May 17, 2018

Antimicrobial properties

Scientific papers regarding black soldier fly larvae antimicrobial properties:

The conducted feeding trials have shown the enormous potential of using Hermetia illucens in organic waste management. The material reduction of human faeces was 51.3 %, while the corresponding WBC was 9 %.
The inactivation of Salmonella Senftenberg and the two types of Salmonella Typhimurium (the widespread DT 178 and the antibiotic multiresistant DT 104) was found to be accelerated in the BSF larvae treatment, while the reduction in the concentration of S. Dublin was as rapid as in the control.

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named
cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein
with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gramnegative bacterium Escherichia coli, fungus Rhizoctonia solani KhuÈn (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens.

The black soldier y Hermetia illucens is used for the bioconversion of organic waste into feed for livestock and aquaculture, and is economically among the most important farmed insects in the world. The larvae can be fed on agricultural waste and even liquid manure, resulting in highly unpredictable pathogen levels and dietary conditions. Here we show that H. illucens larvae express a remarkably
expanded spectrum of antimicrobial peptides (AMPs), many of which are induced by feeding on a diet containing high bacterial loads. The addition of sulfonated lignin, cellulose, chitin, brewer’s grains or sunower oil revealed the diet-dependent expression proles of AMPs in the larvae. The highest number of AMPs and the highest levels of AMP expression were induced by feeding larvae on diets supplemented
with protein or sunower oil. Strikingly, the diet-dependent expression of AMPs translated into diet dependent proles of inhibitory activities against a spectrum of bacteria, providing an intriguing example for the emerging eld of nutritional immunology. We postulate that the ne-tuned expression of the expanded AMP repertoire mediates the adaptation of the gut microbiota to the digestion of unusual diets, and this feature could facilitate the use of H. illucens for the bioconversion of organic waste.

The antibacterial effects of larval extract from Hermetia illucens, commonly known as the black soldier fly, have been demonstrated in vitro. In this study, gas chromatography–mass spectrometry analysis identified the active compound within this larval extract as hexanedioic acid. The antibacterial effects of hexanedioic acid were investigated in mice infected with Klebsiella pneumoniae. After administration of hexanedioic acid, infected mice showed decreased lung bacterial loads and lower rates of body weight loss compared to those in the infection‐only control group. Based on lung bacterial loads, oral hexanedioic acid treatment showed better protection than intraperitoneal treatment. Histopathology confirmed that daily administration of hexanedioic acid for 10 days showed zero toxicity to the kidneys or livers of mice. Therefore, hexanedioic acid may be a novel antibacterial agent.

Methicillin-resistant Staphylococcus aureus (MRSA), are the most frequent cause of sepsis, which urgently demanding new drugs for treating infection. Two homologous insect CSαβ peptides-DLP2 and DLP4 from Hermetia illucens were firstly expressed in Pichia pastoris, with the yields of 873.5 and 801.3 mg/l, respectively. DLP2 and DLP4 displayed potent antimicrobial activity against Gram-positive
bacteria especially MRSA and had greater potency, faster killing, and a longer postantibiotic effect than vancomycin. A 30-d serial passage of MRSA in the presence of DLP2/DLP4 failed to produce resistant mutants. Macromolecular synthesis showed that DLP2/DLP4 inhibited multi-macromolecular synthesis especially for RNA. Flow cytometry and electron microscopy results showed that the cell cycle was
arrested at R-phase; the cytoplasmic membrane and cell wall were broken by DLP2/DLP4; mesosomelike structures were observed in MRSA. At the doses of 3‒7.5 mg/kg DLP2 or DLP4, the survival of mice challenged with MRSA were 80‒100%. DLP2 and DLP4 reduced the bacterial translocation burden over 95% in spleen and kidneys; reduced serum pro-inflammatory cytokines levels; promoted
anti-inflammatory cytokines levels; and ameliorated lung and spleen injury. These data suggest that DLP2 and DLP4 may be excellent candidates for novel antimicrobial peptides against staphylococcal infections.

The antibacterial activity of the extracts of whole black soldier fly larvae (Hermetia illucens) was evaluated and was tested against six strains of plant pathogens. The extracts were prepared by homogenization after mixing grinded larvae with 0.01% acetic acid at 4 °C for 12 hr. Two methods, agar well diffusion and growth curve assay, were used to study the antibacterial activity against these pathogens. In our study, the antibacterial properties of the extracts were demonstrated by growth inhibition of all six tested bacterial pathogens. Inhibition zone assay and fluorescence assay confirmed that larval extracts have significant antibacterial activity against bacterial pathogens. The data provide further evidence that larval extracts play a role in the defense against microorganisms.

