AMPs in H. illucens [56]. The induction of AMPs by different bacteria had no significant effect on the overall antimicrobial activity of the bacteria. The immune response of H. illucens is nonspecific, and different induction sources can induce the insect AMPs to produce a peptide with a broad spectrum of antimicrobial activity and are not specific to a particular antigen-inducing substance [57,58]. The AMPs of H. illucens have a broad spectrum of antimicrobial activity and are promising agents in aquaculture.

4.2. Effect of Feed Supplementation with Different Concentrations of RP on H. illucens Larvae

4.2.1. Effect of Feed Supplementation with Different Concentrations of RP on the Growth Performance of H. illucens

The first experiment showed that 1.22 × 108 CFU/g of RP significantly improved the digestive capacity and growth of H. illucens. The second experiment explored the effect of different concentrations of RP on the growth and digestibility of H. illucens to arrive at the optimum growth-promoting concentration of RP. RP can provide protein, lipids, essential amino acids, essential fatty acids, vitamins and carotenoids [59,60,61,62,63], which can be used as a substitute for feed ingredients [64]. Feeding RP increases the growth and survival of fairy shrimp (Streptocephalus sirindhornae) [65]. Saejung et al. reported that three types of anaerobic RP increased the growth and survival of S. sirindhornae [66]. RP is rich in essential amino acids and polyunsaturated fatty acids (PUFA), which are extremely important for the growth of crustaceans [60,67]. Our results demonstrate that RP can promote the growth of H. illucens, similar to how they promote the growth of rotifers [68].

4.2.2. Effect of Feed Supplementation with Different Concentrations of RP on the Body Composition of H. illucens

This study showed that adding RP to the feed did not affect the crude protein and ash content of H. illucens, but high concentrations of RP increased the deposition of lipids. RP is rich in amino acids and fatty acids, and high concentrations of RP provide more nutrients for H. illucens feed nutrition, which may alter the lipid metabolism and lead to higher deposition of lipids in H. illucens. RP contains a variety of B vitamins and pantothenic acid, which can be used as coenzymes to participate in the body’s metabolism and promote the animal’s growth and development [66]. These components can promote the metabolism of H. illucens and increase the deposition of lipids in the body.

4.2.3. Induction of AMPs by Feed Supplementation with Different Concentrations of RP

Based on the first experiment, we demonstrated that RP had a better induction effect on the AMPs of H. illucens, and the second experiment explored the induction effect of different concentrations of RP on the AMPs. The results showed that higher concentrations of RP could better induce the expression of AMPs in H. illucens. AMPs were induced in H. illucens by needling injection and injection of bacterial solution [69]. In this study, feed supplementation with RP induced AMP production in H. illucens, which is methodologically simpler than by the needling and injection methods and suitable for large-scale AMP production. The ability of RP to induce the production of AMPs in H. illucens may be attributed to the fact that the metabolic substances produced by RP stimulate H. illucens and cause them to produce stress, resulting in the high production of AMPs. Our results indicate that high concentrations (1.22 × 109 and 1.22 × 1010 CFU/g) of RP induced AMPs to a stable level. Considering the production costs, we suggest that the optimal concentration of AMPs induced by RP is 1.22 × 109 CFU/g.

4.2.4. Effect of Feed Supplementation with RP on the Intestinal Flora of H. illucens

In our study, alpha diversity analysis showed that adding RP to the diet significantly increased the Shannon and Ace diversity indices, which indicates that RP can improve the species richness and evenness of the gut microbiota of H. illucens, resulting in a more diverse and evenly distributed microbiota. Moreover, the effect of different concentrations of RP on the composition of the gut flora varied, with higher concentrations of RP having higher flora diversity. Studies have shown that increasing the diversity of the gut microbiome is better for host health [70].
Adding many feed probiotics can influence the structure of the animal’s intestinal flora and improve the host’s health status [71,72]. The results of gut sequencing in this study show that the dominant flora of H. illucens is distributed in Proteobacteria, Firmicutes and Actinobacteria. Proteobacteria. Firmicutes and Actinobacteria are most commonly found in insects [73]. In this study, the abundance of Proteobacteria and Actinobacteria decreased, and Firmicutes increased in H. illucens fed with high concentrations of RP, which may be the result of feeding RP. Proteobacteria are Gram-negative bacteria, most of which are parthenogenic or exclusively anaerobic [74] and are usually the main component of the insect gut microbiota [73]. A potential disease risk is associated with the increased abundance of Proteobacteria, as it includes many pathogenic bacteria. Most Firmicutes are Gram-positive bacteria, including many probiotic bacteria such as Bacillus and Lactobacillus. The high abundance of Firmicutes and the low abundance of Proteobacteria and Actinobacteria in this study suggest that RP improves the microbial composition of the gut.
At the genus level, some common probiotic bacteria, such as Lactobacillus and Rhodopseudomonas, increased significantly in abundance after feeding RP. Lactobacillus and Rhodopseudomonas are common probiotics used in animal production farming, and Lactobacillus can improve digestibility and immunity in aquatic animals [75]. Rhodopseudomonas can promote the growth of probiotics, regulate water quality and enhance animal resistance [76]. With the increased feeding RP concentration, the abundance of Corynebacterium and Proteus decreased significantly. Except for Corynebacterium diphtheriae, Corynebacterium is mostly conditionally pathogenic and can cause various clinical infections, such as sepsis, endocarditis and pneumonia, and has attracted the attention of scholars abroad [77,78]. Proteus is a genus of conditioned pathogenic bacteria that can cause food poisoning and many diseases under certain conditions [79,80,81]. In this study, feed supplementation with RP increased the abundance of beneficial bacteria and decreased the abundance of harmful bacteria, which may be the result of increased digestibility and disease resistance in H. illucens. Bacteria capable of producing multiple extracellular hydrolytic enzymes can be isolated from selective and enriched media in the gut of nutrient-rich larvae [73]. These isolated strains have multiple hydrolytic capabilities that can provide ecological advantages to hosts grown in polymer-rich diets. Specific hydrolytic activity is associated with a specific phylogenetic group [73]. The improved digestibility of H. illucens may be related to the composition and abundance of the intestinal flora. The intestinal flora may influence the metabolic function of H. illucens, enhancing its digestive capacity and disease resistance. On the one hand, RP influences the nutritional level, material metabolism and immune function of H. illucens. Intestinal flora correlates with physiological status, digestive and absorption functions and disease resistance. Thus, RP indirectly influences the nutritional level, material metabolism and immune function of H. illucens through the regulation of the intestinal flora. In addition, due to the antibacterial effect of AMPs, the production of AMPs in the body of black soldier fly may inhibit the growth of harmful flora, giving probiotics a better living space and indirectly regulating the balance of gut flora. AMPs are also key to ensuring that the H. illucens intestine remains healthy in a putrefactive environment.

