Milan Kozánek

The use of fly larvae for organic waste treatment

The idea of using fly larvae for processing of organic waste was proposed almost 100 years ago. Since then, numerous laboratory studies have shown that several fly species are well suited for biodegradation of organic waste, with the house fly (Musca domestica L.) and the black soldier fly (Hermetia illucens L.) being the most extensively studied insects for this purpose. House fly larvae develop well in manure of animals fed a mixed diet, while black soldier fly larvae accept a greater variety of decaying organic matter. Blow fly and flesh fly maggots are better suited for biodegradation of meat processing waste. The larvae of these insects have been successfully used to reduce mass of animal manure, fecal sludge, municipal waste, food scrapes, restaurant and market waste, as well as plant residues left after oil extraction. Higher yields of larvae are produced on nutrient-rich wastes (meat processing waste, food waste) than on manure or plant residues. Larvae may be used as animal feed or for production of secondary products (biodiesel, biologically active substances). Waste residue becomes valuable fertilizer. During biodegradation the temperature of the substrate rises, pH changes from neutral to alkaline, ammonia release increases, and moisture decreases. Microbial load of some pathogens can be substantially reduced. Both larvae and digested residue may require further treatment to eliminate pathogens. Facilities utilizing natural fly populations, as well as pilot and full-scale plants with laboratory-reared fly populations have been shown to be effective and economically feasible. The major obstacles associated with the production of fly larvae from organic waste on an industrial scale seem to be technological aspects of scaling-up the production capacity, insufficient knowledge of fly biology necessary to produce large amounts of eggs, and current legislation. Technological innovations could greatly improve performance of the biodegradation facilities and decrease production costs.

Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands

SUMMARY Horse flies feed from superficial haematomas and probably rely heavily on the pharmacological properties of their saliva to find blood. Here we describe the first evidence of vasodilators in horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary gland extract and clone and express one of the active peptides (termed vasotab). Physiological tests using crude salivary gland extracts and reverse-phase HPLC fractions demonstrated positive inotropism in isolated rat hearts, vasodilatation of coronary and peripheral vessels, and Na, K-ATPase inhibition. One of the vasoactive fractions was analysed by N-terminal Edman degradation and a 47-amino-acid sequence obtained. A full-length cDNA encoding the peptide was cloned from a phage library using degenerate primer PCR and the peptide expressed in insect cells. A 20-amino-acid signal sequence precedes the mature 56-amino-acid vasotab peptide, which is a member of the Kazal-type protease inhibitor family. The peptide has a unique 7-amino-acid insertion between the third and fourth cysteine residues. The recombinant peptide prolonged the action potential and caused positive inotropism of isolated rat heart myocytes, and may be an ion channel modulator.

Biodegradation of Pig Manure by the Housefly, Musca domestica: A Viable Ecological Strategy for Pig Manure Management

The technology for biodegradation of pig manure by using houseflies in a pilot plant capable of processing 500–700 kg of pig manure per week is described. A single adult cage loaded with 25,000 pupae produced 177.7±32.0 ml of eggs in a 15-day egg-collection period. With an inoculation ratio of 0.4–1.0 ml eggs/kg of manure, the amount of eggs produced by a single cage can suffice for the biodegradation of 178–444 kg of manure. Larval development varied among four different types of pig manure (centrifuged slurry, fresh manure, manure with sawdust, manure without sawdust). Larval survival ranged from 46.9±2.1%, in manure without sawdust, to 76.8±11.9% in centrifuged slurry. Larval development took 6–11 days, depending on the manure type. Processing of 1 kg of wet manure produced 43.9–74.3 g of housefly pupae and the weight of the residue after biodegradation decreased to 0.18–0.65 kg, with marked differences among manure types. Recommendations for the operation of industrial-scale biodegradation facilities are presented and discussed.

Identification of Anticoagulant Activities in Salivary Gland Extracts of Four Horsefly Species (Diptera, Tabanidae)

Anticoagulant activities against the extrinsic and intrinsic coagulation pathways were identified in salivary gland extracts (SGE) prepared from four tabanids (Hybomitra muehlfeldi, Tabanus autumnalis, Haematopota pluvialis, Heptatoma pellucens). All extracts prolonged human plasma clotting time in a dose-dependent manner and inhibited thrombin activity in the chromogenic substrate assay. Horsefly SGE did not inhibit factor Xa. Partial purification of SGE proteins using reversed-phase high-performance liquid chromatography revealed species-specific differences in the elution profiles and range of fractions with anticoagulant activities.

Mortality of therapeutic fish Garra rufa caused by Aeromonas sobria

OBJECTIVE To investigate a case of mass mortality of Garra rufa (G. rufa) from a fish hatchery farm in Slovakia. METHODS Causative bacterial agent was swabbing out of affected fish skin area and subsequently identified using commercial test system. Antibiotic susceptibility was determined by the disk diffusion method. RESULTS Infected G. rufa was characterized by abnormal swimming behaviour, bleeding of skin lesions and local haemorrhages. Despite of using recommended aquatic antibiotic treatment no improvement was achieved and Aeromonas sobria (A. sobria) was identified as a causative agent of fish mortality. Due to massive fish mortality, antibiotic susceptibility of pure isolated culture of A. sobria was evaluated employing eight antibiotics against human infections. A. sobria was resistant only against one antibiotic, namely ampicilin. CONCLUSIONS These results indicate that A. sobria can act as a primary pathogen of G. rufa and may be a potential risk factor for immunodeficient or immunoincompetent patients during the ichthyotherapy.