Eumelanin type pigments are synthesized at all the stages of the life cycle of the fly Hermetia illucens: in the larvae, pre-pupae, pupae and adult flies (dead flies). The greatest content of melanin was recorded in the cuticles. Melanin was present not only in the cuticle, hence it remained in the cuticle after the emergence of the adult fly. It was also found in the insect body in a complex with lipids. In pupae, it is mostly lauric acid that was associated with melanin. Its proportion in the melanin-chitosan complex was 80%. The isolated melanin-chitosan complex of adult flies showed a wide range of antibacterial activity, inhibiting the growth of 21 out of the 25 of the test cultures. The melanin-chitosan complex of empty pupal membranes and alcohol suspension of pupal melanin inhibited twice as smaller number of test cultures and the above activity was absolutely in the pupal chitosan. The largest zone of growth inhibition was recorded with respect to Aspergillus niger, Candida albicans, salmonella, and Staphylococcus aureus. An alcohol suspension of pupal melanin inhibited the growth of 10 test cultures. In this case the greatest activity was shown in relation to Mycobacterium B5 and Acinetobacter sp. 1182.

The aim of this study was to evaluate the effect of full-fat insect meals fed ‘on top’ to broiler chickens on their performance and the microbiota composition in the gastrointestinal tract. A total of 1850 day-old Ross 308 females were used in a set of four independent experiments. The insects Gryllodes sigillatus, Shelfordella lateralis, Gryllus assimilis, Tenebrio molitor and Hermetia illucens were applied in amounts that varied from 0.05 to 0.2%. In general, the application of insect meals to the diets of broilers did not affect their growth performance over the experimental period. However, the 0.2% additions of T. molitor and H. illucens increased feed intake at days 15–35 (P = 0.011) and the entire period of feeding (days 1–35; P = 0.018) (Experiment 3). Moreover, in Experiment 4 the supplementation of 0.2% of
S. lateralis improved body weight gain (days 11–21 and 1–21), feed intake (days 1–10 and 1–21) and feed conversion ratio (days 1–21). The addition of insect meals reduced the pH value of digesta in the crop (Experiments 1 and 2) and in the caeca (Experiment 2). Supplementation with H. illucens caused the most significant effect on the microbiota populations in the crop, ileum and caeca (Experiment 3). However, at the higher levels of S. lateralis addition to the diets of broilers, the counts of selected microbiota in the crop and ileum increased (Experiment 4). These results indicate that the application of the insect full-fat meals in relatively small amounts can affect the microbiota composition in the gastrointestinal tract of broiler chickens.

Larvae of black soldier fly, Hermetia illucens, are scavengers who live in extremely unpleasant and harsh environments, such as manure and compost, populated by bacteria and fungi. Carcasses of dead animals and rotting plants could be degraded by larvae of black soldier fly. These biological characteristics suggest that the larvae of black soldier fly are rich in antimicrobial peptides (AMPs) and other substances which possess the activity to fight against resistant bacterial strains. Wound healing is a complex process consisting of four overlapping but strictly defined phases: hemostasis, inflammatory phase, proliferative phase and the remodelling phase (maturation and epithelialization). All phases of wound healing are dependent on the secretion of a variety of cellular compounds such as growth factors, chemokines, cytokines, proteinases, and extracellular matrix proteins. In vitro process of wound healing could be monitored using these different cellular compounds as molecular markers. Focus of this research is enrichment of extracts from H. illucens larvae in order to obtain purified AMPs and other antibiotic compounds and in vitro monitoring of the impact of extracts from H. illucens larvae on bacterial growth and cytotoxicity of human cells. Extracts showed high capacity of inhibition of bacterial growth, especially species Pseudomonas fluorescens. Moreover, majority of extracts which inhibited bacterial growth did not show citotoxic effect on human cells.

Active antimicrobial peptide HI-3 was isolated and purified from the 5th instar larvae of Hermetia illucens L., and its effects on proliferation, apoptosis and migration of nasopharyngeal carcinoma (CNE2) cells were investigated. The expressions of telomerase reverse transcriptase (hTERT) in CNE2 cells were also studied in vitro to elucidate the mechanism involved in the action of HI-3 on CNE2 cells. Results showed that three fractions (HI-1, HI-2, HI-3) were isolated from the hemolymph of H. illucens larvae. After purified by RP-HPLC, only HI-3 showed the inhibitory activities to four strains of bacteria. It was also showed that HI-3 could effectively inhibit the proliferation of CNE2 cells in a dose- and time- dependent manner. Apoptosis of CNE2 cells was observed in the treatment with 160 μg/ml HI-3, and the early apoptosis rate up to 27.59%. However, no significantly inhibitory effects and apoptosis were found on human umbilical vein endothelial cells (HUV-C). Moreover, HI-3 could significantly reduce the migration ability of CNE2 cells when compared with that of the control. On the other hand, the levels of mRNA and protein of hTERT in the HI-3 treatment were all significantly lower than that of the control. Results indicated that HI-3 could inhibit the proliferation of CNE2 cells and induce the apoptosis of CNE2 cells by down-regulating the telomerase activity in CNE2 cells, while no obvious effect was occurred on HUV-C. It inferred that HI-3 is a potential anti-tumor drug with low toxicity to normal cells.

