Morphofunctional characterization of hemocytes in black soldier fly larvae

In insects, the cell-mediated immune response involves an active role of hemocytes in phagocytosis, nodulation, and encapsulation. Although these processes have been well documented in multiple species belonging to different insect orders, information concerning the immune response, particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens, is still limited. This is a serious gap in knowledge given the high economic relevance of H. illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections. The present study represents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell-mediated immune response of this insect. In particular, in addition to prohemocytes, we identified five hemocyte types that mount the immune response in the larva, and analyzed their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes. These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte-like cells, developing a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph, respectively. These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes, which could thus trophically support other hemocytes during the immune response. Finally, the regulation of the cell-mediated immune response by eicosanoids was investigated.     

黑水虻幼虫及预蛹抗逆性的初步研究

本文初步研究了老熟和预蛹阶段的黑水虻Hermetiaillucens对于酒精毒性、氧胁迫和高渗透压条件下的耐受特性,结果显示黑水虻幼虫和预蛹在三种胁迫因子处于20%～60%(浓度)的较低水平时,其平均死亡率低于20%,表现出良好的抗逆性。高浓度的酒精、矿物油和氯化钠溶液对于黑水虻的生存都会产生负面影响,但是幼虫与预蛹由于其生理形态上的变化,致使其在抗逆性方面表现出显著差异,预蛹阶段的黑水虻对矿物油较为敏感,而幼虫则对高盐环境的耐受性表现较弱。     

黑水虻幼虫处理餐厨垃圾过程中体内养分组成与消化酶活性变化规律的研究

黑水虻Hermetia illucens作为一种新型资源环境昆虫,其幼虫可以处理餐厨垃圾、畜禽粪便、蔬菜残体等各种有机废弃物。幼虫富含蛋白质和油脂,可以作为水产饲料的蛋白来源。本研究探索黑水虻幼虫处理餐厨垃圾过程中其养分组成与消化酶活性变化之间的关系。通过黑水虻幼虫自由取食餐厨垃圾,每日采集样品用于物质养分和消化酶活性的测定。结果显示：黑水虻幼虫粗蛋白含量呈现先下降后上升的变化规律,而幼虫总糖含量呈现先上升后下降的变化规律,粗脂肪含量维持上升的趋势。幼虫处理餐厨垃圾过程中,体内蛋白酶在初期迅速上升,第6天后逐渐下降,而淀粉酶呈现出先缓慢上升再迅速上升最后下降的变化规律。脂肪酶在黑水虻幼虫处理餐厨垃圾前期保持较高的活性然后缓慢下降。同时,通过相关性分析,黑水虻幼虫粗蛋白含量变化与蛋白酶活性没有相关性,而总糖与粗脂肪含量变化分别与淀粉酶、脂肪酶有相关性。因此,部分消化酶活性变化与黑水虻幼虫养分组成具有一定的联系,本研究结果为工厂化养殖黑水虻提供一定的理论基础。     

Black soldier fly larvae show a stronger preference for manure than for a mass-rearing diet

The attention for black soldier fly larvae (BSFL) as an alternative ingredient for food and feed products is on the rise. While many studies have reported the efficiency of BSFL to bio-convert a wide range of organic waste streams into larval biomass, so far, it is unknown whether BSFL prefer certain waste streams over others when they have the possibility to choose. Here, we performed a choice-test experiment to explore the preference of BSFL when exposed to pig manure and a mass-rearing diet consisting of plant by-products currently used for industrial BSFL production. We found that after 1 hr of exposure to both feeds, BSFL strongly preferred pig manure over the mass-rearing diet. The preference for manure became stronger as larval age increased. Our results provide the first evidence that BSFL express a distinct diet preference. Understanding the reasons for the strong preference for manure is relevant for a diverse array of practical applications and to inform the discussion on insect welfare.     

