Hexanedioic acid from Hermetia illucens larvae (Diptera: Stratiomyidae) protects mice against Klebsiella pneumoniae infection

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.     

黑水虻幼虫的耐饥力及饥饿幼虫复食后的生物学特性

【目的】旨在明确黑水虻Hermetia illucens幼虫的耐饥力及饥饿幼虫复食后的发育、生殖力等生物学特性,为评估饥饿对黑水虻后续发育的影响并指导生产实践提供依据。【方法】将不同日龄或不同体重黑水虻幼虫进行不同时长的饥饿处理,测定和分析其耐饥力与日龄、百头重的关系;对7日龄幼虫进行不同时长的饥饿处理后再复喂,测定不同饥饿时长对其预蛹出现时间、总预蛹率、预蛹重、成虫繁殖等的影响。【结果】黑水虻幼虫饥饿后的半数致死时间(LT₅₀)与日龄成指数关系,与体重呈线性相关。7日龄幼虫的耐饥力较强,LT₅₀超过了50 d,饥饿30 d时存活率仍然达到了95%。百头重小于0.5 g的个体,其LT₅₀小于30 d;百头重在2～2.5 g的个体,其LT₅₀在45～70 d。幼虫超过8日龄时或百头重超过4.5 g时,多数个体在饥饿5 d时就提前进入了后续发育阶段。7日龄幼虫饥饿40 d内复食,其预蛹率未受到影响,但预蛹重显著增加;短期饥饿(0～10 d)对其成虫产卵量无显著影响,长期饥饿(30～40 d)使其成虫产卵量显著下降;饥饿长达30 d以上时,卵孵化率也显著降低。【结论】本研究明确了不同体重或不同日龄的黑水虻幼虫都具有一定的耐饥力;饥饿幼虫复食后预蛹重显著增加。     

Novel antibacterial peptides induced by probiotics in Hermetia illucens (Diptera: Stratiomyidae) larvae

There is a need to discover new therapeutic substances due to the emergence of deadly infectious diseases and various antibiotic resistance. We focused on the larvae that are utilized as a medical insect for the treatment of skin damage in Europe and America. This study was to investigate the pharmacological activities of novel antibacterial peptides isolated from Hermetia illucens larvae against the Klebsiella pneumoniae and Shigella dysenteriae. The larvae were immunized by probiotics (Lactobacillus casei) for 24 h. The hemolymph from the immunized larvae was fractionated through reverse‐phase chromatography. Peptides were purified using HPLC and the coomassie blue staining, and identified using Nano‐LC‐ESI‐MS/MS system. Antibacterial activities of the peptides were evaluated by turbidometric assay, liquid broth dilution assay, resazurin assay, and agar disk diffusion method. The minimum inhibitory concentrations (MICs) of the peptides were measured as 150 μg/mL through the turbidometric, liquid broth dilution, and resazurin assays. The peptides effectively inhibited their growth/proliferation as well as the survival rate of the tested bacteria. Furthermore, the immunized larvae exhibited overexpression of the peptides compared to non‐immunized larvae. These results demonstrate that the peptides induced by H. illucens exert strong antibacterial activity against Gram‐negative bacteria. The results suggest that the activation of the humoral immunity induced by immunization functioned to enhance the production of antibacterial peptides from the insect and their antibacterial properties. This study indicates the potential of the peptides produced from larvae as antibacterial peptide substance for the development of novel antibacterial drugs.     

Detection of antimicrobial substances from larvae of the black soldier fly,Hermetia illucens (Diptera: Stratiomyidae)

Maggots have become highly successful in the treatment of non‐healing wounds and multidrug‐resistant pathogen infections. The main objective of this study was to extract antibacterial substances from larvae of the black soldier fly, Hermetia illucens. To induce immune responses, we septically injured the larvae with a contaminated needle. Lyophilized H. illucens larvae were homogenized and extracted with acidic methanol. We examined the antifungal and antibacterial effects of the low molecular weight antimicrobial factors within the larval extract on the growth of a broad range of microorganisms, including Gram‐positive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), and Gram‐negative Pseudomonas aeruginosa. Furthermore, we isolated the anti‐MRSA substances from the larval extract using high performance liquid chromatography. These investigations revealed that the larval extract possessed a broad‐spectrum of antibacterial activity, demonstrating that secretions of H. illucens larvae prove useful in the fight against MRSA and can potentially be a source of novel antibiotic‐like compounds for infection control.     