We investigate the effects of the immune function (HI titer) in broilers fed diets containing Hermetia illucens (H. illucens) peptide extract over a 40-day period. Twenty-four broiler chicks (Arbor Acres, 1 d old) were divided into four groups and fed different diets (control, 0.1%, 0.5%, and 1% H. illucens peptide extract). To evaluate HI titer, all broilers were vaccinated with H9H2 vaccine subcutaneously on the lateral thorax, according to the manufacturer’s recommendations. Similar HI titer was observed with 1% H. illucens peptide extract treatment compared to the control after 40 days (p>0.01). Groups fed 0.5% H. illucens peptide extract demonstrated the most effective immune effects (p<0.01), followed by groups fed 0.1% H. illucens peptide extract. In conclusion, using 0.1% or 0.5% H. illucens peptide extract before or after vaccination improved HI titer immune function in broilers.

To evaluate the effects of feeding a Hermetia illucens (HI) larvae meal on the different intestinal traits of hens, and to determine the toxic elements’ concentration in the insect meal and diets, 162 hens were randomly allotted to three groups. The control received a corn-soybean meal-based diet (SBM); the HI25 and HI50 groups received two diets in which the 25% and 50% of the dietary protein were replaced by the HI protein, respectively. The duodenal and jejunal villi height and villi/crypt were higher (p < 0.01) in the SBM than in the HI groups. The ileal villi height was higher (p < 0.05) in the SBM and HI25 groups than the HI50. The HI50 group exhibited a lower duodenal maltase activity. The intestinal alkaline phosphatase (IAP) activity linearly decreased in the duodenum and jejunum as the dietary insect meal inclusion increased. The HI50 group had a higher acetate and butyrate level than the SBM. The levels of cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As)in the diets and insect meal were lower than the maximum values established by the EU Commission. The 25% soybean protein replacement with Hermetia illucens larvae meal in the diet of laying hens was more suitable and closer to the optimal level than 50%.

Black soldier fly (BSF) larvae and pre-pupae could be satisfactorily raised on household organic waste and used as poultry feed, offering a potential sustainable way to recycle untapped resources of waste. The present study was conducted to determine if whole (non-defatted) BSF larvae and pre-pupae raised on experimental household waste could substitute soybean meal and oil as ingredients for laying hen diets. While no significant differences in feed intake and the egg-laying rate of hens were observed throughout the experiment, egg weight and eggshell thickness were greater in the pre-pupae-fed group than in the other groups. Moreover, although diversity of the cecal microbiota was significantly higher in the pre-pupae-fed than in the control group, no significant differences in bacterial genera known to cause food poisoning were observed when comparing the treatment groups. Nonetheless, Lactobacillus and Bifidobacterium populations were significantly lower in the treatment than in the control group. Fat content in BSF was possibly related with the changes in the cecal microbiota. Hence, since BSF fat was deficient in essential fatty acids, special attention should be paid to the fat content and its fatty acid composition in the case of regular inclusion of BSF larvae and pre-pupae oil as an ingredient in poultry diets.

This study evaluated the effects of dietary insect meal from Hermetia illucens larvae on autochthonous gut microbiota of rainbow trout (Oncorhynchus mykiss). Three diets, with increasing levels of insect meal inclusion (10%, 20%, and 30%) and a control diet without insect meal were
tested in a 12-week feeding trial. To analyze the resident intestinal microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME pipeline were used. The number of reads taxonomically classified according to the Greengenes database was 1,514,155. Seventy-four Operational Taxonomic Units (OTUs) at 97% identity were identified. The core of adhered intestinal microbiota, i.e., OTUs present in at least 80% of mucosal samples and shared regardless of the diet, was constituted by three OTUs assigned to Propiobacterinae, Shewanella, and Mycoplasma genera, respectively. Fish fed the insect-based diets showed higher bacterial diversity with a reduction in Proteobacteria in comparison to fish fed the fishmeal diet. Insect-meal inclusion in the diet increased the gut abundance of Mycoplasma, which was attributed the ability to produce lactic and acetic acid as final products of its fermentation. We believe that the observed variations on the autochthonous intestinal microbiota composition of trout are principally due to the prebiotic properties of fermentable chitin.

