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.     

黑水虻幼虫蛋白及其酶解产物的抗氧化活性研究

为了探究黑水虻Hermetia illucens幼虫蛋白及酶解产物的抗氧化活性,以鲜活黑水虻幼虫冻虫为原料,采用碱提酸沉法提取黑水虻蛋白,通过碱性蛋白酶、菠萝蛋白酶、风味蛋白酶、木瓜蛋白酶对其蛋白质溶液进行酶解,分别从2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸（ABTS）自由基、羟自由基、1,1-二苯基-2-三硝基苯肼（DPPH）自由基3个方面对黑水虻蛋白及其酶解液的抗氧化能力进行测定。结果表明：黑水虻幼虫蛋白及4种酶解后的蛋白肽具有较好的抗氧化活性,其中黑水虻幼虫蛋白对ABTS自由基、羟自由基、DPPH自由基的半抑制浓度（IC50）分别为2.91、0.232、0.764 mg/mL,而酶解过后的蛋白肽具有更强的抗氧化活性,对ABTS自由基、羟自由基、DPPH自由基的最低半抑制浓度（IC50）分别为0.295、0.082、0.354 mg/mL。本研究初步证明了酶解制备抗氧化肽的可行性,为昆虫蛋白资源的利用和无抗饲料的研制提供了新的研究思路。     

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.     

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。     

Establishment of highly efficient transgenic system for black soldier fly (Hermetia illucens)

The black soldier fly (BSF), Hermetia illucens, is a promising insect for mitigating solid waste problems as its larvae are able to bioconvert organic waste into valuable biomass. We recently reported a high-quality genome assembly of the BSF; analysis of this genome sequence will further the understanding of insect biology and identify genes that can be manipulated to improve efficiency of bioconversion. To enable genetic manipulation of the BSF, we have established the first transgenic methods for this economically important insect. We cloned and identified the ubiquitous actin5C promoter (Hiactin5C-p3k) and 3 endogenous U6 promoters (HiU6:1, HiU6:2, and HiU6:3). The Hiactin5C promoter was used to drive expression of a hyperactive variant of the piggyBac transposase, which exhibited up to 6-fold improvement in transformation rate when compared to the wild-type transposase. Furthermore, we evaluated the 3 HiU6 promoters using this transgenic system. HiU6:1 and HiU6:2 promoters provided the highest knockdown efficiency with RNAi and are thus promising candidates for future Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) development. Overall, our findings provide valuable genetic engineering toolkits for basic research and genetic manipulation of the BSF.     

亮斑扁角水虻对有机废弃物的应用及其肠道微生物研究进展

有机废弃物的安全处理与二次利用是当今社会发展进程中的一大难题,亮斑扁角水虻Hermetia illucens强大的生物转化能力可有效解决此类问题：一方面,亮斑扁角水虻可大量消耗有机废弃物,有效治理环境污染;另一方面,转化后的虫体营养价值高,广泛应用于养殖领域。然而,亮斑扁角水虻的生物转化作用机制目前尚不明确,可转化的有机废弃物范围及其转化效率的影响因素仍有待探讨。本文结合近年来关于亮斑扁角水虻领域的研究,主要从亮斑扁角水虻对有机废弃物的生物转化作用、转化效率的影响因素、虫沙的应用和肠道微生物的功能4个方面进行综述,同时对未来需要解决的问题进行了展望,以期为亮斑扁角水虻的综合开发利用提供参考依据。     

Effects of food substrates and moxidectin on development of black soldier fly,Hermetia illucens

Black soldier fly is a common and widely distributed saprophagous species that has an excellent potential for being used for biological conversion of organic wastes on an industrial scale. The main goal of the reported study was expanding the list of wastes suitable for utilization by this species. We compared larval growth on cull potatoes, horse manure and cafeteria food waste in 100‐L bins in a greenhouse. We also conducted laboratory experiments to investigate whether black soldier fly larvae are affected by the presence of moxidectin, a common endectocide used to treat an array of domestic animals and readily excreted in faeces, in their food substrates. Feeding on potatoes resulted in slower growth, and the final size of potato‐fed larvae was smaller compared to the larvae fed on cafeteria waste. Nevertheless, potatoes supported substantial biomass accumulation, and could be a valuable option for rearing fly larvae for commercial feed production. Larvae feeding on horse manure gained very little weight and eventually failed to pupate. Moxidectin had a strong negative effect on larval survivorship; however, ca. 30% of larvae reared in the substrate containing a realistic field concentration of moxidectin still survived to adulthood. Our findings confirm that using black soldier fly larvae is a promising technology for recycling organic wastes, including those of plant origin.     

