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.     

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.     

Rational design and characterization of bioplastics from Hermetia illucens prepupae proteins

In this study proteins extracted from prepupae of Hermetia illucens, also known as black soldier fly, are investigated as promising base for a new type of bioplastics for agricultural purposes. Design of experiments techniques are employed to perform a rational study on the effects of different combination of glycerol as plasticizer, citric acid as cross-linking agent and distilled water as solvent on the capability of proteins to form a free-standing film through casting technique, keeping as fixed the quantity of proteins. Glycerol shows interesting properties as plasticizer contributing to the formation of homogenous and free-standing film. Moreover, mechanical and thermal characterizations are performed to estimate the effect of increasing amounts of proteins on the final properties and thickness of the specimens. Proteins derived from H. illucens can be successfully employed as base for bioplastics to be employed for agricultural purposes.     

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.     

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.     

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.     

Effects of dietary protein and carbohydrate on life‐history traits and body protein and fat contents of the black soldier fly Hermetia illucens

We investigate how the black soldier fly Hermetia illucens L. (Diptera: Stratiomyidae) responds to dietary protein (P) and carbohydrate (C) contents and the P:C ratio in terms of both immature and adult life‐history traits, as well as effects on larval body composition. Nine chicken‐feed based diets varying in their P:C ratio are formulated. We test three protein concentrations (10%, 17% and 24%) and three carbohydrate concentrations (35%, 45% and 55%) and their combinations. All nine diets support the complete development and reproduction of this species. Survival is high on all diets. Development time, larval yield, larval crude fat and egg yield are more influenced by P and C contents than by the P:C ratio. Low contents result in a shorter development time. Larval yield is higher on diets with higher C‐contents. Pupal development is faster on a low dietary P‐content for all three C‐contents. Egg yield only increases when P‐content increases, although it also varies with the P:C ratio. Larval crude protein content is similar on all nine diets but increases when C‐content is low (10%) in P10 and P17. Larval crude fat content is high at P24‐diets irrespective of C‐content. We conclude that a high macronutrient content combined with a low P:C ratio positively affects H. illucens performance. The diet P17:C55 supports the highest larval and adult performance and results in a high larval body protein content and an intermediate crude fat content.     

Semiochemical and physical stimuli involved in host recognition by Telenomus podisi (Hymenoptera: Scelionidae) toward Euschistus heros (Heteroptera: Pentatomidae)

The egg parasitoid, Telenomus podisi, was shown to recognize its host, Euschistus heros, through both chemical and physical cues. These were determined in short-range bioassays. The cues comprised fertile and infertile host eggs, egg extracts, crude extracts of adult males and females and successive dilutions of the synthetic racemic mixture of methyl 2,6,10-trimethyltridecanoate, a male-produced pheromone of E. heros. Some of the treatments used induced a selection process in T. podisi, through host recognition behaviour, indicating a consistent response of this species to E. heros. The possibility that some of these compounds could be exploited as long-range kairomones, by T. podisi, is discussed. Using GC analysis, an unidentified compound (Rt 24.8 min), possibly from the egg adhesive material, was detected from E. heros egg extracts. Its retention time compared to the methyl 2,6,10-trimethyltridecanoate showed a lower volatility, suggesting that the egg adhesive may act as a short-range kairomone. Physical cues appeared to be an important component in the host selection process by T. podisi. The parasitoid discriminated infertile eggs which are differently shaped and smaller than fertile eggs.     

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.     

THE PHYLOGENETIC POSITION OF ANEMIA COLIMENSIS

The unique leaf structure of the genus Anemia with its upright fertile basal pinnae has been interpreted as derived from the typical fern leaf with unmodified basal pinnae. Reported herein for the first time is the leaf morphology of a species, Anemia colimensis Mickel, which shows the most primitive condition known in the genus and clearly confirms the course of evolution. The fertile pinnae are but little modified in size, lamination, and posture. Although A. colimensis has characters of two subgenera, it is shown to be a primitive element in subgen. Anemiorrhiza and not a member of subgen. Coptophyllum as previously reported.     

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.     

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.     

