An attacin antimicrobial peptide, Hill_BB_C10074, from Hermetia illucens with anti-Pseudomonas aeruginosa activity.

BACKGROUND: There is a global need to develop new therapies to treat infectious diseases and tackle the rise in antimicrobial resistance. To date, the larvae of the Black Solider Fly, Hermetia illucens, have the largest repertoire of antimicrobial peptides derived from insects. Antimicrobial peptides are of particular interest in the exploration of alternative antimicrobials due to their potent action and reduced propensity to induce resistance compared with more traditional antibiotics. RESULTS: The predicted attacin from H. illucens, Hill_BB_C10074, was first identified in the transcriptome of H. illucens populations that had been fed a plant-oil based diet. In this study, recombinant Hill_BB_C10074 (500 µg/mL), was found to possess potent antimicrobial activity against the serious Gram-negative pathogen, Pseudomonas aeruginosa. Sequence and structural homology modelling predicted that Hill_BB_C10074 formed a homotrimeric complex that may form pores in the Gram-negative bacterial outer membrane. In vitro experiments defined the antimicrobial action of Hill_BB_C10074 against P. aeruginosa and transmission electron microscopy and electrochemical impedance spectroscopy confirmed the outer membrane disruptive power of Hill_BB_C10074 which was greater than the clinically relevant antibiotic, polymyxin B. CONCLUSIONS: Combining predictive tools with in vitro approaches, we have characterised Hill_BB_C10074 as an important insect antimicrobial peptide and promising candidate for the future development of clinical antimicrobials.

Suppression of Methicillin-Resistant Staphylococcus aureus and Reduction of Other Bacteria by Black Soldier Fly Larvae Reared on Potato Substrate.

Larvae of black soldier flies, Hermetia illucens, are increasingly used for biological conversion of animal and plant wastes into ingredients of animal feeds on an industrial scale. The presence of pathogenic microorganisms in harvested larvae may be a serious problem for wide-scale adoption of this technology. Fortunately, black soldier fly larvae may have some antimicrobial properties. Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium associated with various environments that can be pathogenic to humans and farmed animals. We tested whether black soldier fly larvae suppress MRSA on potato substrate. Autoclaved potatoes containing black soldier fly larvae (P+BSFL), potatoes inoculated with MRSA and containing black soldier fly larvae (P+MRSA+BSFL), and potatoes inoculated with MRSA (P+MRSA) were incubated in glass jars. Substrate samples were taken after 3 and 7 days of incubation and plated on Trypticase soy agar (TSA) and Staphylococcus medium 110 agar (SA) to quantify total bacteria and MRSA, respectively. DNA was extracted from potato substrates on both days and sequenced to assess bacterial and fungal diversity using 515F/806R and internal transcribed spacer (ITS) 1/2 primers, respectively, and QIIME 2.0 software. Both total bacterial and MRSA-specific CFU were reduced in the presence of black soldier fly larvae, with a larger reduction for the latter. Twenty-five bacterial genera and 3 fungal genera were detected. Twenty bacterial genera were shared among the treatments and the days, but their relative abundances often varied. Among the most abundant genera, only Enterococcus and Lactococcus were universally present. Our findings confirm antimicrobial properties of black soldier fly larvae. IMPORTANCE Larvae of black soldier flies, Hermetia illucens, may be used to provide an environmentally sustainable and economically viable method for biological conversion of animal and plant wastes into ingredients of animal feeds on an industrial scale. However, contamination of harvested larvae by pathogenic microorganisms inhabiting decaying substrates may be a serious problem for wide-scale adoption of this technology. Fortunately, black soldier fly larvae may have some antimicrobial properties, including suppression of several common pathogens. Our study showed that such a suppression applies to methicillin-resistant Staphylococcus aureus, which is a ubiquitous bacterium pathogenic to animals (including humans).

Sterilization of Lucilia sericata (Diptera: Calliphoridae) Eggs for Maggot Debridement Therapy.