Microplastics affected black soldier fly (Hermetia illucens) pupation and short chain fatty acids

Microplastics (MPs) are microscopic particles that are now found in almost all ecosystems where they may exert adverse effects on various organisms, including insects. In this study, 20 black soldier fly (Hermetia illucens) larvae (BSFL) were exposed to MPs, in the form of polypropylene (PP-MPs; 55 ± 4 µm) at 0.22% (26,972,507 particles/kg), versus a control (no added MPs) in triplicate for two weeks. After two weeks, final lengths, weights, percentage of pupation, fatty acid composition of BSFL and substrate reduction percentage were measured. Survival, lengths/weights and percentage substrate reduction in the PP-MP group were not significantly different from the control. However, percentage of pupation was significantly lower in the PP-MP treatment (at 65.2%) compared to the control (at 83.8%). Among the measured fatty acids, only propionic and butyric acid changed, and these short-chain fatty acids (SCFAs) significantly increased from 0.12% and 0.17% to 0.14 and 0.19%, respectively, in the PP-MP-exposed group. This study indicates that exposure to environmentally relevant levels of PP-MPs can substantially delay development, while the increase in the SCFA may indicate a change in the gut microbiota.     

黑水虻幼虫和预蛹的饲料价值研究进展

黑水虻Hermetia illucens L.是一种全世界广泛分布的资源昆虫,幼虫在自然界以动物粪便、腐烂的有机物,如腐肉、腐烂的水果、蔬菜和植物性垃圾为食,能够被用于畜禽粪便的无害化处理。黑水虻幼虫在将粪便转化成昆虫生物量的同时,减少了粪便的污染。利用畜禽粪便饲养的黑水虻幼虫干重达到42%～43%,其中粗蛋白含量占干重的42%～44%,脂肪含量占干重的31%～35%,灰份占干重的11%～15%,钙质占干重的4.8%～5.1%,磷占干重的0.60%～0.63%。使用粪便饲养的幼虫和预蛹,含有丰富的必需氨基酸和矿物质,是家禽、家畜和鱼类养殖的良好饲料来源。文章综述利用不同动物粪便饲养的黑水虻幼虫和预蛹的饲料价值及其幼虫和预蛹用于鸡、猪和鱼类饲料添加剂的研究进展。     

Characteristics and nutrient function of intestinal bacterial communities in black soldier fly (Hermetia illucens L.) larvae in livestock manure conversion

The potential utility of black soldier fly larvae (BSFL) to convert animal waste into harvested protein or lipid sources for feeding animal or producing biodiesel provides a new strategy for agricultural waste management. In this study, the taxonomic structure and potential metabolic and nutrient functions of the intestinal bacterial communities of BSFL were investigated in chicken and swine manure conversion systems. Proteobacteria, Firmicutes and Bacteroidetes were the dominant phyla in the BSFL gut in both the swine and chicken manure systems. After the larvae were fed manure, the proportion of Proteobacteria in their gut significantly decreased, while that of Bacteroidetes remarkably increased. Compared with the original intestinal bacterial community, approximately 90 and 109 new genera were observed in the BSFL gut during chicken and swine manure conversion, and at least half of the initial intestinal genera found remained in the gut during manure conversion. This result may be due to the presence of specialized crypts or paunches that promote microbial persistence and bacteria–host interactions. Ten core genera were found in all 21 samples, and the top three phyla among all of the communities in terms of relative abundance were Proteobacteria, Firmicutes and Bacteroidetes. The nutrient elements (OM, TN, TP, TK and CF) of manure may partly affect the succession of gut bacterial communities with one another, while TN and CF are strongly positively correlated with the relative abundance of Providencia. Some bacterial taxa with the reported ability to synthesize amino acids, Rhizobiales, Burkholderia, Bacteroidales, etc., were also observed in the BSFL gut. Functional analysis based on genes showed that intestinal microbes potentially contribute to the nutrition of BSFL and the high-level amino acid metabolism may partly explain the biological mechanisms of protein accumulation in the BSFL body. These results are helpful in understanding the biological mechanisms of high-efficiency nutrient conversion in BSFL associated with intestinal microbes.     