5. Conclusions

In conclusion, compared to the other strains, RP is more suitable as a feed additive to improve the growth performance, digestibility and induction of AMPs in H. illucens. High concentrations (1.22 × 109–1.22 × 1010 CFU/g) of RP are more effective in promoting growth and microbial composition of the gut of H. illucens, improving digestibility and induction of AMPs.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ani13172722/s1. Figure S1: The inhibition circles of each feed group; Table S1: Raw sequencing information of the microbiota composition in the H. illucens intestine.

Author Contributions

Y.M.: methodology, writing—original draft preparation, data curation, visualization, investigation. X.Z.: data curation, software, investigation. Z.Z.: data curation, visualization, investigation. J.L. (Jiajun Li): investigation, validation. P.Z.: investigation, figures. J.L. (Juntao Li): investigation, data collection. J.X. (Jiarui Xu): investigation, data analysis. J.X. (Jianan Xian): conceptualization, methodology, writing—reviewing and editing, funding acquisition. Y.L.: conceptualization, methodology, writing—reviewing and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Hainan Provincial Natural Science Foundation of China (No. 322RC763), Central Public-Interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630052019013) and Social Public-Interest Scientific Institution Reform Special Fund (No. ITBBQDF2023008).

Institutional Review Board Statement

The animal protocol utilized in this study was authorized by the Animal Ethics Committee of Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (Protocol code ITBB20220209 and date of February 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The partial data analyzed for this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. He, S.W.; Wang, G.H.; Yue, B. TO17: A teleost antimicrobial peptide that induces degradation of bacterial nucleic acids and inhibits bacterial infection in red drum, Sciaenops ocellatus. Fish Shellfish Immunol. 2018, 72, 639–645. [Google Scholar]
  2. Hansen, M.P.; Hoffmann, T.C.; McCullough, A.R.; van Driel, M.L.; Del Mar, C.B. Antibiotic resistance: What are the opportunities for primary care in alleviating the crisis? Front. Public Health 2015, 3, 35. [Google Scholar]
  3. Tu, J.; Wu, G.; Zuo, Y.; Zhao, L.; Wang, S. ZL-2, a cathelicidin-derived antimicrobial peptide, has a broad antimicrobial activity against gram-positive bacteria and gram-negative bacteria in vitro and in vivo. Arch. Pharmacal Res. 2015, 38, 1802–1809. [Google Scholar]
  4. Kraemer, S.A.; Ramachandran, A.; Perron, G.A.O. Antibiotic pollution in the environment: From microbial ecology to public policy. Microorganisms 2019, 7, 180. [Google Scholar]
  5. Klare, I.; Konstabel, C.; Badstübner, D.; Werner, G.; Witte, W. Occurrence and spread of antibiotic resistances in Enterococcus faecium. Int. J. Food Microbiol. 2003, 88, 269–290. [Google Scholar]
  6. Brorsen, B.W.; Lehenbauer, T.; Ji, D.; Connor, J. Economic impacts of banning subtherapeutic use of antibiotics in swine production. J. Agric. Appl. Econ. 2002, 34, 489–500. [Google Scholar]
  7. Hull, R.; Katete, R.; Ntwasa, M. Therapeutic potential of antimicrobial peptides from insects. Biotechnol. Mol. Biol. Rev. 2012, 7, 31–47. [Google Scholar]
  8. Natalia, G.; Andrey, Y.; Anastasia, K.; Dmitry, T.; Evdokia, P.; Tatyana, S.; Domenico, B.; Camillo, R.; Sergey, C.; Surajit, B. Natural antimicrobial peptide complexes in the fighting of antibiotic resistant biofilms: Calliphora vicina medicinal maggots. PLoS ONE 2017, 12, e0173559. [Google Scholar]
  9. Mouammine, A.; Pages, S.; Lanois, A.; Gaudriault, S.; Jubelin, G.; Bonabaud, M.; Cruveiller, S.; Dubois, E.; Roche, D.; Legrand, L.; et al. An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence. Sci. Rep. 2017, 7, 43670. [Google Scholar]
  10. Zhao, Y.; Xie, Z.; Feng, J.; Li, W.; Cao, Z. Pharmacological characterization of human beta-defensins 3 and 4 on potassium channels: Evidence of diversity in beta-defensin-potassium channel interactions. Peptides 2018, 108, 14–18. [Google Scholar]
  11. Wiesner, J.; Vilcinskas, A. Antimicrobial peptides: The ancient arm of the human immune system. Virulence 2010, 1, 440–464. [Google Scholar] [CrossRef]
  12. Hancock, R.E.W.; Lehrer, R. Cationic peptides: A new source of antibiotics. Trends Biotechnol. 1998, 16, 82–88. [Google Scholar] [CrossRef] [PubMed]