响应面法优化黑水虻幼虫蛋白质提取工艺

【目的】对黑水虻Hermetia illucens幼虫蛋白质进行不同提取方法的比较,选择最优提取方法,并确定其最优工艺参数,为黑水虻幼虫蛋白提取与资源利用提供依据。【方法】以黑水虻幼虫为原料,分别采用碱提法、酶提法、盐提法和Tris-HCl缓冲液提法对黑水虻幼虫蛋白质进行提取,并比较分析。通过单因素试验分别确定NaOH质量浓度、液料比、提取温度及提取时间4个因素的较优水平。在单因素试验结果基础上,按照Box-Behnken响应面试验设计进行响应面优化试验。【结果】提取黑水虻幼虫蛋白质的4种方法中碱提法的提取率最高,最佳提取条件为：NaOH质量浓度2.44 g/100mL,液料比22 mL/g,提取温度53℃,提取时间2 h。提取率验证试验结果为88.49%,与预测值相对误差为0.28%。【结论】响应面模型拟合度高,优化出的最佳提取工艺可行。     

黑水虻抗菌肽HI-3对RAW264.7细胞免疫调控作用研究

为了明确黑水虻Hermetia illucensL.抗菌肽HI-3对RAW264.7细胞的免疫调控作用,拓展黑水虻抗菌肽在免疫领域的应用潜能,本文研究了抗菌肽HI-3对RAW264.7细胞形态、增殖、吞噬功能、分泌NO和细胞因子以及抗氧化活性的影响。结果表明,20、40、80、160、320μg/mL的抗菌肽HI-3对RAW264.7细胞形态及增殖指数均无显著影响,但均能显著增强RAW264.7细胞的吞噬功能,且随着HI-3浓度增加吞噬能力逐渐增强,当HI-3浓度达到320μg/mL时RAW264.7细胞的吞噬能力最强(P<0.01);而在160μg/mL以下处理浓度时,对NO含量无显著性影响(P>0.05);抗菌肽HI-3可以上调RAW264.7细胞分泌IL-6、TNF-α和IL-1β,下调抑炎因子IL-10的分泌量,且具有一定的浓度依赖性,浓度达到320μg/mL时效果最为显著(P<0.01);另外,抗菌肽HI-3亦能够显著提高RAW264.7细胞超氧化物歧化酶(SOD)活力和总抗氧化能力(T-AOC)含量,尤其是320μg/mL时,效果最为明显(P<0.0...     

低含水量饲料对黑水虻生长发育的影响

【目的】了解低含水量饲料对黑水虻Hermetia illucens生长发育的影响。【方法】在27℃、RH 80%、光周期14L∶10D的饲养条件下,以含水量70%人工饲料饲养黑水虻为对照,比较饲料含水量降为30%和50%时黑水虻幼虫的存活率、幼虫体重、雌雄成虫体长、羽化率以及不同发育阶段持续时间等生物学参数与对照组的差异。【结果】30%和50%含水量饲料饲养幼虫平均体重与对照幼虫相比显著降低（P<0.05）,30%和50%含水量两个处理间体重亦有显著差异（P<0.05）。黑水虻幼虫不能在含水量30％的人工饲料中发育至预蛹,大部分幼虫至13 d时死亡。50%含水量饲料饲养幼虫比对照延迟5 d出现预蛹,滞后14 d结束预蛹,预蛹过程耗时18 d,比对照延长8 d;50%含水量饲料饲养雌雄虫比对照成虫体长显著缩短（P<0.05）,雌雄成虫羽化时间亦比对照延后5 d,雌雄成虫羽化历期均为18 d,比对照增加5 d;50%含水量饲料饲养黑水虻自卵孵化至蛹全部羽化为成虫所需时间比对照延长10.67 d,所需时间为55.67 d,而对照仅需45.00 d;但是50%含水量饲料饲养幼虫存活率和成虫羽化率与对照相比差异均不显著（P>0.05）。【结论】含水量低于70%的人工饲料不利于黑水虻的生长发育。     