黑水虻卵胚胎发育过程及在生产上的应用

黑水虻作为近年来广受关注的资源昆虫,其规模化应用的基础理论研究成为热点。本文对其胚胎发育过程进行研究,利用显微镜对资源昆虫黑水虻卵孵化发育过程进行观察并拍照,对胚胎各个时期的发育时间和形态进行记录。试验发现,在32℃下,黑水虻卵的发育历期为51 h。黑水虻的胚胎发育经过卵裂期、胚盘胚带形成期、原肠形成期、胚体分节期、体壁形成期、器官形成期6个典型时期。试验对孵化过程中的15个具体发育时期的特征和发育时间进行记录,形成了一套虫卵镜检技术应用于实际生产。在生产上,通过镜检技术对每一批次待孵化虫卵进行镜检观察,可初步判定该批次待孵化虫卵所处的发育阶段和死亡率,预估虫卵孵化时间和孵化率,为规模化稳定培育黑水虻苗种提供理论依据。     

Development,growth and metabolic rate of Hermetia illucens larvae

The larvae of Hermetia illucens are known to successfully bio‐convert a vast range of organic substrates into high protein and fat biomass, but little is known about the larval instars. During this research, larval head capsules and biomass growth were measured daily and the specific metabolic rate of larger instars were considered. The head capsule measurements revealed that H. illucens pass through 6 actively feeding larval stadia before entering the last nonfeeding but migrating 7th stadium. Larval growth follows a sigmoid curve with slowly accelerating growth in the earlier stadia and decelerating growth in the latest stadia. In contrast, development was fast until reaching stadium 6 and then slowed down. Accordingly, the specific metabolic rate was high in instars 3, 4 and 5 and reduced in instars 6 and 7.     

Isolation of Hermetia illucens larvae core gut microbiota by two different cultivation strategies

Hermetia illucens larvae (black soldier fly larvae, BSFL) convert efficiently organic waste to high quality biomass. To gain knowledge on the specific functions of gut microbes in this process it is a prerequisite to culture members of the core gut microbiota. Two different cultivation strategies were applied here for this purpose, a dilution-to-extinction cultivation and direct plating using six different media to culture aerobic heterotrophic bacteria. A total of 341 isolates were obtained by the dilution-to-extinction cultivation and 138 isolates by direct plating from guts of BSFL reared on chicken feed. Bacterial isolates were phylogenetically identified at the genus level by 16S rRNA gene sequencing (phylotyping) and differentiated at the strain level by genomic fingerprinting (genotyping). The main proportion of isolates was assigned to Proteobacteria, Firmicutes (Bacilli), and Actinobacteria. Predominant genera discussed in literature as member of a potential BSFL core gut microbiota, Providencia, Proteus, Morganella, Enterococcus, Bacillus, and members of the family Enterobacteriaceae, were isolated. A high intra-phylotype diversity was obtained by genomic fingerprinting which was especially enhanced by the dilution-to-extinction cultivation. This study showed that the application of different cultivation strategies including a dilution-to-extinction cultivation helps to culture a higher diversity of the BSFL gut microbiota and that genomic fingerprinting gives a better picture on the genetic diversity of cultured bacteria which cannot be covered by a 16S rRNA gene sequence based identification alone.

     

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.     

黑水虻幼虫肠道和体表产酶细菌的分离和鉴定

从野外收集和室内饲养的黑水虻Hermetia illucens L.幼虫体表和肠道分离出同时具蛋白质和有机磷分解能力的细菌10株,其中编号为T2、T4、T6、T7和T8的菌株分离自体表,编号为S14、S15、S16、S19和S20的菌株分离自肠道。克隆细菌的16S rDNA和DNA促旋酶B亚基编码基因gyrB对10株细菌进行了鉴定。通过16S rDNA序列确定10株菌为芽孢杆菌属,根据gyrB基因以及BLAST结果构建系统发育树,将10株细菌鉴定为枯草芽孢杆菌。然后将10株菌的gyrB基因以B.subtilis subsp.subtilisstr.168和模式菌株B.subtilis subsp.subtilis BCRC10255相应基因序列为参照构建系统发育树,分析10株菌在种的水平上的亲缘关系,发现10株菌存在亲缘关系差异,没有重复菌株。     