  13. Shumo, M.; Osuga, I.M.; Khamis, F.M.; Tanga, C.M.; Fiaboe, K.K.M.; Subramanian, S.; Ekesi, S.; Huis, A.V.; Borgemeister, C. The nutritive value of black soldier fly larvae reared on common organic waste streams in Kenya. Sci. Rep. 2019, 9, 10110. [Google Scholar] [CrossRef] [PubMed]
  14. Liu, X.; Chen, X.; Wang, H.; Yang, Q.; Rehman, K.U.; Li, W.; Cai, M.; Li, Q.; Mazza, L.; Zhang, J. Dynamic changes of nutrient composition throughout the entire life cycle of black soldier fly. PLoS ONE 2017, 12, e0182601. [Google Scholar] [CrossRef]
  15. Zhan, S.; Fang, G.; Cai, M.; Kou, Z.; Xu, J.; Cao, Y.; Bai, L.; Zhang, Y.; Jiang, Y.; Luo, X. Genomic landscape and genetic manipulation of the black soldier fly Hermetia illucens, a natural waste recycler. Cell Res. 2020, 30, 50–60. [Google Scholar] [CrossRef]
  16. Park, K.; Yun, E.Y.; Park, S.W.; Goo, T.W. The antimicrobial activity of bacterial-challenged black soldier fly, Hermetia illucens. J. Life Sci. 2016, 26, 1409–1414. [Google Scholar] [CrossRef]
  17. Osama, E.; Zhou, D.; Song, Q.; Aziz, S.A.; Cai, M.; Zheng, L.; Yu, Z.; Zhang, J.; Humberto, L.M. Screening, expression, purification and functional characterization of novel antimicrobial peptide genes from Hermetia illucens (L.). PLoS ONE 2017, 12, e0169582. [Google Scholar]
  18. Xu, J.; Luo, X.; Fang, G.; Zhan, S.; Wu, J.; Wang, D.; Huang, Y. Transgenic expression of antimicrobial peptides from black soldier fly enhance resistance against entomopathogenic bacteria in the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 2020, 127, 103487. [Google Scholar] [CrossRef]
  19. Alvarez, D.; Kevin, A. Treilhou, MichelTene, NathanCastillo, DenisSauvain, Michel. Prospecting peptides isolated from black soldier fly (Diptera: Stratiomyidae) with antimicrobial activity against Helicobacter pylori (Campylobacterales: Helicobacteraceae). J. Insect Sci. 2019, 19, 17. [Google Scholar] [CrossRef]
  20. Lee, K.S.; Yun, E.Y.; Goo, T.W. Antimicrobial activity of an extract of Hermetia illucens larvae immunized with Lactobacillus casei against Salmonella Species. Insects 2020, 11, 704. [Google Scholar] [CrossRef]
  21. Park, S.I.; Kim, J.W.; Yoe, S.M. Purification and characterization of a novel antibacterial peptide from black soldier fly (Hermetia illucens) larvae. Dev. Comp. Immunol. 2015, 52, 98–106. [Google Scholar] [CrossRef] [PubMed]
  22. Lee, D.H.; Chu, K.B.; Kang, H.J.; Lee, S.H.; Quan, F.S. Peptides in the hemolymph of Hermetia illucens larvae completely inhibit the growth of Klebsiella pneumonia in vitro and in vivo. J. Asia-Pac. Entomol. 2020, 23, 36–43. [Google Scholar] [CrossRef]
  23. Sheppard, D.C.; Tomberlin, J.K.; Joyce, J.A.; Kiser, B.C.; Sumner, S.M. Rearing methods for the black soldier fly (Diptera: Stratiomyidae). J. Med. Entomol. 2002, 39, 695–698. [Google Scholar] [CrossRef] [PubMed]
  24. Zokaeifar, H.; Balcázar, J.L.; Saad, C.R.; Kamarudin, M.S.; Sijam, K.; Arshad, A.; Nejat, N. Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 2012, 33, 683–689. [Google Scholar] [CrossRef]
  25. Yeh, S.; Chang, C.; Chang, C.; Liu, C.; Cheng, W. Dietary sodium alginate administration affects fingerling growth and resistance to Streptococcus sp. and iridovirus, and juvenile non-specific immune responses of the orange-spotted grouper, Epinephelus coioides. Fish Shellfish Immunol. 2008, 25, 19–27. [Google Scholar] [CrossRef]
  26. Hirwitz, W.; Latimer, G. Official methods of analysis of AOAC International (16th edn). Trends Food Sci. Technol. 1995, 6, 382. [Google Scholar]
  27. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef]
  28. Martin, M. Cut adapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 2011, 17, 10–12. [Google Scholar] [CrossRef]
  29. Edgar, R.C. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 2013, 10, 996–998. [Google Scholar] [CrossRef] [PubMed]
  30. Edgar, R.C.; Haas, B.J.; Clemente, J.C.; Quince, C.; Knight, R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 2011, 27, 2194–2200. [Google Scholar] [CrossRef]
  31. Park, S.I.; Chang, B.S.; Yoe, S.M. Detection of antimicrobial substances from larvae of the black soldier fly, Hermetia illucens (Diptera: Stratiomyidae). Entomol. Res. 2014, 44, 58–64. [Google Scholar] [CrossRef]
  32. Xiaoqi, Z.; Caiyun, X.; Hemin, N.; Xiao, Z.; Xiaona, C.; Xiaohui, H.; Huan, S.; Jungang, W.; Hong, S. The biological properties of an antimicrobial peptide extracted from Tenebrio molitor. Chin. J. Appl. Entomol. 2015, 52, 712–720. [Google Scholar] [CrossRef]