武汉亮斑扁角水虻幼虫肠道微生物对其成虫产卵行为的影响

研究武汉亮斑扁角水虻幼虫肠道微生物对武汉亮斑水虻成虫产卵行为的影响。首先使用LB(Luria-Bertani)培养基对武汉亮斑水虻幼虫肠道微生物进行分离纯培养,筛选得到23株肠道微生物。以灭菌的LB液体培养基为阴性对照,使用已证实引诱产卵效果好的来自武汉亮斑水虻卵表的属于戈登氏菌属的细菌(Gordonia sihwensis)FE06为阳性对照,利用筛选到的武汉亮斑水虻幼虫肠道微生物摇瓶发酵液对武汉亮斑水虻成虫进行产卵行为实验。发现有5株武汉亮斑水虻幼虫肠道微生物(BSFLG01、BSFLG03、BSFLG04、BSFLG05、BSFLG06)对武汉亮斑水虻产卵有显著的积极促进作用,其中3株(BSFLG03、BSFLG05、BSFLG06)对水虻成虫产卵的引诱作用明显强于所设立的阳性对照FE06;菌株BSFLG07为一种毕赤酵母菌(Pichia kudriavzevii),对武汉亮斑水虻产卵有消极影响。经菌落培养特征、菌体形态比较、16SrDNA或18SrDNA序列测定和发育树比对分析,鉴定出这5种具有显著引诱武汉亮斑水虻产卵作用的微生物分别为:枯草芽胞杆菌(Bacillus subtilis)BSFLG01、一种沙雷氏菌(Serratiasp.)BSFLG03、库德毕赤酵母(Pichia kudriavzevii)BSFLG04、皱落假丝酵母(Candida rugosas)BSFLG05、粘质沙雷氏菌(Serratia marcescens)BSFLG06。     

Nutritional composition of black soldier fly larvae feeding on agro-industrial by-products

Black soldier fly (BSF) larvae, Hermetia illucens L. (Diptera: Stratiomyidae), bio-convert organic side streams into high-quality biomass, the composition of which largely depends on the side stream used. In the present study, BSF larvae were reared on feed substrates composed of dried brewers’ spent grains, each supplemented with either water, waste brewer’s yeast, or a mixture of waste brewer’s yeast and cane molasses to obtain 12 different substrates: barley/water, barley/yeast, barley/yeast/molasses, malted barley/water, malted barley/yeast, malted barley/yeast/molasses, malted corn/water, malted corn/yeast, malted corn/yeast/molasses, sorghum-barley/water, sorghum-barley/yeast, and sorghum-barley/yeast/molasses. The crude protein, fat, ash, and mineral contents of the BSF larvae fed each feed substrate were quantified by chemical analyses. The effect of substrate, supplementation, and their interaction on crude protein, fat, and ash contents of BSF larval body composition was significant. Calcium, phosphorus, and potassium were the most abundant macrominerals in the larvae and their concentrations differed significantly among substrates. These findings provide important information to support the use of BSF larval meal as potential new source of nutrient-rich and sustainable animal feed ingredients to substitute expensive and scarce protein sources such as fishmeal and soya bean meal.     

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。     

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.     

Dynamics of the intestinal bacterial community in black soldier fly larval guts and its influence on insect growth and development

Black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is a prominent insect for the bioconversion of various organic wastes. As a saprotrophic insect, the BSF inhabits microbe-rich environments. However, the influences of the intestinal microorganisms on BSF growth and development are not very clear. In this study, the dynamics of the intestinal bacterial community of BSF larvae (BSFL) were analyzed using pyrosequencing. Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were the most prevalent bacterial phyla in the intestines of all larval instars. The dynamic changes in bacterial community compositions among different larval instars were striking at the genus level. Klebsiella, Clostridium, Providencia, and Dysgonomonas were the relatively most abundant bacteria in the 1st- to 4th-instar BSFL, respectively. Dysgonomonas and Providencia also dominated the 5th- and 6th-instar larvae, at ratios of 31.1% and 47.2%, respectively. In total, 148 bacterial strains affiliated with 20 genera were isolated on different media under aerobic and anaerobic conditions. Among them, 6 bacteria, BSF1–BSF6, were selected for further study. The inoculation of the 6 isolates independently into germ-free BSFL feeding on an artificial diet showed that all the bacteria, except BSF4, significantly promoted BSF growth and development compared with the germ-free control. Citrobacter, Dysgonomonas, Klebsiella, Ochrobactrum, and Providencia promoted BSF development significantly by increasing the weight gains of larvae and pupae, as well as increasing the prepupae and eclosion rates. In addition, Citrobacter, Klebsiella and Providencia shortened the BSF life cycle significantly. The results illustrate the promotive effects of intestinal bacteria on BSF growth and development.     