Maggot debridement therapy (MDT) is a therapy with the medical use of sterile fly larvae of certain species, particularly those within the Calliphoridae family including green bottle fly, Lucilia sericata (Meigan, Diptera: Calliphoridae), for treating chronically infected wounds and ulcers. Lucilia sericata flies were maintained under insectary conditions, and the eggs were sterilized using three treatments: hydrogen peroxide solutions, used as a hand disinfectant (Treatment 1-T1), hydrogen peroxide, surface disinfectant (Treatment 2-T2), and SaniHigene (Treatment 3-T3) and the control (without treatment). All three treatment caused the complete sterilization of eggs, and no bacterial colonies were found on the blood agar culture. The egg hatching rate after 72 h was much higher than after 24 h. Egg mortality in hydrogen peroxide solutions, T1 and T2, was 3-4% and less than in solution T3 (13%). Owing to less mortality and more sterility of the eggs, the aforementioned solutions are suggested to be appropriate for sterility in maggot therapy.

Lactobacillus plantarum in black soldier fly (Hermetica illucens) meal modulates gut health and immunity of freshwater crayfish (Cherax cainii).

Probiotic supplements are being used to improve the growth and immune performance of aquaculture species over the last couple of decades. In recent times, black soldier fly (BSF) is considered as one of the promising sources of alternative protein to fishmeal protein in aqua-diets. Since the freshwater crayfish, marron (Cherax cainii), a Western Australian's native and iconic freshwater crayfish species, grows fairly slow under commercial farming environment, this study was aimed to investigate the supplemental effect of BSF and BSF with probiotic bacteria Lactobacillus plantarum (BSFLP) on overall health and immune performance of marron after 56 days of feeding under laboratory conditions. The post-trial data revealed insignificant influences of any diets on growth performance, however, both BSF and BSFLP based diets significantly improved some haemolymph parameters and gut health of marron. High throughput sequence data revealed that both BSF and BSFLP diets significantly improved the diversity of microbial communities including some beneficial bacteria for crustaceans in the hindgut of marron. Further analysis showed that both BSF and BSFLP diets upregulated the expression of some genes in the gut tissue and haemocytes associated with the innate immune response of marron at 48 h post injection. The up-regulation of some immune genes in BSFLP diet group was found significantly linked to OTU abundance for Lactobacillus. The findings of this study could be helpful for improving overall health status of marron.

The microbial safety of seaweed as a feed component for black soldier fly (Hermetia illucens) larvae.

Farmed insects can offer an environmentally sustainable aquafeed or livestock feed ingredient. The value of black soldier fly (Hermetia illucens) (BSF) larvae could be improved by enrichment in omega-3 through the dietary inclusion of seaweed. However, the industry practice of drying seaweed at low temperatures to retain nutritional properties may benefit the survival of human pathogenic bacteria, particularly if the seaweed has been harvested from contaminated water. Here we have demonstrated that E. coli and E. coli O157:H7 died-off in seaweed dried at 50 °C, although both were detected in the dried powder following 72 h storage. V. parahaemolyticus fell below the level of detection in stored seaweed after drying at ≥ 50 °C, but L. monocytogenes remained detectable, and continued to grow in seaweed dried at ≤60 °C. Therefore, drying seaweed at low temperatures risks pathogen carry-over into insects destined for animal feed. BSF larvae reared on an artificially contaminated seaweed-supplemented diet also became contaminated by all four bacteria present in the supplement. Water quality at seaweed harvesting sites, seaweed desiccation, and insect rearing practices, represent critical points where development of regulatory standards could achieve targeted control of pathogenic hazards.

Adaptive Divergence in a Defense Symbiosis Driven from the Top Down.