Alternative treatment of gluteofemoral fistulas using honey: a case report

Perianal fistula is a common disease that affects particular patients with inflammatory bowel disease. Honey has been used as a natural remedy for centuries for the treatment of various disorders. We report a patient with persistent fistulas, in whom conventional medical and surgical therapy failed. In this case, most of fistulas in gluteofemoral region were completely healed and closed after 6 months of treatment with honey. In addition, honey reduced inflammation, pain and induration of affected region. This medical approach positively affected patients mental condition and also improved his quality of life.

Anticoagulant activities in salivary glands of tabanid flies.

Abstract Tabanid flies are telmophages (pool feeders), taking frequent and rapid bloodmeals from many different individual hosts. To investigate how they accomplish this intermittent feeding strategy, we examined the anticoagulant activities in salivary gland extracts (SGE) from 19 species representing six genera: Atylotus, Chrysops, Haematopota, Heptatoma, Hybomitra and Tabanus (Diptera: Tabanidae). Standard coagulation screen assays were used to determine thrombin time, prothrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. SGE of most species (except Chrysops spp.) considerably prolonged human plasma clotting time in a dose‐dependent manner, and showed potent and specific antithrombin activity in the chromogenic substrate assay. Heptatoma pellucens displayed the strongest anticoagulant activity. Specific anti‐factor Xa activity in tabanid SGE was not detected. Electrophoretic profiles of SGE proteins differed between genera and species. Overall, the results suggest that tabanids have evolved at least two antihaemostatic strategies.

On the pathogens of the spruce bark beetle Ips typographus (Coleoptera: Scolytinae) in the Western Carpathians

Pathogens of spruce bark beetle Ips typographus were compared at eleven sites in Slovakia (the Western Carpathians). Seven different pathogen species were recorded: protozoan species Chytridiopsis typographi, Gregarina typographi, Malamoeba scolyti, nematodes of the genus Neoparasitylenchus and one unidentified nematode species. Presence of the entomopathogenic fungus Beauveria bassiana was also observed as well as parasitization by hymenopteran parasitoids. High infection rate of G. typographi and the nematode Neoparasitylenchus was detected in almost all sites and lethal impact of M. scolyti by massive infections was observed. The beetles from seven sites were screened for endosymbiotic bacteria Wolbachia, however, with no positive result.

A Behavioral Method for Separation of House Fly (Diptera: Muscidae) Larvae from Processed Pig Manure

ABSTRACT A behavioral method applicable in biodegradation facilities for separation of house fly (Musca domestica L.) larvae from processed pig manure is presented. The method is based on placing a cover over the larval rearing tray, while escaping larvae are collected in collection trays. Separation units must be placed in a dark room to avoid negative phototactic responses of the larvae. After 24 h of separation, over 70% of the larvae escaped from processed manure and were collected in collection trays. Most of the larvae pupated within 48 h after separation. Mean weight of pupae recovered from manure residue was not significantly different from mean weight of pupae of separated individuals. Eclosion rate of pupae recovered from manure residue was significantly lower than eclosion of separated individuals, and was strongly related to separation success. Factors responsible for escape behavior of larvae are discussed.

The Effect of Salivary Gland Extract of Lucilia sericata Maggots on Human Dermal Fibroblast Proliferation within Collagen/Hyaluronan Membrane In Vitro: Transmission Electron Microscopy Study

BACKGROUND: Lucilia sericata maggots are applied to chronic wounds to aid healing when conventional treatments have failed. After their application into a necrotic wound, they potentially influence wound healing with a combination of specific proteinases that are involved in the remodeling of extracellular matrix. These proteases cause changes in fibroblast adhesion and spread upon extracellular matrix protein surfaces, affecting integrity of the protein surfaces—especially fibronectin—while maintaining cell viability. OBJECTIVE: This study focused on in vitro monitoring of the effect of homogenate substances prepared from maggot salivary gland of L sericata on the ultrastructure of human neonatal fibroblasts. METHODS: Collagen/hyaluronan membrane was used as the synthetic substitute of extracellular matrix. The cultured human neonatal fibroblasts B–HNF-1 were seeded on the surface of the collagen/hyaluronan membrane and cultured with maggot salivary gland extract (SGE) at a concentration of 2.4 glands/1 mL. RESULTS: The authors observed increased cell metabolism and protein production (euchromatic nucleus, voluminous nuclear membrane, large reticular nuclei, distended and filled cisterns of rough endoplasmic reticulum, Golgi apparatus with saccules, and vesicles packed with fine fibrillar material) after incubating the cells in culture medium with SGE. CONCLUSION: The authors believe that increased cell metabolism and protein production corresponded with formation of microfibrillar net used for migration of fibroblasts in culture, but mainly for proper production of extracellular matrix. The authors suggest that their results may help explain the effect of SGE on wound healing and support implementation of maggot therapy into human medicine.