Antibacterial effect of extracts of Hermetia illucens (Diptera: Stratiomyidae) larvae against Gram‐negative bacteria

Larvae of the black soldier fly, Hermetia illucens are well‐known fly larvae that inhabit many countries around the world. Antimicrobial agents derived from the larvae may be among the substances that are produced in the body for their survival. This study was carried out to identify the antimicrobial effects of H. illucens larvae that commonly inhabit animal waste and food waste. To evaluate the pharmacological effects of H. illucens larvae extracts, the larvae were extracted by various organic solvents, and their antibacterial effects were determined by antimicrobial methods, such as agar disk diffusion and turbidometric assays. The methanol extracts (ME) indicated antibacterial effects against the proliferation of Klebsiella pneumoniae, Neisseria gonorrhoeae and Shigella sonnei. However, antibacterial effects were not induced in Gram‐positive bacteria such as Bacillus subtilis, Streptococcus mutans and Sarcina lutea. The bacterial growth treated with ME was strongly inhibited from 20 mg/mL in a dose‐dependent manner compared with other extracts, and antibacterial activity gradually decreased after 24 h. Moreover, the minimal inhibitory concentration (MIC) values of ME against Klebsiella pneumoniae, Neisseria gonorrhoeae and Shigella sonnei for 12 h were measured as 44.74 mg/mL, 43.98 mg/mL and 43.96 mg/mL, respectively. These results demonstrate that ME of H. illucens larvae not only has antibacterial activity which strongly inhibits the growth and proliferation of the bacteria but also unique properties which effectively block the viability of the bacteria.     

No neonates without adults

With the potential to process the world's agricultural and food waste, provide sustainable fodder for livestock, aquaculture, and pet animals, as well as act as a source of novel biomolecules, the black soldier fly, Hermetia illucens, has been launched into the leading position within the insects as feed industry. Fulfilment of these goals, however, requires mass-rearing facilities to have a steady supply of neonate larvae, which in-turn requires an efficient mating process to yield fertile eggs; yet, little is known about adult reproductive behavior, nor what physiological factors lead to its emergence. Moreover, fertile egg production tends to be highly variable in colony. Therefore, this review brings together what is currently known of the organismal biology of H. illucens, compiling information on adult morphology, physiology, biogeography, genomics, and behavioral ecology. As a holistic synthesis, it highlights several directions of interest for research to follow.     

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.     

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.     

Beneficial effect of supplementing an artificial diet for Amblyseius swirskii with Hermetia illucens haemolymph

Artificial diets have been developed to sustain the mass rearing of a wide range of arthropod natural enemies, with varying success. In some cases, such diets can be optimized using insect‐derived materials, such as haemolymph. In this study, we examined the effect of supplementing haemolymph of the black soldier fly, Hermetia illucens, to a basic artificial diet for the phytoseiid mite Amblyseius swirskii. The survival, development and reproduction of the predatory mite were assessed when fed on artificial diets composed of honey, sucrose, tryptone, yeast extract and egg yolk, supplemented with 5%, 10%, or 20% of H. illucens pre‐pupal haemolymph. Developmental time from larva to adult was shorter for males and females offered artificial diets supplemented with 20% haemolymph vs. the basic diet. The oviposition rate and total fecundity of females reared on the basic diet were substantially lower than those of females supplied with the enriched diets. The intrinsic rate of increase was highest on the diet containing 20% haemolymph, followed by those containing 10% and 5% haemolymph. In a subsequent diet‐switching experiment, mites fed on the basic diet in their juvenile stages were switched upon adulthood to diet enriched with different concentrations of H. illucens haemolymph. The females that were fed with the enriched diets from the adult stage on had higher oviposition rates and fecundities than those maintained on the basic diet, but their reproductive parameters were not significantly affected by the concentration of the haemolymph in the artificial diet. In conclusion, supplementing artificial diets with black soldier fly haemolymph significantly improved their nutritional value for A. swirskii. Our findings indicate the potential of using H. illucens as a cheap source for haemolymph in artificial diets, as the fly can be cost‐effectively produced at a large scale on organic waste materials.     