黑水虻抗菌肽HI-3对人结肠癌HCT-8细胞 谷氨酰胺和谷氨酸代谢通路的影响

【目的】研究黑水虻Hermetia illucens抗菌肽HI-3对人结肠癌HCT-8细胞氨基酸代谢的影响,以丰富对其抑癌机理的认识。【方法】采用CCK-8法测定不同浓度(80, 160和320 μg/mL)抗菌肽HI-3对HCT-8细胞的抑制率;利用GC-MS进行HCT-8细胞代谢物测定,通过基于R软件的通路分析找出氨基酸含量差异最显著的氨基酸代谢通路并筛选出该通路靶标酶。320 μg/mL HI-3处理HCT-8细胞后,利用酶活性检测试剂盒测定靶标酶谷氨酰胺酶(GLS)活性;利用RT-qPCR和Western blot技术分别对HCT-8细胞的GLS基因进行mRNA及蛋白表达水平的测定;利用生化试剂盒和ELISA试剂盒检测HCT-8细胞内谷氨酰胺和谷氨酸代谢通路涉及的重要代谢物谷氨酰胺(Gln)、谷氨酸(Glu)、谷胱甘肽(GSH)、α-酮戊二酸(α-KG)和ATP含量的变化。【结果】浓度为80, 160和20 μg/mLHI-3对HCT-8细胞的抑制率分别为33.85%±3.50%, 46.26%±0.90%和55.53%±1.70%,且抑制率随HI 3浓度升高而增大。320 μg/mL HI-3处理对谷氨酰胺和谷氨酸代谢通路的影响最大,其中氨基酸代谢物含量与阴性对照组(0 μg/mL HI-3)相比差异最为显著;这一通路中的靶标酶GLS活性及其GLS的mRNA和蛋白表达水平均极显著低于阴性对照组;另外与此通路相关的重要代谢物Gln, Glu, GSH, α-KG和ATP含量与阴性对照组相比亦显著减少。【结论】浓度为320 μg/mL黑水虻抗菌肽HI-3对HCT-8细胞谷氨酰胺和谷氨酸代谢通路影响最为显著,并能通过阻碍该通路来显著抑制HCT-8细胞的增殖。     

黑水虻幼虫抗菌肽HI-3对结肠癌HCT-8细胞凋亡影响及其机制研究

黑水虻作为一种全球广泛分布的昆虫,因生长环境决定了其丰富的抗菌肽来源,目前针对黑水虻抗菌肽的研究已逐渐从抗菌转向了抗癌。本文研究黑水虻抗菌肽HI-3对HCT-8细胞凋亡影响,探索其可能的抑癌机制,旨在为后续黑水虻抗菌肽抗肿瘤应用的研究提供作用靶点。采用流式细胞仪检测抗菌肽HI-3对HCT-8细胞凋亡率、线粒体膜电位、钙离子及活性氧(Reactive oxygen species, ROS)水平变化的影响;酶标仪检测抗菌肽HI-3对半胱天冬酶(Caspase)-3和Caspase-9活力的影响;并通过荧光定量PCR技术和蛋白芯片技术检测细胞凋亡相关基因和蛋白表达情况。结果表明,抗菌肽HI-3可以诱导HCT-8细胞凋亡,且随处理浓度增加,凋亡率亦增加,当浓度达到400μg/mL时,HCT-8细胞凋亡率与阴性对照组及其他处理组相比均有显著性差异(P<0.05);HI-3处理后HCT-8细胞内线粒体膜电位、钙离子浓度及ROS水平均随处理浓度增加而升高,当浓度达到400μg/mL时达到最高,各处理浓度下3组数值与阴性对照组相比均存在显著差异(P<0.05);随着HI-3处理浓度增加H...     