The objective of this experiment was to test the effects of supplementation of defatted black soldier fly (Hermetia illucens) larvae (BSFL) meal in beagle dogs. A total of nine healthy female beagles (initial body weight 12.1±1.76 kg) were fed grain-based diets with three levels of BSFL meal (0, 1% or 2%) in a 42-day feeding trial. At the end of week 6 of the experiment, all dogs were intraperitoneally challenged with Escherichia coli lipopolysaccharide (LPS) at 100 μg/kg of body weight. Albumin concentration was linearly increased as increasing BSFL meal level (P<0.05). A linear increase (P<0.05) in calcium concentration was observed when increasing dietary BSFL meal. Although dietary treatments did not affect the digestibility of ether extract, the digestibility of dry matter and crude protein were linearly increased as increasing the level of BSFL meal. The concentration of tumor necrosis factor-α was linearly decreased but glutathione peroxidase (GPx) concentration was linearly increased when increasing the level of BSFL meal at 6 h after challenge (P<0.05). In addition, there were quadratic increases in concentrations of GPx and superoxide dismutase with increasing dietary BSFL meal level at 3 h after challenge (P<0.05). These findings from the present study demonstrate that BSFL meal can be supplemented in the diet to convert beneficial effects to beagle dogs indicated as improved digestibility of dry matter and crude protein and antiinflammatory and anti-oxidative capacity.

Background: Insects, such as Hermetia illucens larvae, are rich in chitin and proteins, and represent a suitable feed ingredient replacement for animals. However, little is known about the effect of administering H. Illucens larvae on intestinal microbiota, bacterial metabolite profiles, and mucosal immune status in animals. This study aimed to investigate the effects of administering H. illucens larvae on colonic microbiota and bacterial metabolites production in finishing pigs. Seventy-two crossbred (Duroc × Landrace × Large White) female pigs (initial body weight, 76.0 ± 0.
52 kg) were randomly allocated to three different dietary treatments: a control diet (Control group) and two diets corresponding to 4% (H1 group) and 8% (H2 group) H. illucens larvae inclusion levels, respectively. Each treatment consisted of eight pens (replicates), with three pigs per pen. After 46 days of feeding, eight pigs per treatment (n = 8) were slaughtered, and the colonic digesta and mucosa were collected for microbial composition and microbial fermentation products, and genes expression analyses.

Results: The results showed that the H1 diet significantly increased the abundance of Lactobacillus, Pseudobutyrivibrio, Roseburia, and Faecalibacterium compared with those in the control group (P < 0.05), with a decrease in the abundance of Streptococcus. The numbers of Lactobacillus, Roseburia, and Clostridium cluster XIVa were significantly greater in the H1 group than in the control group (P < 0.05). Meanwhile, H2 diet increased the number of Clostridium cluster XIVa compared with the control group (P < 0.05). For colonic metabolites, total short chain fatty acids, butyrate, and isobutyrate concentrations were significantly higher in the H1 group than those in the control group (P < 0.05); the H1 treatment caused a striking decrease in protein fermentation compared with the control group, as the concentrations of total amines, cadaverine, tryptamine, phenol, p-cresol, and skatole were significantly lower (P < 0.05). Additionally, H2 diet also increased butyrate concentration compared with control group (P < 0.05), while decreased the concentrations of phenol, p-cresol, and skatole (P < 0.05). Pigs in the H1 group down-regulated the expression of TLR-4 and pro-inflammatory cytokines (IFN-γ) compared with pigs in the control group (P < 0.05), and up-regulated anti-inflammatory cytokine (IL-10) and intestinal barrier genes (ZO-1, occludin, and mucin-1). H2 diet up-regulated the expression of ZO-1 compared with control group (P < 0.05). Furthermore, the changes in the colonic mucosal gene expression were associated with changes in the bacterial composition and their metabolites.

Conclusions: Collectively, dietary inclusion of Hermetia illucens larvae may enhance mucosal immune homeostasis of pigs via altering bacterial composition and their metabolites. These findings provide a new perspective on insect meal as a sustainable protein source rich in nutrient ingredients for swine.

The gut microbiota of insects contains a wide range of organisms that protect them against the attack of pathogens by releasing various types of bioactive compounds. In the present study, we report the isolation and identification of the fungus Chrysosporium multifidum as a component of the microbiota from the larval gut of Hermetia illucens. Extract from the broth culture of C. multifidum showed moderate activity on a strain of methicillin resistant Staphylococcus aureus (MRSA). The bioguided isolation of the extract resulted in the characterization of six α-pyrone derivatives (1-6) and one diketopiperazines (7), among them 5,6-dihydro-4-methoxy-6-(1-oxopentyl)-2H-pyran-2-one (4) showed the best activity (IC50 = 11.4 ± 0.7 µg/ml and MIC = 62.5 μg/ml).

The aim of this study was to evaluate the in vitro antibacterial activity of
Black soldier fly (BSF) larva extract. The BSF larva was extracted using methanol and then tested for antibacterial activity using agar diffusion method (zone growth inhibition). The antibacterial activity was conducted against Salmonella sp. and Escherichia coli, two important bacterial strains
in poultry, using six dilution levels (10 mg/ml, 20 mg/ml, 40 mg/ml, 80 mg/ml, 160 mg/ml and 320 mg/ml). All the results were subjected analyze using t-test method. Based on the diameter of the inhibition zone, the BSF larva extract has a strong (P<0.05) antibacterial activity against Salmonella sp. and E. coli when the concentration used 320 mg/ml. In addition, BSF larva extract also contain high amount of lauric acid (49.18%), a saturated fatty acid that has been proven to proposes as antibacterial agent. Therefore, it could be concluded that the BSF larva extract could be used as a candidate for antibacterial substances.