Intersex is required for female sexual development in Hermetia illucens

Sex-determination pathways are extremely diverse. Understanding the mechanism of sex determination in insects is important for genetic manipulation of the pest population and for breeding of economically valuable insects. Although sex determination has been well characterized in the model species Drosophila melanogaster, little is known about this pathway in Stratiomyidae. In the present study, we first identified the Drosophila intersex (ix) homolog in Hermetia illucens, also known as the black soldier fly, which belongs to the Stratiomyidae family and which is an important insect for the conversion of various organic wastes. Phylogenetic analyses and multiple sequence alignment revealed that Hiix is conserved compared with Drosophila. We showed that Hiix is highly expressed in internal genitalia. Disruption of the Hiix gene using CRISPR/Cas9 resulted in female-specific defects in external genitalia and abnormal and undersized ovaries. Taken together, our study furthers our understanding of sex determination in insects and could facilitate breeding of H. illucens.     

Lipid nutritional indices,regioisomeric distribution,and thermal properties of Tenebrio molitor and Hermetia illucens larvae fat

The fatty acid (FA) profile, nutritional index, and thermal properties of lipids from Tenebrio molitor and Hermetia illucens larvae were studied. T. molitor and H. illucens larvae had high lipid contents (respectively 28.8% and 42.6%), saturated (25.0% and 55.8%), monounsaturated (MUFA) (39.2% and 28.3%), and polyunsaturated (PUFA) fatty acids (35.8% and 15.9%). Both larvae fats contained beneficial ω-3, ω-6, and ω-9 FA. For T. molitor and H. illucens, the lipid nutritional indices were atherogenicity indices 0.68 and 2.75, thrombogenicity indices 0.58 and 0.74, and health-promoting indices 3.51 and 0.80 hypocholesterolemic/hypercholesterolemic acid ratio 1.38 and 0.23, suggesting the nutritional superiority of T. molitor larvae fat. Regioisomeric distribution analysis showed that PUFA in H. illucens larvae fat are concentrated on the sn-1,3 positions, whereas those in T. molitor larvae fat are distributed in all three positions. The thermal stability and crystallisation profiles differed for both larvae fats and demonstrated their potential use in thermally processed foods.     

Correlation of fruit tree rhizosphere soils with entomopathogenic fungi

Entomopathogenic fungi are microorganisms that control the density of host insects in nature; they are being studied as environmentally friendly alternatives to chemical insecticides for controlling insect pests. The main habitat of entomopathogenic fungi is soil, and the correlation between the distribution of entomopathogenic fungi and the physicochemical characteristics of soils planted with different trees, including vine (outdoor, greenhouse, and greenhouse shine musket), apple, peach, and pear, were analyzed. The entomopathogenic fungi of the genera Beauveria, Metarhizium, and Purpureocillium investigated in this study were all found in soil samples from vine-greenhouse, apple, and peach trees. Purpureocillium and Beauveria abundances were positively correlated with soil properties; however, Metarhizium abundances were not correlated with soil properties. The Metarhizium isolates discovered in this study showed pathogenicity to cotton aphids (an agricultural pest) and can be employed as sources for biological studies in the future. This study provides data on the diversity and abundance of entomopathogenic fungi related with soil properties, as well as the molecular, biological, and insecticidal characteristics of Metarhizium isolates.     

Diptera of forensic importance in the Iberian Peninsula: larval identification key

A revision of the species and families of sarcosaprophagous flies (Diptera: Calliphoridae, Sarcophagidae, Muscidae, Fanniidae, Drosophilidae, Phoridae, Piophilidae and Stratiomyidae) suitable for forensic purposes in the Iberian Peninsula is presented. Morphological characteristics that allow the accurate identification of third instars of the species present in the Iberian Peninsula are described and presented in the form of a diagnostic key. For larval Calliphoridae, characteristics such as the spines of the body segments were useful for the genus Calliphora whereas features of the anal segment and the cephalopharyngeal skeleton were useful for larvae of Lucilia. Identification of three Chrysominae species present in the Iberian Peninsula is included. For larval Sarcophagidae, characters such as the arrangement and shape of spiracular openings, structures of the anal segment and the cephalopharyngeal skeleton were used for the first time. A new record of Sarcophaga cultellata Pandellé, from a human corpse, is also included as well as recent incursions into the European cadaveric entomofauna such as Synthesiomyia nudiseta (van der Wulp) and Hermetia illucens (Linnaeus). This work provides useful new information that could be applied to forensic investigations in the Iberian Peninsula and in southern Europe.     