Comparative structural bioinformatics analysis of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> chemotaxis proteins within <Emphasis Type="Italic">Bacillus subtilis</Emphasis> group

Chemotaxis is a process in which bacteria sense their chemical environment and move towards more favorable conditions. Since plant colonization by bacteria is a multifaceted process which requires a response to the complex chemical environment, a finely tuned and sensitive chemotaxis system is needed. Members of the Bacillus subtilis group including Bacillus amyloliquefaciens are industrially important, for example, as bio-pesticides. The group exhibits plant growth-promoting characteristics, with different specificity towards certain host plants. Therefore, we hypothesize that while the principal molecular mechanisms of bacterial chemotaxis may be conserved, the bacterial chemotaxis system may need an evolutionary tweaking to adapt it to specific requirements, particularly in the process of evolution of free-living soil organisms, towards plant colonization behaviour. To date, almost nothing is known about what parts of the chemotaxis proteins are subjected to positive amino acid substitutions, involved in adjusting the chemotaxis system of bacteria during speciation. In this novel study, positively selected and purified sites of chemotaxis proteins were calculated, and these residues were mapped onto homology models that were built for the chemotaxis proteins, in an attempt to understand the spatial evolution of the chemotaxis proteins. Various positively selected amino acids were identified in semi-conserved regions of the proteins away from the known active sites.     

A FERTILE SPECIES OF TRIPHYLLOPTERIS FROM THE EARLY CARBONIFEROUS (MISSISSIPPIAN) OF SOUTHWESTERN VIRGINIA

The first occurrence of attached fertile structures to foliage assignable to the form genus Triphyllopteris Schimper is reported from the early Carboniferous Price Formation of southwestern Virginia. Sporangium-bearing branch systems consisting of a basal undivided foliar region and a distal much-divided fertile region are described as a new species T. uberis Skog and Gensel. Ovoid fusiform sporangia, 2.0 mm long and 0.5 mm wide and containing trilete spores, terminate ultimate divisions of the branch systems and collectively form masses up to 4 cm in all dimensions. T. uberis is compared to previously known sporangiate organs of Late Devonian and early Carboniferous age, many of which lack attached foliage. Similarities in the overall branching pattern of many of these non-synangiate, sporangiate organs of early Carboniferous age is noted. The possible mode of dispersal and affinities of the plant bearing T. uberis fertile foliage is discussed.     

Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

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.     

Influence of very low doses of mediators on fungal laccase activity - nonlinearity beyond imagination

Laccase, an enzyme responsible for aerobic transformations of natural phenolics, in industrial applications requires the presence of low-molecular substances known as mediators, which accelerate oxidation processes. However, the use of mediators is limited by their toxicity and the high costs of exploitation. The activation of extracellular laccase in growing fungal culture with highly diluted mediators, ABTS and HBT is described. Two high laccase-producing fungal strains, Trametes versicolor and Cerrena unicolor, were used in this study as a source of enzyme. Selected dilutions of the mediators significantly increased the activity of extracellular laccase during 14 days of cultivation what was distinctly visible in PAGE technique and in colorimetric tests. The same mediator dilutions increased demethylation properties of laccase, which was demonstrated during incubation of enzyme with veratric acid. It was established that the activation effect was assigned to specific dilutions of mediators. Our dose-response dilution process smoothly passes into the range of action of homeopathic dilutions and is of interest for homeopaths.     

STUDIES OF PALEOZOIC MARATTIALEANS: AN EARLY PENNSYLVANIAN SPECIES OF THE FERTILE FERN SCOLECOPTERIS

Remains of the fossil Marattiales are very rare in Lower Pennsylvanian sediments. The present report describes a new species of the fertile fern foliage Scolecopteris from the Lewis Creek, Kentucky locality (Lower or lower Middle Pennsylvanian). Scolecopteris conicaulis n. sp. has radial synangia composed of a ring of 4–7 elongate, exannulate sporangia. Most features of the synangia of S. conicaulis were previously hypothesized to be primitive in Scolecopteris based on geologically younger species. Supposed primitive characters include the large synangium pedicel with fiber core, an outer-facing sporangial wall lacking differentiation or zonation, and large spores. The anatomy of the sporangium walls, pinnule morphology, and general spore type support an association with the Minor group of Scolecopteris. The new species is similar in several important features to Scolecopteris (Cyathotrachus) altus, the only other anatomically preserved fertile marattialean known from this early time, and indicates a considerably earlier origin for fertile foliage of this type.     

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.