Defensin‐like peptide3 from black solder fly: Identification,characterization, and key amino acids for anti‐Gram‐negative bacteria

An antibacterial peptide was isolated from a black soldier fly, Hermetia illucens. The molecular mass of this peptide was established as 4247.37 by matrix‐assisted laser desorption/ionization‐time of flight mass (MALD‐TOF MS) spectrometry. The amino acid sequence of the mature peptide was determined by N‐terminal sequencing using Edman degradation, combined with cDNA sequencing of the previously reported defensin‐like peptide (DLP) 3. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP3 had potent activity against Gram‐positive and negative bacteria, but DLP4 had only anti‐Gram‐positive activity as previously reported. Recombinant DLP3 and DLP4 were overexpressed in Escherichia coli, and antibacterial activities were identical to DLPs purified from H. illucens hemolyph. In silico analysis revealed that only six amino acid sequences were different between DLP3 and DLP4, but antibacterial activity against Gram‐negative bacteria differed. Therefore these amino acid variants may be key amino acids (Gly‐10, Val‐18, Met‐23, Arg‐25, Asp‐32, Arg‐40) related to killing Gram‐negative bacteria.     

Black soldier fly (Hermetia illucens) larvae oil as an alternative fat ingredient to soybean oil in laying hen diets

ObjectiveThe objective of this study was to determine whether dietary black soldier fly (Hermetia illucens, HI) larvae oil (HILO) could serve as an alternative fat source to soybean oil (SBO) in laying hen diets.MethodsWe randomly assigned 25-week-old Hy-line Brown laying hens (n = 144) to receive (n = 6 hens/group; eight replicates) a control or an experimental diet in which SBO was replaced with 50% (50HILO) or 100% HILO (100HILO).ResultsDietary HILO did not negatively affect body weight or productive performance during the study. The eggs also had similar quality parameters, proximate composition, and cholesterol levels. However, the yolk color index was significantly higher (p<0.01) in the 100HILO than in the other groups. Dietary HILO significantly altered the composition of fatty acids (FAs) in abdominal fat and eggs. Total saturated fatty acids (SFAs) and total polyunsaturated FAs (PUFAs) were significantly increased and decreased in the 50HILO and 100HILO groups, respectively, compared with those in the control group (p<0.001 and p<0.0001, respectively). Specifically, the medium-chain FAs lauric and myristic acids were remarkably increased in the abdominal fat of laying hens fed HILO (p<0.0001), whereas only myristic acid increased in eggs (p<0.0001). Undesirable heavy metal (aluminum, fluorine, arsenic, lead, mercury, and cadmium) concentrations were below permissible limits in eggs.ConclusionWe considered that HILO could be an alternative dietary fat to SBO for laying hens with maintained productive performance and good egg quality.     

Black soldier fly (Diptera: Stratiomyidae) larval heat generation and management

Mass production of black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), larvae results in massive heat generation, which impacts facility management, waste conversion, and larval production. We tested daily substrate temperatures with different population densities (i.e., 0, 500, 1000, 5000, and 10 000 larvae/pan), different population sizes (i.e., 166, 1000, and 10 000 larvae at a fixed feed ratio) and air temperatures (i.e., 20 and 30 °C) on various production parameters. Impacts of shifting larvae from 30 to 20 °C on either day 9 or 11 were also determined. Larval activity increased substrate temperatures significantly (i.e., at least 10 °C above air temperatures). Low air temperature favored growth with the higher population sizes while high temperature favored growth with low population sizes. The greatest average individual larval weights (e.g., 0.126 and 0.124 g) and feed conversion ratios (e.g., 1.92 and 2.08 g/g) were recorded for either 10 000 larvae reared at 20 °C or 100 larvae reared at 30 °C. Shifting temperatures from high (30 °C) to low (20 °C) in between (∼10-d-old larvae) impacted larval production weights (16% increases) and feed conversion ratios (increased 14%). Facilities should consider the impact of larval density, population size, and air temperature during black soldier fly mass production as these factors impact overall larval production.     