In our study, a novel bioactive polysaccharide was identified in the larvae of the black soldier fly (BSF) (Hermetia illucens) as a molecule that activates the mammalian innate immune response. We attempted to isolate this molecule, which was named dipterose‐BSF, by gel‐filtration and anion‐exchange chromatography, followed by nitric oxide (NO) production in mouse RAW264.7 macrophage cells as a marker of immunomodulatory activity. Dipterose‐BSF had an average molecular weight of 1.47 × 105 and consisted of ten monosaccharides. Furthermore, in vitro assays demonstrated that dipterose‐BSF enhanced the expression of proinflammatory cytokines and interferon β (IFNβ) in RAW264.7 cells. The inhibition of Toll‐like receptor 2 (TLR2) and 4 (TLR4) significantly attenuated NO production by dipterose‐BSF, indicating that dipterose‐BSF stimulates the induction of various cytokines in macrophages via the TLR signaling pathway. This observation was analogous with the activation of nuclear factor kappa B in RAW264.7 cells after exposure to dipterose‐BSF. Our results suggest that dipterose‐BSF has immunomodulatory potential through activating the host innate immune system, which allows it to be a novel immunomodulator for implementation as a functional food supplement in poultry, livestock, and farmed fish.

This study investigated the efects of replacement of fshmeal (FM) with poultry by-product (PBM) protein, supplemented with black soldier fy, Hermetia illucens (HI) larvae on growth, histomormhology, immunity and resistance to Vibrio harveyi in juvenile barramundi. Two hundred and twenty five barramundi averaging 3.51±0.03g were randomly allocated into three groups and fed isonitrogenous and isocalorifc diets containing diferent levels of PBM supplemented with HI as follows: Control (FM based diet), 45PBM+HI (45% PBM supplemented with 10% HI), and 90PBM+HI (90% PBM supplemented with 10% HI) for 6 weeks. Results showed that dietary inclusion of 45PBM+HI signifcantly improved the growth performance than control whereas growth inhibition occurred in the
90PBM+HI. The 45PBM+HI groups demonstrated signifcant increases in histometric measurements (villus and enterocyte width, and microvilli height) and acidic mucins. The impaired growth in 90PBM+HI groups was further associated with multifocal necrosis in the liver, an upregulation of the stress related genes (HSP70 and HSP90) and increase in the levels of liver enzymes. When 45PBM+HI was fed, survival against V. harveyi increased signifcantly and also an increase in serum immunity and immune-related genes in the head kidney was observed after infection.

Extensive use of antibiotics has caused the microbial resistance to rise drastically within the last few decades, and new approaches are therefore needed to develop effective antibacterial substances. In this study, we
identified peptide in the hemolymph of Hermetia illucens larvae using reverse-phase chromatography, HPLC and Nano-LC-ESI-MS/MS system. We investigated the antibacterial effect of HP/F9 peptides against Klebsiella
pneumoniae in vitro and in vivo. The peptide effectively inhibited the growth of K. pneumoniae in vitro and completely removed K. pneumoniae from the lungs of mice. Importantly, peptides (22,000 Da, HP/F9) successfully reduced lung inflammation upon K. pneumoniae infection. These results indicate that the HP/F9 peptide from H. illucens larva can effectively protect the mouse from K. pneumoniae infection. HP/F9 could be a new candidate for the development of effective antibacterial substance.

In the present research, bacterial diversity was studied during a 6-month feeding trial utilizing zebrafish (Danio rerio) fed Hermetia illucens reared on different substrates with an emphasis on fish gut bacterial diversity. A polyphasic approach based on viable counting, PCR-DGGE and metagenomic 16S rRNA gene amplicon target sequencing was applied. Two different H. illucens groups were reared on coffee by-products (C) or a mixture of vegetables (S). Viable counts showed a wide variability based on substrate. PCR-DGGE and Illumina sequencing allowed the major and minor bacterial taxa to be detected. Both samples of larvae and their frass reared on the S substrate showed the highest richness and evenness of bacterial communities, whereas zebrafish (ZHC) fed H. illucens reared on substrate
C and zebrafish (ZHS) fed H. illucens reared on substrate S had the lowest bacterial richness and evenness. A stimulating effect of bioactive compounds from coffee by-products on the occurrence of Lactobacillaceae and Leuconostoccaceae in H. illucens reared on substrate C has been hypothesized. Zebrafish gut samples originating from the two feeding trials
showed complex microbial patterns in which Actinobacteria and Alteromonadales were always detected, irrespective of the diet used. Enterobacteriaceae in fish guts were more abundant in ZHS than in ZHC, thus suggesting an influence of the bioactive compounds (chlorogenic and caffeic acids) in the substrate on Enterobacteriaceae in fish guts. ZHC
showed a higher abundance of Clostridia than did ZHS, which was likely explained by stimulating activity on the bacteria in this class by the bioactive compounds contained in H. illucens reared on substrate C. An influence of the microbiota of H. illucens or insect-derived bioactive compounds on the gut microbiota of zebrafish has been suggested. The presence of bacteria consistently associated with zebrafish guts has been found irrespective of the diet, thus attesting to the likely stability of the core fish microbiota.