黑水虻消化道形态及组织结构的观察

本文比较了不同发育阶段黑水虻Hermetia illucens消化道的形态学差异,掌握了幼虫消化系统的组织学特征。利用体视镜观察黑水虻5龄幼虫、预蛹及成虫的消化道形态,利用光学显微镜和扫描电镜观察幼虫消化道各段(前肠、中肠、后肠)的显微及超微结构。结果表明：黑水虻幼虫及预蛹的消化道均由前肠(食道和前胃)、中肠及后肠组成,从幼虫到成虫,消化道的长度不断缩短。与幼虫和预蛹相比,成虫消化道形态变化明显,前胃消失,出现了嗉囊及胃盲囊,中肠进一步缩短,后肠分化为回肠、结肠和直肠。组织学观察结果显示,幼虫的唾液腺开口于口腔,由膨大的管状腺体和腺管组成。食道由特化为角质刺突的内膜层及发达的肌层组成,其末端延伸至前胃。前胃膨大为球状,包括三层组织结构。根据上皮细胞形态的差异,中肠可分为四个区段。后肠薄,肠腔内褶丰富,肠壁可见数量较多的杆状细菌。马氏管开口于中、后肠交界处,包括4支盲管,管内壁密布微绒毛。黑水虻消化道形态随发育阶段的变化,反映了各阶段摄食及消化生理的差异。幼虫消化道各段具有各自典型的组织学特征,其前、中、后肠可能分别承担了食物接纳与初步消化、消化与吸收以及重吸收功能。本研究结果为进一步了...     

Review of insect pathogen risks for the black soldier fly (Hermetia illucens) and guidelines for reliable production

Black soldier fly [BSF; Hermetia illucens L. (Diptera: Stratiomyidae)] larvae are very effective in transforming low-grade food waste into valuable high-end proteins and fat, in intensive production facilities. The production output of this species is growing quickly, but upscaling brings risks to the health status of the reared insects. Until now, not a single major case of disease outbreak caused by a pathogen in a BSF production unit has been reported. This contrasts with data on other species of mass-produced insects, which have experienced various disease outbreaks, indicating that BSFs are comparatively resistant to insect diseases. Further, there are no records of natural infections caused by entomopathogens in BSF. In this review, the known entomopathogens of Diptera, especially BSF, and their potential risks for causing disease in these insects are summarized.     

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。     

Effects of aeration rate and feed on growth,productivity and nutrient composition of black soldier fly (Hermetia illucens L.) larvae

Black soldier fly larvae (Hermetia illucens L.) are widely cultivated as bioconversion agents. These larvae are known to be highly sensitive to changes in the external environment, such as temperature and relative humidity. Therefore, a rearing hive equipped with an instrumentation system known as the Modular Fly Hive was designed to cultivate black soldier larvae. This study aimed to determine the effects of aeration rate (0 – 0.84 m³/s.) and feed type (coconut endosperm waste and soybean curd) on growth and productivity of black soldier fly cultivated in Modular Fly Hives as well as well as feed digestibility and nutrient composition of the larval biomass. Increasing aeration rate decreased the average temperature and relative humidity within the hives. The results showed that the larvae demonstrated a sigmoidal growth curve for all types of feed and the type of feed significantly affects the average wet weight and length of the larvae. The greatest dry biomass productivity (52.85 ± 3.85 g/m²/day) was observed when the larvae were cultivated using soybean curd residue with an aeration rate of 0.84 m³/s, while the lowest dry biomass productivity (35.27 ± 9.72 g/m²/day) was observed when the larvae were cultivated using coconut endosperm waste without aeration. Proximate analysis revealed that the larval biomass had total protein, lipid, ash and carbohydrate content in the range of 37.20 – 48.60%, 9.61 – 20.02%, 4.80 – 6.40%, 33.86 – 38.70%, respectively. Amino acid in the biomass were dominated by glutamic acid (11.11–12.30%), aspartic acid (8.25 – 10.35%), leucine (8.09 – 8.57%), and lysine (6.74 – 8.14%). Lipid isolated from the larval biomass were mainly composed of lauric acid (28.35–61.68%), linoleic acid (6.27 – 30.29%), palmitic acid (7.62 – 15.23%), and myristic acid (5.05 – 14.34%).     

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).