不同废弃物对黑水虻幼虫生长、营养组成及生产性能影响研究

本文以黑水虻Hermetia illucens为研究对象,探索不同废弃物对其幼虫生长、营养组成及生产性能的影响。结果表明,黑水虻幼虫可将餐厨垃圾（FW）、厨余垃圾（KW）、蔬菜废弃物（VW）、猪粪（PM）、牛粪（DM）、鸡粪（CM）等转化成优质蛋白质和脂肪。以FW和KW作为基质养殖10 d后,幼虫体长（19.0 mm、17.1 mm）与单头重（171.54 mg、136.82 mg）显著高于其它处理。虫粉粗蛋白含量受废弃物种类影响相对较小。不同废弃物处理虫粉均显示出良好氨基酸谱。尽管各处理虫粉总必需氨基酸含量低于鱼粉和豆粕,但FW、KW和CM处理虫粉缬氨酸含量高于鱼粉和豆粕,VW处理虫粉异亮氨酸和缬氨酸含量高于鱼粉和豆粕,PM处理虫粉组氨酸和缬氨酸含量高于鱼粉和豆粕。以FW和KW作为基质养殖10 d后,尽管虫粉不饱和脂肪酸和必需脂肪酸比例低于采食PM和DM的处理,但远高于其它处理的虫粉粗脂肪含量（42.13%、37.49%）,虫粉不饱和脂肪酸和必需脂肪酸含量也最高;料虫比（6.06、7.90）显著低于其它处理,幼虫转化率（59.40%、52.07%）显著高于其它处理;废弃物减量化率（89.53%、84.46%）仅低于VW处理（94.86%）。利用黑水虻幼虫处理FW和KW,幼虫生长性能和营养组成优于其它废弃物处理,且生产性能最好。     

Enhancement of fruit byproducts through bioconversion by Hermetia illucens (Diptera: Stratiomyidae)

Bioconversion is a biological process by which organic materials are converted into products with higher biological and commercial value. During its larval stage the black soldier fly Hermetia illucens is extremely voracious and can feed on a wide variety of organic materials. To study the impact of different fruit byproducts on the insect's growth, final larval biomass, substrate reduction, bioconversion parameters, and larval nutritional composition, 10 000 black soldier fly larvae (BSFL) were reared on 7.0 kg of one of three substrates (strawberry, tangerine, or orange) or on a standard diet as a control. The results highlight that BSFL can successfully feed and grow on each of these diets, though their development time, growth rate, and final biomass were differently impacted by the substrates, with strawberry being the most suitable. The lipid and protein contents of BSFL were similar among larvae fed on different substrates; however, major differences were detected in ash, micronutrient, fiber, fatty acid, and amino acid contents. Overall, the results indicate that fruit waste management through the BSFL bioconversion process represents a commercially promising resource for regional and national agrifood companies. Our study offers new perspectives for sustainable and environmentally friendly industrial development by which fruit byproducts or waste might be disposed of or unconventionally enhanced to create secondary products of high biological and economic value, including BSFL biomass as animal feed or, in perspective, as alternative protein source for human nutrition.     