1. The aim of the present study was to investigate the effect of partial (50%) or total replacement of soybean oil (SO) by black soldier fly larvae (BSFL) fat on the growth performance, coefficients of apparent nutrient digestibility, selected internal organ weights and length, pancreatic enzyme activity and gastrointestinal tract (GIT) microecology modulation, as well as microbiota activity, physiological and immunological responses in young turkey poults.2. A total of 216, seven day old female turkeys (B.U.T 6) were randomly distributed to three dietary treatments using six replicate pens per group with 12 birds per pen. The following design of the trial was applied: SO 100% soybean oil; BSFL50 a 50/50 combination of SO and BSFL fat; or 100% BSFL fat (total replacement of SO).3. The use of BSFL fat did not affect the growth performance, nutrient digestibility, GIT morphology, or quality of the breast and thigh muscles. However, reduced trypsin activity was noticed in the BSFL100 group, but this had no effect on digestibility. Total replacement of SO reduced proliferation of potentially pathogenic bacteria, i.e., Enterobacteriaceae spp., as well as decreasing levels of IL-6, while partial substitution lowered the TNF-α concentration.4. The replacement of commonly used SO by BSFL fat can be successfully applied in young turkey poult nutrition. BSFL fat may be considered an antimicrobial agent and support immune responses.

This study was conducted to investigate whether dietary supplementation of black soldier fly (Hermetia illucens Linnaeus) pulp (BSFP) affects growth performance, antioxidant capacity and intestinal health of juvenile mirror carp (Cyprinus carpio var. specularis). A total of 270 juvenile mirror carp (13.68 ± 0.02 g) were randomly allotted to five dietary treatments, BSFP0, BSFP25, BSFP50, BSFP75 and BSFP100, in which BSFP was included in the basal diet at 0, 43.7, 87.3, 131 and 174.7 g/kg, respectively. Then, fish were fed to apparent satiation for 8 weeks. Fish growth performance and nutrient utilization were not different among the five groups (p > .05). Increasing BSFP dietary content significantly decreased whole‐body lipid content but increased kidney index (p < .05). The general relative intestine length was significantly higher in the BSFP100 group than the BSFP0 group (p < .05). Increasing BSFP dietary content significantly increased serum catalase activity and decreased malonaldehyde content (p < .05). The intestinal villus height, villus area and muscle layer thickness were significantly lower in the BSFP100 group than the BSFP0 group (p < .05). No significant differences in the activity of intestinal trypsin, lipase and amylase were observed among all groups (p > .05). In conclusion, this study indicated that BSFP at the level below 131 g/kg could be added in diet of juvenile mirror carp without any negative effect on growth performance and intestinal health. Dietary BSFP inclusion at low levels decreased the whole‐body lipid content and increased the antioxidant activity of juvenile mirror carp.

The larvae of black soldier fly/BSF (Hermetia illucens) seems to be a considerable material as an alternative to antibiotic growth promoters (AGPs) since they are rich in antimicrobial peptides (AMPs) and lauric acid that can improve the health and immune response. This study aimed to evaluate the effect of BSF larvae meal as a replacement of fish meal on production performances, health status, and immune response of quails. The experimental design using completely randomized design consisted of 3 treatments of dose of BSF larvae meal i.e., R0: ration without supplementation of BSF larvae meal; R1: ration supplemented with 6.57% BSF larvae meal to substitute 50% fish meal, and R2: ration supplemented with 13.15% BSF larvae meal to substitute 100% fish meal. The parameters observed were production performances, hematological profile, macrophage phagocytic activity and capacity, and antibody titer against the avian influenza virus. A completely randomized design was arranged with 3 treatments and 5 replications and data collected were analyzed by using one-way analysis of variance (ANOVA). The results showed that ration supplemented with 13.15% BSF larvae meal demonstrated significant effects on the enhancement of egg production and egg mass (p<0.05). Based on hematological profile, the health status of experimental quails was unaltered by the treatments, but immune response of quails fed ration supplemented with 13.15% BSF larvae meal was found to be significantly increased as revealed by the increased average macrophage phagocytic activity and capacity, as well as a higher antibody titer against avian influenza virus (p<0.05). It is concluded that black soldier fly (BSF) larvae meal can be used as an alternative substitution to fish meal up to 13.15%, based on the improvement of immune response and the increase in egg production of quails.