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。     

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。     

Effective bioconversion of farmed chicken products by black soldier fly larvae at commercially relevant growth temperatures

Global population growth and an increasing demand for meat has driven the intensification of livestock production. On poultry farms, the accumulation of waste such as faeces, carcasses and unsellable eggs creates environmental and health hazards that need to be mitigated. The larvae of the black soldier fly (Hermetia illucens; BSFL) offer a potential solution to the problems of waste management on poultry farms. BSFL consume large quantities of organic waste and convert it into larval biomass, which can then be processed for use as livestock feeds or biofuels. This makes BSFL an ideal candidate for value-added waste management on chicken farms. Here, we examined the development and nutrient profile of BSFL given five different diet treatments: poultry feed (control), chicken meat, chicken egg, chicken manure and a mixture of equal parts chicken meat, egg and manure. Chicken meat, egg and mixed diets were all found to be suitable feedstocks for BSFL, but the manure-only treatment was associated with a high failure rate of larval development. Mixing manure with other poultry waste streams ameliorated the negative impacts of manure on BSFL. Larvae reared on chicken meat, egg and the mixed diet had equal or higher mean crude protein (CP) (39.9%, 33.8% and 31.5%, respectively) and crude lipid (CL) contents (30.1%, 29.00% and 28.7%, respectively), compared with BSFL reared exclusively on chicken feed (CP: 30.9%, CL: 23.8%), demonstrating the suitability of these waste-stream diets for the potential animal feed quality of the BSFL. We discuss how BSFL bioconversion could be implemented to address environment management issues on poultry farms.     

A comparison in product‐value potential in four treatment strategies for food waste and faeces – assessing composting,fly larvae composting and anaerobic digestion

Municipalities are expected to provide solid waste management, which is funded by tax revenue or/and waste treatment fees. In many low‐ and middle‐income countries, municipalities struggle to provide an adequate level of service, and in these places, the informal sector plays a major role in the collection and treatment of solid waste. In contrast to the plastic and metal fraction, the organic fraction is not managed by the informal sector, primarily because it has low or no financial value and treatment would cost more than the possible revenue. If the organic fraction could be converted to valuable products, the treatment could bear its own cost and this could act as an incentive to collect and treat this fraction. In this study, the potential product value generated through four treatment strategies treating food waste and faeces was compared in a Swedish context: (i) thermophilic composting; (ii) black soldier fly treatment (BSF treatment); (iii) anaerobic digestion (AD); and (iv) BSF treatment followed by AD (BSF + AD). In order to assess the AD strategies, the biomethane potentials of the substrates were assessed. Food waste had the highest biomethane potential, while BSF‐treated faeces had the lowest (417 and 188 NmL g VS^?1, respectively). Thermophilic composting yielded the lowest value product (organic fertilizer; 26 € t^?1 treated food waste) and BSF treatment + AD the highest total value of products (animal feed, vehicle gas and organic fertilizer; 215 € t^?1 treated food waste). The treatment costs were not taken into account here; the total value gives an indication of the cost margin for the different strategies studied. In places with an existing AD plant, BSF treatment + AD strategy is the most economically viable. In places where no such plant exists, BSF treatment is likely to be the most economically favourable treatment.     

First report of the apparent metabolisable energy value of black soldier fly larvae fat used in broiler chicken diets

In the available literature, there are limited data about the energetic value of insect-derived products. In particular, insect fat cannot be used in practical broiler nutrition due to the lack of precise apparent metabolisable energy (AME) value. Therefore, the present study aimed to investigate the AME and apparent metabolisable energy corrected to zero nitrogen balance (AME_N) levels of Hermetia illucens larvae fat for broiler chickens of various ages. A total of 400 1-day-old male Ross 308 chicks were randomly allotted to four dietary groups (10 replicate pens per treatment; 10 birds per pen). The following treatments were applied: HI0 – basal diet without dietary fat inclusion, HI03 – basal diet enriched with 30 g/kg H. illucens larvae fat, HI06 – basal diet enriched with 60 g/kg H. illucens larvae fat, and HI09 – basal diet enriched with 90 g/kg H. illucens larvae fat. Broilers had ad libitum access to mash form feed and water. Excreta samples were collected on d 14, d 28, and d 35. To determine the AME and AME_N values of H. illucens larvae fat, the simple linear regression method was used. The results show that the AME and AME_N values of H. illucens larvae fat for broiler chickens are 9 049 kcal/kg (37.86 MJ/kg) and 9 019 kcal/kg (37.74 MJ/kg), respectively. Additionally, because the birds’ age significantly (P < 0.001) affected the AME and AME_N levels, the implementation of H. illucens larvae fat to broiler diets should be considered in each nutritional period using the recommended regression model AME = 2 559.758 + 62.989 × fat inclusion (%) + 7.405 × day of age and AME_N = 2 543.2663 + 62.8649 × fat inclusion (%) + 7.3777 × day of age. The present data emphasised that the H. illucens larvae fat metabolisable energy is similar to that of soybean oil.     