  33. Schiavone, A.; Cullere, M.; De Marco, M.; Meneguz, M.; Biasato, I.; Bergagna, S.; Dezzutto, D.; Gai, F.; Dabbou, S.; Gasco, L.; et al. Partial or total replacement of soybean oil by black soldier fly larvae (Hermetia illucens L.) fat in broiler diets: Effect on growth performances, feed-choice, blood traits, carcass characteristics and meat quality. Ital. J. Anim. Sci. 2017, 16, 93–100. [Google Scholar] [CrossRef]
  34. Nijdam, D.; Rood, T.; Westhoek, H. The price of protein: Review of land use and carbon footprints from life cycle assessments of animal food products and their substitutes. Food Policy 2012, 37, 760–770. [Google Scholar] [CrossRef]
  35. Zhang, Y.; Ji, T.; Jiang, Y.; Zheng, C.; Yang, H.; Liu, Q. Long-term effects of three compound probiotics on water quality, growth performances, microbiota distributions and resistance to Aeromonas veronii in crucian carp Carassius auratus gibelio. Fish Shellfish Immunol. 2022, 120, 233–241. [Google Scholar] [CrossRef] [PubMed]
  36. Yu, J.; Zhou, Y.; Wen, Q.; Wang, B.; Gong, H.; Zhu, L.; Lan, H.; Wu, B.; Lang, W.; Zheng, X.; et al. Effects of faecal microbiota transplantation on the growth performance, intestinal microbiota, jejunum morphology and immune function of laying-type chicks. Anim. Prod. Sci. 2022, 62, 321–332. [Google Scholar] [CrossRef]
  37. Ramos, M.A.; Weber, B.; Gonçalves, J.F.; Santos, G.A.; Rema, P.; Ozório, R.O.A. Dietary probiotic supplementation modulated gut microbiota and improved growth of juvenile rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2013, 166, 302–307. [Google Scholar] [CrossRef]
  38. Lee, S.H.; Ingale, S.L.; Kim, J.S.; Kim, K.H.; Lokhande, A.; Kim, E.K.; Kwon, I.K.; Kim, Y.H.; Chae, B.J. Effects of dietary supplementation with Bacillus subtilis LS 1–2 fermentation biomass on growth performance, nutrient digestibility, cecal microbiota and intestinal morphology of weanling pig. Anim. Feed Sci. Technol. 2014, 188, 102–110. [Google Scholar] [CrossRef]
  39. Merrifield, D.L.; Dimitroglou, A.; Bradley, G.; Baker, R.T.M.; Davies, S.J. Probiotic applications for rainbow trout (Oncorhynchus mykiss Walbaum) I. Effects on growth performance, feed utilization, intestinal microbiota and related health criteria. Aquacult. Nutr. 2010, 16, 504–510. [Google Scholar] [CrossRef]
  40. Kannan, M.; Vitenberg, T.; Ben-Mordechai, L.; Khatib, S.; Opatovsky, I. Effect of yeast supplementation on growth parameters and metabolomics of black soldier fly larvae, Hermetia illucens (L.) (Diptera: Stratiomyidae). J. Insects Food Feed 2023, 1–12. [Google Scholar] [CrossRef]
  41. Hidalgo, M.C.; Skalli, A.; AbellÁN, E.; Arizcun, M.; Cardenete, G. Dietary intake of probiotics and maslinic acid in juvenile dentex (Dentex dentex L.): Effects on growth performance, survival and liver proteolytic activities. Aquacult. Nutr. 2006, 12, 256–266. [Google Scholar] [CrossRef]
  42. Li, Y.; Yang, Y.; Song, L.; Wang, J.; Hu, Y.; Yang, Q.; Cheng, P.; Li, J. Effects of dietary supplementation of Lactobacillus plantarum and Bacillus subtilis on growth performance, survival, immune response, antioxidant capacity and digestive enzyme activity in olive flounder (Paralichthys olivaceus). Aquacult. Fish. 2021, 6, 283–288. [Google Scholar] [CrossRef]
  43. Suzer, C.; Çoban, D.; Kamaci, H.O.; Saka, Ş.; Firat, K.; Otgucuoğlu, Ö.; Küçüksari, H. Lactobacillus spp. bacteria as probiotics in gilthead sea bream (Sparus aurata, L.) larvae: Effects on growth performance and digestive enzyme activities. Aquaculture 2008, 280, 140–145. [Google Scholar] [CrossRef]
  44. Liu, H.; Wang, S.; Cai, Y.; Guo, X.; Cao, Z.; Zhang, Y.; Liu, S.; Yuan, W.; Zhu, W.; Zheng, Y.; et al. Dietary administration of Bacillus subtilis HAINUP40 enhances growth, digestive enzyme activities, innate immune responses and disease resistance of tilapia, Oreochromis niloticus. Fish Shellfish Immunol. 2017, 60, 326–333. [Google Scholar] [CrossRef]
  45. Duan, Y.; Zhang, Y.; Dong, H.; Wang, Y.; Zheng, X.; Zhang, J. Effect of dietary Clostridium butyricum on growth, intestine health status and resistance to ammonia stress in pacific white shrimp Litopenaeus vannamei. Fish Shellfish Immunol. 2017, 65, 25–33. [Google Scholar] [CrossRef]
  46. Hoseinifar, S.H.; Soleimani, N.; Ringoe, E. The effects of fructooligosaccharide on growth performance, haemato-immunological parameters, gut microbiota and stress resistance of common carp (Cyprinus carpio) fry. Br. J. Nutr. 2014, 112, 1296–1302. [Google Scholar] [CrossRef]