Stable expression of human growth hormone over 50 generations in transgenic insect larvae

Developments in insect transgenesis using transposons combined with available mass rearing technology for insects such as the Medfly, Ceratitis capitata, provide opportunity for the production of protein for industrial, agricultural and healthcare purposes on a very large scale. In this study, we report the germ-line transformation and expression of a cDNA encoding human growth hormone (hGH) in transgenic Drosophila using the Minos transposon. Production and secretion of a bioactive hGH into the haemolymph of transgenic larvae was demonstrated by immunoblot analysis, ELISA and a proliferation bioassay. Stable expression of hGH was observed over 50 generations. The results indicate that mass reared transgenic diptera with a rapid period of larval growth could provide cost effective production systems for the manufacture of therapeutic and other high value proteins.     

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.     

Effect of the rearing diet on gene expression of antimicrobial peptides in Hermetia illucens (Diptera: Stratiomyidae)

Insect proteins have been proposed for human and animal food production. Safeguarding the health status of insects in mass rearing allows to obtain high-quality products and to avoid severe economic losses due to entomopathogens. Therefore, new strategies for preserving insect health must be implemented. Modulation of the insect immune system through the diet is one such strategy. We evaluated gene expression of two antimicrobial peptides (one defensin and one cecropin) in Hermetia illucens (L.) (Diptera: Stratiomyidae) reared on different diets. Analyses were performed on prepupae and 10-day-old larvae reared on cereal- and municipal organic waste-based diets and on only prepupae reared on a cereal-based diet supplemented with sunflower, corn, or soybean oil. The inclusion of sunflower oil at different points in the cereal-based diet was also evaluated. Moreover, diet-driven differences in the inhibitory activity of the hemolymph were tested against Escherichia coli DH5α and Micrococcus yunnanensis HI55 using diffusion assays in solid media. Results showed that a municipal organic waste-based diet produced a significant overexpression of antimicrobial peptides only in prepupae. Inclusion of vegetable oils caused an upregulation of at least one peptide, except for the corn oil. Higher expression of both genes was observed when sunflower oil was added 5 days before pupation. All hemolymph samples showed an inhibitory activity against bacteria colonies. Our results suggest that municipal organic waste-based diet and vegetable oil-added diet may successfully impact the immune system of H. illucens. Such alternatives may also exist for other species of economic interest.     

Increasing cell biomass in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

Background  

Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.     

一株地膜降解真菌的筛选及其降解性能分析

【目的】分离并鉴定具有聚乙烯材料降解能力的微生物菌株,探究其降解农用地膜的效能,为地膜的微生物降解途径提供支撑。【方法】以线性低密度聚乙烯粉末为唯一碳源的培养物中分离出1株具有降解聚乙烯材料能力的真菌,利用分子生物学方法结合菌株的培养性状对该菌株进行鉴定,通过观察聚乙烯粉末降解情况和测定地膜失重率,结合红外扫描、高分辨场发射扫描电子显微镜分析该菌株对农用地膜的降解效果。【结果】筛选获得1株具有农用地膜降解效果的真菌菌株PT1,经鉴定为桔青霉(Penicillium citrinum),桔青霉PT1菌株能以重均分子量(Mw)2000和400000的聚乙烯粉末作为唯一碳源生长,经红外扫描、电镜观察发现桔青霉PT1可侵蚀传统聚乙烯地膜。桔青霉PT1能快速利用聚酯类生物降解地膜生长,35 d地膜失重率达50%左右。【结论】本文筛选到具有地膜降解特性的桔青霉PT1真菌,丰富了降解聚乙烯材料的微生物类群,同时也为废弃农用地膜的处理提供了环保的处理途径。     

Systematic examination of polymorphism in amyloid fibrils by molecular-dynamics simulation

Amyloid fibrils often exhibit polymorphism. Polymorphs are formed when proteins or peptides with identical sequences self-assemble into fibrils containing substantially different arrangements of the β-strands. We used atomistic molecular-dynamics simulation to examine the thermodynamic stability of a amyloid fibrils in different polymorphic forms by performing a systematic investigation of sequence and symmetry space for a series of peptides with a range of physicochemical properties. We show that the stability of fibrils depends on both sequence and the symmetry because these factors determine the availability of favorable interactions between the peptide strands within a sheet and in intersheet packing. By performing a detailed analysis of these interactions as a function of symmetry, we obtained a series of simple design rules that can be used to determine which polymorphs of a given sequence are most likely to form thermodynamically stable fibrils. These rules can potentially be employed to design peptide sequences that aggregate into a preferred polymorphic form for nanotechnological purposes.     