Most studies of adaptive radiation in animals focus on resource competition as the primary driver of trait divergence. The roles of other ecological interactions in shaping divergent phenotypes during such radiations have received less attention. We evaluate natural enemies as primary agents of diversifying selection on the phenotypes of an actively diverging lineage of gall midges on tall goldenrod. In this system, the gall of the midge consists of a biotrophic fungal symbiont that develops on host-plant leaves and forms distinctly variable protective carapaces over midge larvae. Through field studies, we show that fungal gall morphology, which is induced by midges (i.e., it is an extended phenotype), is under directional and diversifying selection by parasitoid enemies. Overall, natural enemies disruptively select for either small or large galls, mainly along the axis of gall thickness. These results imply that predators are driving the evolution of phenotypic diversity in symbiotic defense traits in this system and that divergence in defensive morphology may provide ecological opportunities that help to fuel the adaptive radiation of this genus of midges on goldenrods. This enemy-driven phenotypic divergence in a diversifying lineage illustrates the potential importance of consumer-resource and symbiotic species interactions in adaptive radiation.

Case Report: Atypical Cutaneous Manifestations of Tularemia after Horsefly Bite.

Tularemia is an infectious zoonosis caused by Francisella tularensis, an aerobic, noncapsulated, Gram-negative coccobacillus. It is more common in the northern hemisphere, and there are sporadic reports in non-endemic areas. The bacterium is usually transmitted by the bite or feces of a tick or other arthropods such as mosquitoes and horseflies. We report a case of an Italian patient with tularemia after a horsefly bite.

Solving a Bloody Mess: B-Vitamin Independent Metabolic Convergence among Gammaproteobacterial Obligate Endosymbionts from Blood-Feeding Arthropods and the Leech Haementeria officinalis.

Endosymbiosis is a common phenomenon in nature, especially between bacteria and insects, whose typically unbalanced diets are usually complemented by their obligate endosymbionts. While much interest and focus has been directed toward phloem-feeders like aphids and mealybugs, blood-feeders such as the Lone star tick (Amblyomma americanum), Glossina flies, and the human body louse (Pediculus humanus corporis) depend on obligate endosymbionts which complement their B-vitamin-deficient diets, and thus are required for growth and survival. Glossiphoniid leeches have also been found to harbor distinct endosymbionts housed in specialized organs. Here, we present the genome of the bacterial endosymbiont from Haementeria officinalis, first of a glossiphoniid leech. This as-yet-unnamed endosymbiont belongs to the Gammaproteobacteria, has a pleomorphic shape and is restricted to bacteriocytes. For this bacterial endosymbiont, we propose the name Candidatus Providencia siddallii. This symbiont possesses a highly reduced genome with high A+T content and a reduced set of metabolic capabilities, all of which are common characteristics of ancient obligate endosymbionts of arthropods. Its genome has retained many pathways related to the biosynthesis of B-vitamins, pointing toward a role in supplementing the blood-restricted diet of its host. Through comparative genomics against the endosymbionts of A. americanum, Glossina flies, and P. humanus corporis, we were able to detect a high degree of metabolic convergence among these four very distantly related endosymbiotic bacteria.

Expression of lucifensin in Lucilia sericata medicinal maggots in infected environments.

Lucifensin, a novel larval defensin, is one of the antibacterial agents of medicinal maggots involved in maggot therapy. The goal of this study was to examine lucifensin expression in various larval tissues during Lucilia sericata development and in maggots exposed to a variety of infectious environments in vitro. In situ hybridisation revealed lucifensin expression in the salivary glands of all larval stages. Expression was occasionally detected in a few cells of the fat body and in the grease coupler of the salivary glands. Expression of lucifensin in the salivary glands was initiated 5-6 h after hatching from the egg. Maximum expression was reached about 24 h after hatching, remained strong during the second and third instars and declined at the end of the third instar, before the wandering stage. Expression of lucifensin was also investigated in maggots after oral ingestion of certain pathogens regularly found in infected chronic wounds. No differences were detected in the salivary glands after stimulation by wound bacterial isolates. However, lucifensin expression was strongly stimulated in the fat body by the presence of Staphylococcus aureus and Pseudomonas aeruginosa. Our data suggest that certain infectious environments increase lucifensin expression only in the fat body, whereas its production and antimicrobial activity in excretion/secretion products are not affected.

Selective antimicrobial activity of maggots against pathogenic bacteria.