  47. Yu, M.C.; Li, Z.J.; Lin, H.Z.; Wen, G.L.; Ma, S. Effects of dietary medicinal herbs and Bacillus on survival, growth, body composition, and digestive enzyme activity of the white shrimp Litopenaeus vannamei. Aquacult. Int. 2009, 17, 377–384. [Google Scholar] [CrossRef]
  48. Seenivasan, D.C.; Subramanian, R.; Muralisankar, T.; Bhavan, P. Influence of combined probiotics Lactobacillus sporogenes and Bacillus subtilis on survival, growth, biochemical changes and energy utilization performance of Macrobrachium Rosenberger (De Man 1879) post larvae. J. Ecobiotechnol. 2012, 4, 29–34. [Google Scholar]
  49. Ovie, S.O.; Ibiyo, L.; Babalola, T.; Eze, S.S. The effects of varying levels of yeast (Saccharomyces cerevisiae) on the growth and body composition of Heterobranchus longifilis fingerlings. Zoologist 2013, 10, 34–39. [Google Scholar]
  50. Darafsh, F.; Soltani, M.; Abdolhay, H.A.; Shamsaei Mehrejan, M. Improvement of growth performance, digestive enzymes and body composition of Persian sturgeon (Acipenser persicus) following feeding on probiotics: Bacillus licheniformis, Bacillus subtilis and Saccharomyces cerevisiae. Aquacult. Res. 2020, 51, 957–964. [Google Scholar] [CrossRef]
  51. Rocha, I.F.; Maller, A.; de Cássia Garcia Simão, R.; Kadowaki, M.K.; Angeli Alves, L.F.; Huergo, L.F.; da Conceição Silva, J.L. Proteomic profile of hemolymph and detection of induced antimicrobial peptides in response to microbial challenge in Diatraea saccharalis (Lepidoptera: Crambidae). Biochem. Biophys. Res. Commun. 2016, 473, 511–516. [Google Scholar] [CrossRef]
  52. Slocinska, M.; Marciniak, P.; Rosinski, G. Insects antiviral and anticancer peptides: New leads for the future? Protein Pept. Lett. 2008, 15, 578–585. [Google Scholar] [CrossRef]
  53. Sahoo, A.; Swain, S.; Behera, A.; Sahoo, G.; Mahapatra, P.; Panda, S. Antimicrobial peptides derived from insects offer a novel therapeutic option to combat biofilm: A systematic review. Front. Microbiol. 2021, 12, 661195. [Google Scholar] [CrossRef] [PubMed]
  54. Rizzo, A.; Losacco, A.; Carratelli, C.R. Lactobacillus crispatus modulates epithelial cell defense against Candida albicans through Toll-like receptors 2 and 4, interleukin 8 and human β-defensins 2 and 3. Immunol. Lett. 2013, 156, 102–109. [Google Scholar] [CrossRef]
  55. Xie, X.S.; Dong, J.Z.; Li, J.; Li, Y.L.; Wang, Y.Y.; Li, X.F.; Zhang, Y.N. Analysis of the antimicrobial activity of antimicrobial peptides extracted from immunized Tenebrio molitor induced by different microbial treatments against 26 pathogens. Agric. Sci. Technol. 2010, 11, 1–4+9. [Google Scholar] [CrossRef]
  56. Choi, W.; Choi, H.-J.; Goo, T.; Quan, F. Novel antibacterial peptides induced by probiotics in Hermetia illucens (Diptera: Stratiomyidae) larvae. Entomol. Res. 2017, 48, 237–247. [Google Scholar] [CrossRef]
  57. Zhang, W.; Tettamanti, G.; Bassal, T.; Heryanto, C.; Eleftherianos, I.; Mohamed, A. Regulators and signalling in insect antimicrobial innate immunity: Functional molecules and cellular pathways. Cell Signal. 2021, 83, 110003. [Google Scholar] [CrossRef]
  58. Zdbicka-Barabas, A.; Bulak, P.; Polakowski, C.; Bieganowski, A.; Waśko, A.; Cytrynska, M. Immune response in the larvae of the black soldier fly Hermetia illucens. Invertebr. Surviv. J. 2017, 14, 9–17. [Google Scholar]
  59. Patthawaro, S.; Lomthaisong, K.; Saejung, C. Bioconversion of agro-industrial waste to value-added product lycopene by photosynthetic bacterium Rhodopseudomonas faecalis and its carotenoid composition. Waste Biomass Valoriz. 2020, 11, 2375–2386. [Google Scholar] [CrossRef]
  60. Saejung, C.; Ampornpat, W. Production and nutritional performance of carotenoid-producing photosynthetic bacterium Rhodopseudomonas faecalis PA2 grown in domestic wastewater intended for animal feed production. Waste Biomass Valoriz. 2019, 10, 299–310. [Google Scholar] [CrossRef]
  61. Saejung, C.; Puensungnern, L. Evaluation of molasses-based medium as a low cost medium for carotenoids and fatty acid production by photosynthetic bacteria. Waste Biomass Valoriz. 2020, 11, 143–152. [Google Scholar] [CrossRef]
  62. Saejung, C.; Salasook, P. Recycling of sugar industry wastewater for single-cell protein production with supplemental carotenoids. Environ. Technol. 2020, 41, 59–70. [Google Scholar] [CrossRef]
  63. Saejung, C.; Thammaratana, T. Biomass recovery during municipal wastewater treatment using photosynthetic bacteria and prospect of production of single cell protein for feedstuff. Environ. Technol. 2016, 37, 3055–3061. [Google Scholar] [CrossRef] [PubMed]