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.     

肠道共生细菌预发酵鸡粪对黑水虻生长发育的影响

以利用黑水虻Hermetia illucens L.肠道共生细菌不同菌株培养液预发酵的鸡粪为培养料,研究了添加不同菌株的发酵鸡粪对黑水虻幼虫生长发育的影响。结果表明,添加不同菌株的预发酵鸡粪能显著增加预蛹和蛹的重量,显著提高化蛹率并增加雌、雄成虫体长,缩短预蛹所需时间,但对幼虫存活率、成虫羽化率、成虫寿命没有显著影响。同时,不同菌株的作用效果有所不同。     

Factors affecting the bioconversion of Philippine tung seed by black soldier fly larvae for the production of protein and oil-rich biomass

A systematic study on the use of Philippine tung (Reutealis trisperma) seed as a substrate for the cultivation of black soldier fly larvae (Hermetia illucens) was performed. The characteristics of Reutealis trisperma seed from two different locations: West Java and Papua, were determined. The seed has a relatively high oil (37.6–39.2%, dry weight) and protein content (14.9–28.2%, dry weight). The effect of cake content in the substrate (0–20%, wet weight), moisture content in the substrate (50–70%, wet weight), feeding rate (50–100?mg/larva/d), lighting condition (dark-light) and substrate depth in a rearing container (4–10?cm) was performed. An optimum prepupal biomass productivity of 123.4?g/m²/d was obtained (20%, wet weight of cake content in the substrate, 60%, wet weight of moisture content in the substrate, 100?mg/larva/d, dark, 6?cm substrate depth). The protein and oil content of the biomass were also determined to evaluate the effect of Reutealis trisperma seed as a substrate for the cultivation of black soldier fly larvae to produce protein and oil-rich biomass. The oil content in the biomass was also extracted and the fatty acid composition was identified. The prepupal biomass has a relatively high amount of protein (45%, dry weight) and oil content (26.6%, dry weight) and is suitable for cattle feed application.     

Improvement in bioconversion efficiency and reduction of ammonia emission by introduction of fruit fermentation broth in a black soldier fly larvae and kitchen waste conversion system

The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is an insect commonly used for the bioconversion of various organic wastes. Not only can the BSF convert organic waste into macromolecular organic substances, such as insect proteins, but it can also lessen the pollution associated with these waste products by reducing ammonia emissions, for example. In this study, we measured the effects of adding fruit fermentation broth (Fer) and commercial lactic acid bacteria fermentation broth (Em) to kitchen waste (KW), as deodorizing auxiliary substances, on the growth performance of black soldier fly larvae (BSFL), the intestinal flora structure of BSFL, the ammonia emission from the KW substrate, and the microbial community structure of the KW substrate. We found that the addition of Fer or Em increased the body weight of BSFL after 6 d of culture, increasing the growth rate by 9.96% and 7.96%, respectively. The addition of Fer not only reduced the pH of the KW substrate but also increased the relative abundance of probiotics, such as Lactobacillus, Lysinibacillus, and Vagococcus, which inhibited the growth of ammonifiers such as Bacillus, Oligella, Paenalcaligenes, Paenibacillus, Pseudogracilibacillus, and Pseudomonas, resulting in the reduction of ammonia emission in the KW substrate. Moreover, the addition of Fer or Em significantly increased the relative abundances of Bacteroides, Campylobacter, Dysgonomonas, Enterococcus, and Ignatzschineria in the gut of BSFL and increased the species diversity and richness in the KW substrate. Our findings provide a novel way to improve the conversion rate of organic waste and reduce the environmental pollution caused by BSF.