Hermetia illucens larvae meal (HILM) are rich in proteins and chitin, and represent an innovative feed ingredient for animals. However, little is known about the intestinal bacteria and immune homeostasis response of HILM as a fishmeal replacement on weanling piglets. Thus, this study aimed to investigate the changes in specific ileal and cecal bacterial populations and their metabolic profiles, and ileal immune indexes in weanling piglets fed with a diet containing HILM. A total of 128 weanling piglets were fed either a basal diet or 1 of 3 diets with 1%, 2%, and 4% HILM (HI0, HI1, HI2, and HI4, respectively). Each group consisted of 8 pens (replicates), with 4 pigs per pen. After 28 d of feeding, 8 barrows per treatment were euthanized, the ileal and cecal digesta, and ileal mucosa were collected for analyzing bacterial population and metabolic profiles, and immune indexes, respectively. Results showed that HILM increased (P < 0.05, maximum in HI2) the number of Lactobacillus and Bifidobacterium in the ileum and cecum, but quadratically decreased (P < 0.05, minimum in HI2) the number of Escherichia coli. In the cecum, the number of Firmicutes, RuminococcusClostridium cluster IV, and Prevotella showed a quadratic response to increasing (P < 0.05, maximum in HI2) HILM levels. Lactate and butyrate concentrations in the ileum and cecum were quadratically increased (P < 0.05, maximum in HI2) with increasing HILM levels. In the cecum, the amines, phenol, and indole compounds concentrations were quadratically decreased (P < 0.05, minimum in HI2) with increasing HILM levels, while total short-chain fatty acids and acetate concentrations were quadratically increased (P < 0.05, maximum in HI2). In the ileum, the TLR4NF-κBMyD88, and TNF-α mRNA expressions were quadratically decreased (P < 0.05, minimum in HI2) with increasing HILM levels, while the mRNA expression of IL-10, barrier function (MUC1ZO-1Occludin, and Claudin-2), and development-related genes (IGF-1GLP-2, and EGF) was quadratically increased (P < 0.05, maximum in HI2). Furthermore, the changes in the mucosal gene expression were associated with changes in the bacterial populations and their metabolites. Collectively, these results showed that a diet supplemented with 2% HILM affected specific bacterial populations and metabolic profiles, and maintained ileal immune status. These findings provide new insights into the use of insect meal as a suitable alternative protein source for swine feeding.

This study was undertaken to determine the effects of feed supplementation with Hermetia illucens (HI) on the fecal score, blood profiles, immune response, and small intestinal morphology in weaned pigs. A total of 24 weaned pigs (Landrace×Yorkshire×Duroc; 5.86±0.13 kg) were randomly allotted to 4 treatments and 6 replicates on the basis of initial body weight. The experiment was accomplished over 0-14 days. The dietary treatments included a corn-soybean meal diet supplemented with 0, 1, 2, and 3% HI. A linear response to increasing dietary HI was observed for the number of monocytes (p<0.01) and eosinophils (p<0.05), whereas red blood cells tended to decrease with increasing HI levels. Plasma TNF-α levels were also determined to linearly decrease with HI supplementation (p=0.07). Moreover, a linearly decreasing tendency (p=0.06) was observed in the fecal score with increasing dietary levels of HI. Weaned pigs fed diets supplemented with increasing dietary concentrations of HI showed linearly improved (p<0.05) duodenal villus height during the study period. Taken together, these results indicate the beneficial effects of HI on diarrhea reduction, immune response, and small intestinal morphology in weaned pigs.

This research was conducted to determine the antibacterial activity of BSF extract invitro on the growth of Salmonella typhimurium, E. coli and Pseudomonas aureginosa. The experiment was carried out according to the Completely Randomized Design (CRD) consisting of six treatments and three replications for each treatment. The treatments were different concentration levels of BSF extract, i.e. 75, 125, 175, 225, 275 and 325 Chloramphenicol with concentration of 30 µg.discs paper-1 was used as a positive control and dimethyl sulfoxide (DMSO) as a negative control. BSF extract was made using maceration extraction method. The results of this study indicated that the antibacterial activity of BSF extract increased (P<0.05) in line with the increase level of BSF extract concentration. The average diameter of the inhibition zone for Salmonella typhimurium, E. coli and Pseudomonas aureginosa was 11.77 ± 0.03 mm, 11.15 ± 0.05 mm, and 11.15 ± 0.23 mm respectively, which was categorized as strong inhibition zone. In conclusion, the concentration of BSF extract of 325 is an effective concentration to inhibit the growth of the bacteria Salmonella typhimurium, E. coli and Pseudomonas aureginosa.