  64. Delamare-Deboutteville, J.; Batstone, D.J.; Kawasaki, M.; Stegman, S.; Salini, M.; Tabrett, S.; Smullen, R.; Barnes, A.C.; Hülsen, T. Mixed culture purple phototrophic bacteria is an effective fishmeal replacement in aquaculture. Water Res. X 2019, 4, 100031. [Google Scholar] [CrossRef] [PubMed]
  65. Saejung, C.; Chaiyarat, A.; Sanoamuang, L.-O. Effects of algae, yeast and photosynthetic bacteria diets on survival and growth performance in the fairy shrimp, Streptocephalus sirindhornae (Branchiopoda, Anostraca). Crustaceana 2018, 91, 1505–1522. [Google Scholar] [CrossRef]
  66. Saejung, C.; Chaiyarat, A.; Sanoamuang, L.O. Optimization of three anoxygenic photosynthetic bacteria as feed to enhance growth, survival, and water quality in fairy shrimp (Streptocephalus sirindhornae) cultivation. Aquaculture 2021, 534, 736288. [Google Scholar] [CrossRef]
  67. Patthawaro, S.; Saejung, C. Production of single cell protein from manure as animal feed by using photosynthetic bacteria. MicrobiologyOpen 2019, 8, e913. [Google Scholar] [CrossRef]
  68. Pinoargote, G.; Flores, G.; Cooper, K.; Ravishankar, S. Effects on survival and bacterial community composition of the aquaculture water and gastrointestinal tract of shrimp (Litopenaeus vannamei) exposed to probiotic treatments after an induced infection of acute hepatopancreatic necrosis disease. Aquacult. Res. 2018, 49, 3270–3288. [Google Scholar] [CrossRef]
  69. Xia, Q.; Zhao, Q.F.; Liao, Y.; Zhu, W.; Yu, G.H.; Chen, Y.F.; Song, M.Y. Black soldier fly antimicrobial peptides induced conditions optimization and research of crude extracts activity. J. Environ. Entomol. 2013, 35, 44–48. [Google Scholar]
  70. Li, T.; Long, M.; Ji, C.; Shen, Z.; Gatesoupe, F.J.; Zhang, X.; Zhang, Q.; Zhang, L.; Zhao, Y.; Liu, X.; et al. Alterations of the gut microbiome of largemouth bronze gudgeon (Coreius guichenoti) suffering from furunculosis. Sci. Rep. 2016, 6, 30606. [Google Scholar] [CrossRef]
  71. Daniels, C.; Hoseinifar, S.H. Prebiotic applications in shellfish. Aquacult. Nutr. 2014, 1, 401–418. [Google Scholar] [CrossRef]
  72. Valipour, A.; Nedaei, S.; Noori, A.; Khanipour, A.A.; Hoseinifar, S.H. Dietary Lactobacillus plantarum affected on some immune parameters, air-exposure stress response, intestinal microbiota, digestive enzyme activity and performance of narrow clawed crayfish (Astacus leptodactylus, Eschscholtz). Aquaculture 2019, 504, 121–130. [Google Scholar] [CrossRef]
  73. Callegari, M.; Jucker, C.; Fusi, M.; Leonardi, M.; Daffonchio, D.; Borin, S.; Savoldelli, S.; Crotti, E. Hydrolytic profile of the culturable gut bacterial community associated with Hermetia illucens. Front. Microbiol. 2020, 11, 1965. [Google Scholar] [CrossRef] [PubMed]
  74. Stackebrandt, E.; Murray, R.G.E.; Trüper, H.G. Proteobacteria classis nov., a name for the phylogenetic taxon including the “purple bacteria and their relatives”. Int. J. Syst. Bacteriol. 1988, 42, 281–295. [Google Scholar]
  75. Mohapatra, S.; Chakraborty, T.; Kumar, V.; DeBoeck, G.; Mohanta, K.N. Aquaculture and stress management: A review of probiotic intervention. J. Anim. Physiol. Anim. Nutr. 2013, 97, 405–430. [Google Scholar] [CrossRef]
  76. Liu, R.; Wu, W.; Xu, X.; Wang, Y.; Yu, T.; Wang, J.; Zheng, Q.; Changru, X.; Wu, P. Rhodopseudomonas palustris in effluent enhances the disease resistance, TOR and NF-κB signalling pathway, intestinal microbiota and aquaculture water quality of Pelteobagrus vachelli. Aquacult. Res. 2020, 51, 3959–3971. [Google Scholar] [CrossRef]
  77. Batalla, A.S.; Blanchardière, A.; Vergnaud, M.; Dargère, S.; Verdon, R. Endocardite recidivante a Corynebacterium striatum compliquant une osteite. Med. Mal. Infect. 2010, 41, 160–163. [Google Scholar] [CrossRef]
  78. Wong, K.Y.; Chan, Y.C.; Wong, C.Y. Corynebacterium striatum as an emerging pathogen. J. Hosp. Infect. 2010, 76, 371–372. [Google Scholar] [CrossRef]
  79. Petrás, G.; Lányi, B.; Bognár, S.; Tóth, L. Microflora of the department of acute respiratory diseases and the spreading of Proteus strains. Orv. Hetil. 1972, 113, 1399–1403. [Google Scholar]
  80. Sánchez, F.; Herranz, P.; Gil, J.; Peréz-Navarro, A. The efficacy of oral ciprofloxacin in the treatment of Proteus mirabilis infections of the lower respiratory tract. Ann. Med. Intern 1989, 6, 605. [Google Scholar]