Mucoadhesive Bilayered Patches for Administration of Sumatriptan Succinate

The purpose of this study was to develop and optimize formulations of mucoadhesive bilayered buccal patches of sumatriptan succinate using chitosan as the base matrix. The patches were prepared by the solvent casting method. Gelatin and polyvinyl pyrrolidone (PVP) K30 were incorporated into the patches, to improve the film properties of the patches. The patches were found to be smooth in appearance, uniform in thickness, weight, and drug content; showed good mucoadhesive strength; and good folding endurance. A 3² full factorial design was employed to study the effect of independent variables viz. levels of chitosan and PVP K30, which significantly influenced characteristics like swelling index, in-vitro mucoadhesive strength, in vitro drug release, and in-vitro residence time. Different penetration enhancers were tried to improve the permeation of sumatriptan succinate through buccal mucosa. Formulation containing 3% dimethyl sulfoxide showed good permeation of sumatriptan succinate through mucosa. Histopathological studies revealed no buccal mucosal damage. It can be concluded that buccal route can be one of the alternatives available for administration of sumatriptan succinate.     

Fresh and Dry Mass Estimates of <Emphasis Type="Italic">Hermetia illucens</Emphasis> (Linnaeus, 1758) (Diptera: Stratiomyidae) Larvae Associated with Swine Decomposition in Urban Area of Central Amazonia

Information on biomass is substantial for calculating growth rates and may be employed in the medicolegal and economic importance of Hermetia illucens (Linnaeus, 1758). Although biomass is essential to understanding many ecological processes, it is not easily measured. Biomass may be determined by directly weighing or indirectly through regression models of fresh/dry mass versus body dimensions. In this study, we evaluated the association between morphometry and fresh/dry mass of immature H. illucens using linear, exponential, and power regression models. We measured width and length of the cephalic capsule, overall body length, and width of the largest abdominal segment of 280 larvae. Overall body length and width of the largest abdominal segment were the best predictors for biomass. Exponential models best fitted body dimensions and biomass (both fresh and dry), followed by power and linear models. In all models, fresh and dry biomass were strongly correlated (>75%). Values estimated by the models did not differ from observed ones, and prediction power varied from 27 to 79%. Accordingly, the correspondence between biomass and body dimensions should facilitate and motivate the development of applied studies involving H. illucens in the Amazon region.     

Unveiling the biotransformation mechanism of indole in a Cupriavidus sp. strain

Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base‐pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base‐pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones. Candidates are proteins with homology to FinO, a factor that promotes base pairing between the FinP antisense sRNA and the traJ mRNA to control F plasmid transfer. Recent papers have shown that the Salmonella enterica FinO‐domain protein ProQ binds a large suite of sRNAs, including the RaiZ sRNA, which represses translation of the hupA mRNA, and the Legionella pneumophila protein RocC binds the RocR sRNA, which blocks expression of competence genes. Here we discuss what is known about FinO‐domain structures, including the recently solved Escherichia coli ProQ structure, as well as the RNA binding properties of this family of proteins and evidence they act as chaperones. We compare these properties with those of Hfq. We further summarize what is known about the physiological roles of FinO‐domain proteins and enumerate outstanding questions whose answers will establish whether they constitute a second major class of RNA chaperones.     

Comparative evaluation of Aspergillus niger strains for endogenous pectin-depolymerization capacity and suitability for d-galacturonic acid production

Pectinaceous agricultural residues rich in d-galacturonic acid (d-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are well characterized, this is not the case for the production of pectinases. We, therefore, systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, and N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion, and a suitable morphology. Furthermore, d-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study, therefore, presents a robust initial strain selection to guide future process improvement of d-GalA production from agricultural residues and identifies a high-performance base strain for further genetic optimizations.

     

In Vivo Immobilization of Fusion Proteins on Bioplastics by the Novel Tag BioF

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-β-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.