Maggot therapy, also known as biosurgery, is an ancient method for the healing of chronic infected wounds. Although clinicians have reported on the beneficial activities of the Lucilia sericata larvae that have been used for healing chronic wounds, the selectivity of this therapy against the different pathogenic micro-organisms that are found in chronic wounds has never been analysed. In the present study, we have investigated the in vitro activities of larval excreta/secreta both against selected bacterial strains that frequently occur in chronically infected wounds, and against bacteria isolated directly from the larvae and their excreta/secreta. Additionally, the antibacterial activities were investigated in in vivo studies, by comparing bacterial diversity in wounds before and after the application of L. sericata larvae. In conclusion, larval therapy is highly recommended, particularly for the treatment of wounds infected with Gram-positive bacteria, like Staphylococcus aureus, but less so for wounds infected with Gram-negative bacteria, especially Proteus spp. and Pseudomonas spp. strains. Bacteria from the genus Vagococcus were resistant to the maggot excreta/secreta.

Discovery of Fusarium solani as a naturally occurring pathogen of sugarbeet root maggot (Diptera: Ulidiidae) pupae: prevalence and baseline susceptibility.

The fungus Fusarium solani (Mart.) Sacc. was discovered as a native entomopathogen of the sugarbeet root maggot, Tetanops myopaeformis (Röder), in the Red River Valley of North Dakota during the 2004 sugarbeet production season. This is the first report of a native pathogen affecting the pupal stage of T. myopaeformis. Forty-four percent of larvae collected from a field site near St. Thomas (Pembina Co.) in northeastern North Dakota during May and June of 2004 were infected with the entomopathogen. The mean LC(50) of F. solani, assessed by multiple-dose bioassays with laboratory-reared pupae, was 1.8x10(6)conidia/ml. After isolation and confirmation of pathogenicity, a pure isolate of the fungus was deposited in the ARS Entomopathogenic Fungal Collection (ARSEF, Ithaca, NY) as ARSEF 7382. Symptoms of F. solani infection included rapid pupal tissue atrophy and failure of adults to emerge. Transverse dissections of infected pupae revealed dense hyphal growth inside puparia, thus suggesting fungal penetration and pathogenicity. Mycelia emerged from pupae after host tissues were depleted. Exposure of older pupae to lethal concentrations caused rapid mortality of developing adults inside puparia. A second, more extensive field survey was conducted during the 2005 cropping season, and F. solani infection was observed in root maggots at most locations, although at lower levels (1-10%) of prevalence than in 2004. Aberrant timing or amounts of rainfall received could have caused asynchrony between pathogen and host during the second year of the experiment.

Natural antibiotic resistance of bacteria isolated from larvae of the oil fly, Helaeomyia petrolei.

Helaeomyia petrolei (oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as Providencia spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as Providencia rettgeri exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nine Providencia strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (Morganella morganii) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 10(8) cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 microg of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene-50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.

Whitefly transmission and efficient ssDNA accumulation of bean golden mosaic geminivirus require functional coat protein.

Bean golden mosaic geminivirus (BGMV) has a bipartite genome composed of two circular ssDNA components (DNA-A and DNA-B) and is transmitted by the whitefly, Bemisia tabaci. DNA-A encodes the viral replication proteins and the coat protein. To determine the role of BGMV coat protein systemic infection and whitefly transmission, two deletions and a restriction fragment inversion were introduced into the BGMV coat protein gene. All three coat protein mutants produced systemic infections when coinoculated with DNA-B onto Phaseolus vulgaris using electric discharge particle acceleration "particle gun." However, they were not sap transmissible and coat protein was not detected in mutant-infected plants. In addition, none of the mutants were transmitted by whiteflies. With all three mutants, ssDNA accumulation of DNA-A and DNA-B was reduced 25- to 50-fold and 3- to 10-fold, respectively, as compared to that of wild-type DNA. No effect on dsDNA-A accumulation was detected and there was 2- to 5-fold increase in dsDNA-B accumulation. Recombinants between the mutated DNA-A and DNA-B forms were identified when the inoculated coat protein mutant was linearized in the common region.