  81. Bahrani, F.K.; Massad, G.; Lockatell, C.V.; Johnson, D.E.; Russell, R.G.; Warren, J.W.; Mobley, H.L. Construction of an MR/P fimbrial mutant of Proteus mirabilis: Role in virulence in a mouse model of ascending urinary tract infection. Infect. Immun. 1994, 62, 3363–3371. [Google Scholar] [CrossRef] [PubMed]
Animals 13 02722 g001
Figure 1. Rarefaction curves of samples.
Figure 1. Rarefaction curves of samples.
Animals 13 02722 g001
Animals 13 02722 g002
Figure 2. Venn diagram analysis of species.
Figure 2. Venn diagram analysis of species.
Animals 13 02722 g002
Animals 13 02722 g003
Figure 3. The percentage of community abundance on phylum level. The phylum with less than 1% relative abundance was collected to other, other = 30.
Figure 3. The percentage of community abundance on phylum level. The phylum with less than 1% relative abundance was collected to other, other = 30.
Animals 13 02722 g003
Animals 13 02722 g004
Figure 4. The percentage of community abundance on genus level. Note: The genus with relative abundance less than 1% was collected to other, other = 30.
Figure 4. The percentage of community abundance on genus level. Note: The genus with relative abundance less than 1% was collected to other, other = 30.
Animals 13 02722 g004
Animals 13 02722 g005
Figure 5. Differences in bacterial beta diversity between the treatment groups and RP group. (A) Principal coordinate analysis (PCA), (B) two-dimensional principal coordinate analysis (PCoA), (C) nonmetric multidimensional scaling (NMDS) analysis.
Figure 5. Differences in bacterial beta diversity between the treatment groups and RP group. (A) Principal coordinate analysis (PCA), (B) two-dimensional principal coordinate analysis (PCoA), (C) nonmetric multidimensional scaling (NMDS) analysis.
Animals 13 02722 g005
Animals 13 02722 g006
Figure 6. Student’s t-test bar plot on phylum level between control and RP-A and RP-B groups. (A) Analysis of differences on phylum level bewteen Control groupand RP-A group, (B) Analysis of differences on phylum level bewteen Control groupand RP-B group, (C) Analysis of differences on phylum level bewteen RP-A groupand RP-B group. Phylums with a p-value less than 0.05 are marked red.
Figure 6. Student’s t-test bar plot on phylum level between control and RP-A and RP-B groups. (A) Analysis of differences on phylum level bewteen Control groupand RP-A group, (B) Analysis of differences on phylum level bewteen Control groupand RP-B group, (C) Analysis of differences on phylum level bewteen RP-A groupand RP-B group. Phylums with a p-value less than 0.05 are marked red.
Animals 13 02722 g006
Table 1. Ingredients and nutrient level of experimental diets (based on dry matter).
Table 1. Ingredients and nutrient level of experimental diets (based on dry matter).
IngredientsContent (%)Proximate CompositionContent (%)
Wheat bran50Crude protein22.68
Soybean meal30Crude lipid3.71
Corn meal20Ash2.38
Table 2. Effects of different strains on the growth performance of H. illucens.
Table 2. Effects of different strains on the growth performance of H. illucens.
ParameterWGR (%)SGR (%/d)
Control416.67 ± 47.26 c32.79 ± 1.80 d
EC426.67 ± 25.17 c33.21 ± 0.96 cd
SA686.67 ± 80.21 ab41.18 ± 2.03 ab
BS443.33 ± 20.82 c33.84 ± 0.77 cd
RP730.00 ± 43.59 a42.31 ± 1.04 a
SC693.33 ± 80.21 ab41.35 ± 2.04 ab
LP546.67 ± 51.32 bc37.29 ± 1.61 bc
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 3. Effects of different strains on crude protein, crude lipid and ash of H. illucens.
Table 3. Effects of different strains on crude protein, crude lipid and ash of H. illucens.
ParameterCrude Protein (%)Crude Lipid (%)Ash (%)
Control53.74 ± 2.0814.40 ± 2.57 b2.61 ± 0.10 b
EC55.13 ± 3.0414.69 ± 0.52 ab2.68 ± 0.09 ab
SA55.36 ± 4.0215.93 ± 1.18 ab2.64 ± 0.05 b
BS58.16 ± 3.7213.96 ± 0.30 b2.70 ± 0.10 ab
RP56.15 ± 3.1715.59 ± 0.62 ab2.78 ± 0.12 ab
SC55.77 ± 3.1517.90 ± 0.33 a2.88 ± 0.02 a
LP56.31 ± 4.1116.34 ± 1.19 ab2.76 ± 0.01 ab
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 4. Effects of different strains on the digestive ability of H. illucens.
Table 4. Effects of different strains on the digestive ability of H. illucens.
Parameterα-Amylase (U/mgprot)Lipase (U/gprot)Trypsin (U/gprot)
Control5.68 ± 1.26 cd10.04 ± 1.87 d476.40 ± 62.90 c
EC3.53 ± 0.85 d7.39 ± 1.41 d424.60 ± 59.40 c
SA6.59 ± 1.29 bc14.47 ± 2.47 bc1458.44 ± 274.45 b
BS6.27 ± 1.09 bc10.29 ± 0.71 d1620.73 ± 107.55 ab
RP12.16 ± 1.75 a22.07 ± 3.31 a1869.22 ± 201.88 a
SC8.41 ± 1.08 b17.57 ± 2.56 b1386.38 ± 255.78 b
LP5.40 ± 1.38 cd11.03 ± 2.65 cd712.85 ± 127.94 c