Wohlfahrtiimonas larvae sp. nov., isolated from the larval gut of Hermetia illucens (Diptera: Stratiomyidae)

A novel, Gram-negative, facultative anaerobic, motile and short rod-shaped bacterium, strain KBL006^T was isolated from the larval gut of Hermetia illucens, Black soldier fly. The 16S rRNA gene sequence of strain KBL006^T showed 96.4 % similarity to that of Wohlfahrtiimonas chitiniclastica S5^T. Strain KBL006^T grew optimally at 30 °C, at pH 8.0 and in the presence of 1–2 % (w/v) NaCl. Oxidase activity and catalase activity were positive. The major fatty acids were C_18:1 ω7c, C_14:0, and C_16:0. The major respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol, and two phospholipids. The G+C content of the genomic DNA was 45.2 mol%. Based on these polyphasic data, strain KBL006^T is considered to represent a novel species in the genus Wohlfahrtiimonas, for which the name Wohlfahrtiimonas larvae sp. nov. is proposed. The type strain is KBL006^T (= KACC 16839^T = JCM 18424^T).     

Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae

To investigate the molecular scavenging capabilities of the larvae of Hermetia illucens, two serine proteases (SPs) were cloned and characterized. Multiple sequence alignments and phylogenetic tree analysis of the deduced amino acid sequences of Hi-SP1 and Hi-SP2 were suggested that Hi-SP1 may be a chymotrypsin- and Hi-SP2 may be a trypsin-like protease. Hi-SP1 and Hi-SP2 3-D homology models revealed that a catalytic triad, three disulfide bonds, and a substrate-binding pocket were highly conserved, as would be expected of a SP. E. coli expressed Hi-SP1 and Hi-SP2 showed chymotrypsin or trypsin activities, respectively. Hi-SP2 mRNAs were consistently expressed during larval development. In contrast, the expression of Hi-SP1 mRNA fluctuated between feeding and molting stages and disappeared at the pupal stages. These expression pattern differences suggest that Hi-SP1 may be a larval specific chymotrypsin-like protease involved with food digestion, while Hi-SP2 may be a trypsin-like protease with diverse functions at different stages.     

Bioconversion of poultry manure and vegetable waste mixes by Hermetia illucens (Diptera: Stratiomyidae)

Development time, survival and final nutrient content of Hermetia illucens L. larvae depends on the substrates in which they develop. Mixing different waste types together can increase the performance and survival of the larvae, as well as their waste reduction. The main objective of this study was to evaluate the effect of different ratios of mixed fruit and vegetable waste with poultry manure on larval development time, size, biomass production, survival, bioconversion and waste reduction. Freshly hatched neonates (90 mg; approx. 6000 individuals) were placed on 12 kg of a mixture of fruit and vegetable waste and fresh, unprocessed poultry manure and held at 28 ± 0.5°C. Inclusion of fruit and vegetable waste varied from 0% to 100% in 10% increments. Initial temperature of the substrate was also measured. The individual mass of larvae increased significantly as more fruit and vegetable waste was included, from less than 81.3 ± 6.6 mg on poultry manure only to an average size of 211.6 ± 6.0 mg at 100% fruit and vegetable waste. After approximately 60% inclusion of fruit and vegetable waste the performance and survival of the larvae increased significantly while development time was reduced. A combination of high fruit and vegetable waste and low initial temperatures resulted in lower development time overall. The mixing of wastes can be applied in industry to further the goals of waste reduction and biomass production while incorporating low-quality wastes like poultry manure.     

Biochemical characterization of digestive enzymes in the black soldier fly,Hermetia illucens (Diptera: Stratiomyidae)

The black soldier fly, Hermetia illucens, is beneficial because its larvae feed on organic materials derived from plants, animals and humans and promote the recycling of food waste and organic materials. We investigated the biochemical properties of digestive enzymes released from the salivary gland and gut of the black soldier fly. Because the gut extracts of the black soldier fly larvae had high amylase, lipase and protease activities, we suggested that the black soldier fly might belong to the polyphagous insect group. In addition, a strong trypsin-like protease activity was observed in the gut extracts of the black soldier fly larvae. Higher activities of leucine arylamidase, α-galactosidase, β-galactosidase, α-mannosidase and α-fucosidase were observed from the gut extracts of the black soldier fly larvae compared with those of house fly larvae. These findings may explain previous reports that the black soldier fly larvae can digest food wastes and organic materials more efficiently than any other known species of fly.