The rapid increase of plant diseases caused by bacterial phytopathogens calls for an urgent search for new antibacterials. Antimicrobial compounds of natural origin stand up as frontiers in the attempts of the antibiotic overuse replacement. With this in mind, the Hermetia illucens (H. illucens) larvae have recently gained attention as a promising approach to fulfill this need. This study aimed to isolate the active constituents of H. illucens larvae fat and to estimate its antimicrobial capacity. We discovered the best composition of extracting solution retaining the pronounced antimicrobial activity of the extract. Using gas chromatography-mass spectrometry (GC-MS), we identified the unique natural array of fatty acids as the major constituents of the acidified water-methanol extract (AWME) as having new antimicrobial potency. In standard turbidimetric assay, the minimum inhibitory concentration (MIC) of the AWME was 0.78 mg/mL after 24 h of incubation for all five tested phytopathogenic bacteria strains: Pantoea agglomerans, Xanthomonas campestris, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium atrosepticum, and Dickeya solani. The minimum bactericidal concentration (MBC) ranged from 0.78 to 1.56 mg/mL against all tested strains after 24 h of incubation. The inhibition zone size of AWME (INZ) at 50 mg/mL concentration was in the range 12.2 ± 0.56 to 19.0 ± 0.28 mm, while zone size for the positive control (penicillin-streptomycin) (5000 IU/mL–5000 µg/mL) was in the scale of 20.63 ± 0.53 to 24.0 ± 0.35 mm as revealed by standard disk diffusion assay. For the first time, our findings indicated the substantial antibacterial potential of AWME of H. illucens larvae fat against these actual phytopathogens, thus paving the way for further research to determine the mechanism of action in crop protection.

This thesis investigates the use of black soldier fly larvae meal (BSFLM) as a novel protein source for inclusion in swine diets including the standardized ileal digestibility (SID) of amino acids (AA) and net energy (NE) contents of BSFLM when fed to growing pigs and the effect of BSFLM on growth performance, gastrointestinal tract development, and immune system robustness when partially replacing animal proteins in nursery pig diets. Both full fat (FF) and defatted (DF) BSFLM had high SID for most AA, although provided less digestible protein overall compared to soybean meal. The FF BSFLM was a better source of available energy. When FF BSFLM was supplemented into nursery diets at an inclusion level of up to 50% of the animal protein content, it supported growth performance and indices of immune system robustness and gut health not different from conventional nursery diets containing in-feed antibiotics as growth promoters.

Antimicrobial peptides (AMPs) play a key role in the innate immunity, the first line of defense against bacteria, fungi, and viruses. AMPs are small molecules, ranging from 10 to 100 amino acid residues produced by all living organisms. Because of their wide biodiversity, insects are among the richest and most innovative sources for AMPs. In particular, the insect Hermetia illucens (Diptera: Stratiomyidae) shows an extraordinary ability to live in hostile environments, as it feeds on decaying substrates, which are rich in microbial colonies, and is one of the most promising sources for AMPs. The larvae and the combined adult male and female H. illucens transcriptomes were examined, and all the sequences, putatively encoding AMPs, were analysed with different machine learning-algorithms, such as the Support Vector Machine, the Discriminant Analysis, the Artificial Neural Network, and the Random Forest available on the CAMP database, in order to predict their antimicrobial activity. Moreover, the iACP tool, the AVPpred, and the Antifp servers were used to predict the anticancer, the antiviral, and the antifungal activities, respectively. The related physicochemical properties were evaluated with the Antimicrobial Peptide Database Calculator and Predictor. These analyses allowed to identify 57 putatively active peptides suitable for subsequent experimental validation studies.

The expressions of antimicrobial peptides (AMPs) in the larvae of the black soldier fly, Hermetia illucens, were significantly increased by pathogen or stimulant induced innate immunity activation. We immunized H. illucens fifth instar larvae with five different Lactobacillus species, that is, Lactobacillus acidophilus, L. brevis, L. casei, L. fermentum, or L. delbrueckii, to induce the mass production of AMPs and selected optimal immune inducers. Antimicrobial activities in hemolymph and H. illucens larvae (HIL) extract were evaluated against three salmonella species (Salmonella pullorum, Salmonella typhimurium, and Salmonella enteritidis). Highest antimicrobial activity was shown by the hemolymph of HIL immunized by L. casei and its activity was closely linked with the inductions of cecropin 1 (HiCec1) and defensin 1 (HiDef1) gene expressions. Furthermore, antimicrobial activity in hemolymph was stable to heat and pH and the growth of three Salmonella species were dramatically suppressed by HIL hemolymph and extract after immunization with L. casei. The minimal inhibitory concentration (MICs) of L. casei-immunized HIL extract against Staphylococcus aureus, Escherichia coli, and Salmonella species ranged from 100~200 µg/100 µL and no cytotoxicity to CaCo-2 and L929 cells were observed in the concentration range 100~40,000 µg/100 µL. Taken together, the present investigation demonstrates that L. casei-immunized HIL extract is a powerful natural antibiotic and preservative that can prevent contamination by Salmonella species.