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 5. Effects of different strains on the concentration of AMPs in H. illucens.
Table 5. Effects of different strains on the concentration of AMPs in H. illucens.
ParameterAntibacterial Peptide (mg/mL)
Control0.03 ± 0.01 c
EC0.05 ± 0.01 ab
SA0.05 ± 0.01 b
BS0.04 ± 0.01 bc
RP0.06 ± 0.01 a
SC0.05 ± 0.01 ab
LP0.04 ± 0.01 bc
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 6. The diameter of the inhibition circles of each feed group.
Table 6. The diameter of the inhibition circles of each feed group.
ParameterDiameter (mm)
NC0
PC15.47 ± 0.96 a
Control8.07 ± 0.91 e
EC11.50 ± 1.30 bcd
SA11.53 ± 0.93 bc
BS9.10 ± 0.92 cde
RP12.80 ± 0.66 b
SC11.47 ± 0.95 bcd
LP8.93 ± 0.47 de
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 7. Effects of different concentrations of RP on the growth performance of H. illucens.
Table 7. Effects of different concentrations of RP on the growth performance of H. illucens.
ParameterWGR (%)SGR (%/d)
R0858.00 ± 50.63 c45.18 ± 1.07 c
R1992.22 ± 40.05 bc47.81 ± 0.73 bc
R21031.11 ± 40.73 b48.51 ± 0.73 b
R31067.78 ± 103.94 ab49.10 ± 1.82 ab
R41202.44 ± 13.83 a51.34 ± 0.21 a
R51213.33 ± 24.04 a51.50 ± 0.37 a
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 8. Effects of different concentrations of RP on crude protein, crude lipid and ash of H. illucens.
Table 8. Effects of different concentrations of RP on crude protein, crude lipid and ash of H. illucens.
ParameterCrude Protein (%)Crude Lipid (%)Ash (%)
R056.52 ± 2.1121.39 ± 1.57 c2.96 ± 0.14
R154.71 ± 2.3723.25 ± 0.56 bc2.93 ± 0.05
R254.10 ± 2.0724.93 ± 0.46 ab2.88 ± 0.05
R352.88 ± 1.1625.11 ± 0.60 ab2.92 ± 0.01
R454.05 ± 1.5426.19 ± 0.56 a3.22 ± 0.68
R553.44 ± 2.7826.59 ± 0.51 a2.83 ± 0.04
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 9. Effects of different concentrations of RP on the digestive ability of H. illucens.
Table 9. Effects of different concentrations of RP on the digestive ability of H. illucens.
Parameterα-Amylase (U/mgprot)Lipase (U/gprot)Trypsin (U/gprot)
R05.24 ± 0.46 b30.54 ± 5.06 c928.28 ± 39.35 c
R15.07 ± 0.75 b39.32 ± 6.14 bc1158.97 ± 79.30 bc
R27.40 ± 0.76 a41.07 ± 6.14 bc1258.97 ± 108.69 abc
R36.84 ± 0.79 a42.53 ± 4.19 b1224.84 ± 189.44 bc
R47.23 ± 0.95 a48.98 ± 9.57 ab1559.56 ± 268.55 a
R56.84 ± 1.08 a59.98 ± 7.41 a1464.06 ± 298.10 ab
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 10. Effects of different concentrations of RP on the concentration of AMPs in H. illucens.
Table 10. Effects of different concentrations of RP on the concentration of AMPs in H. illucens.
ParameterAntibacterial Peptide (mg/mL)
R00.04 ± 0.01 c
R10.07 ± 0.01 b
R20.07 ± 0.01 b
R30.09 ± 0.02 b
R4013 ± 0.01 a
R50.14 ± 0.02 a
Means in the same column with different superscripts are significantly different (p < 0.05).
Table 11. The diversity indices of bacteria communities on OTU levels.
Table 11. The diversity indices of bacteria communities on OTU levels.
GroupShannonSimpsonChaoAce
Control1.71 ± 0.11 b0.36 ± 0.04 a107.29 ± 5.87 a107.90 ± 8.21 b
RP-A1.83 ± 0.08 b0.34 ± 0.03 a107.43 ± 6.35 a109.71 ± 5.22 b
RP-B2.49 ± 0.23 a0.16 ± 0.06 b124.56 ± 9.16 a135.32 ± 11.39 a
Means in the same row with different superscripts are significantly different (p < 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Meng, Y.; Zhang, X.; Zhang, Z.; Li, J.; Zheng, P.; Li, J.; Xu, J.; Xian, J.; Lu, Y. Effects of Microorganisms on Growth Performance, Body Composition, Digestive Enzyme Activity, Intestinal Bacteria Flora and Antimicrobial Peptide (AMP) Content of Black Soldier Fly Larvae (Hermetia illucens). Animals 2023, 13, 2722. https://doi.org/10.3390/ani13172722

AMA Style

Meng Y, Zhang X, Zhang Z, Li J, Zheng P, Li J, Xu J, Xian J, Lu Y. Effects of Microorganisms on Growth Performance, Body Composition, Digestive Enzyme Activity, Intestinal Bacteria Flora and Antimicrobial Peptide (AMP) Content of Black Soldier Fly Larvae (Hermetia illucens). Animals. 2023; 13(17):2722. https://doi.org/10.3390/ani13172722

Chicago/Turabian Style

Meng, Yongqi, Xiuxia Zhang, Zelong Zhang, Jiajun Li, Peihua Zheng, Juntao Li, Jiarui Xu, Jianan Xian, and Yaopeng Lu. 2023. "Effects of Microorganisms on Growth Performance, Body Composition, Digestive Enzyme Activity, Intestinal Bacteria Flora and Antimicrobial Peptide (AMP) Content of Black Soldier Fly Larvae (Hermetia illucens)" Animals 13, no. 17: 2722. https://doi.org/10.3390/ani13172722

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop