The Factors Influencing Seasonal Dynamics and Spatial Distribution of Stable Fly Stomoxys calcitrans (Diptera, Muscidae) within Stables.

The biology of the stable fly is fairly well known, but factors influencing the distribution of adult stable flies within stables are still inadequately investigated. The four experimental stables were located in south western Slovakia. Within each stable, five sticky traps were localized along the stable, and the flies were weekly counted during the flight season of years 2015⁻2017. Seasonal activity and stable fly abundance in relation to temperature, rainfall, light conditions, relative air humidity, and cows per stable were evaluated. The seasonal activity of the stable fly shows one large peak at the end of summer and a second smaller peak just before the end of the flight season. The spatial distribution of stable flies was unique for each stable. All of the environmental variables had significant and mostly positive effect on stable fly abundance. The strongest and most positive effect on stable fly counts was temperature and rainfall five weeks prior to collecting session. Within the stable, cow number, air humidity, and light conditions are the strongest candidates to influence their distribution.

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.

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.

The immatures of lauxaniid flies (Diptera: Lauxaniidae) and their taxonomical implications.

The immature stages of insects can provide valuable data both for taxonomy and phylogeny, but they are well known only for negligible proportion of the described species. Here we describe lauxaniid immatures for 17 species that were reared under laboratory conditions and subjected to morphological investigation. Following species were included in our study: Cnemacantha muscaria, Homoneura biumbrata, Homoneura limnea, Minettia austriaca, Minettia fasciata, Minettia flaviventris, Minettia loewi, Minettia plumicornis, Peplomyza litura, Poecilolycia vittata, Pseudolyciella pallidiventris, Sapromyza apicalis, Sapromyza hyalinata, Sapromyza intonsa, Sapromyza sexpunctata, Sapromyzosoma quadripunctata, Sapromyzosoma quadricincta. SEM images of the eggs are provided along with the illustrations of the cephaloskeleton and brief description of all three larval instars. The cephaloskeleton, as well as external morphology suggest that subgenus Minettia s. str. may not be monophyletic. Species Sapromyza sexpunctata and Sapromyzosoma spp., Pseudolyciella pallidiventris and Poecilolycia. vittata are probably closely related. Sapromyza apicalis, S. hyalinata and possibly also S. intonsa form a separate clade from the previous group. These results clearly support the long-standing suspicion, that genus Sapromyza is not monophyletic. Sapromyza sexpunctata should be considered a separate genus related to Sapromyzosoma. The spines on dorsal surface of labial lobe suggest relationships between Peplomyza and Meiosimyza species. Affinities of Cnemacantha muscaria remain uncertain. However, the extended Malpighian tubules suggest relationship to Homoneura or Minettia.

Growth and Survival of Bagged Lucilia sericata Maggots in Wounds of Patients Undergoing Maggot Debridement Therapy.

Maggot debridement therapy (MDT) is an established method of debridement of nonhealing wounds. Despite intense clinical research about its efficacy and effects of substances produced by the larvae, growth and development of maggots in the wounds remain largely unexplored. In the present study, the bags with larvae (n = 52), which had been used to debride traumatic, ischemic, diabetic and venous ulcers, were collected and examined. Survival, length, width and larval instar of the maggots within each bag were recorded and analyzed with respect to the wound type and duration of the treatment. Survival of maggots after a 48-h cycle of MDT ranged between 63.6 and 82.7%. Maggots in venous ulcers had on average 9-19% higher mortality than maggots within traumatic, ischemic, and diabetic ulcers. Length of larvae after 48 h cycle of MDT reached on average 7.09-9.68 mm, and average width varied between 1.77 and 2.26 mm. Larvae in venous ulcers were significantly smaller after 48 h, but not after 72 h treatment compared to the other wound types. Further studies should be aimed to identify other patient-associated factors which might influence growth and survival of the larvae during maggot debridement therapy.

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.

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.

A behavioral method for separation of house fly (Diptera: Muscidae) larvae from processed pig manure.

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.

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 patient's mental condition and also improved his quality of life.

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.

Effect of saliva from horse fly Hybomitra bimaculata on kinetic properties of Na,K-ATPase: possible role in regulation of relaxation.

The possible involvement of salivary gland extract (SGE) from horse flies in modifying hyperpolarization and relaxation via alterations in functional properties of sarcolemmal Na,K-ATPase in the host tissue was tested in vitro by application of various amounts of SGE from Hybomitra bimaculata.SGE in the amount of 3 µg proteins representing approximately the equivalent of one salivary gland of Hybomitra bimaculata induced a stimulatory effect on Na,K-ATPase at all ATP concentrations applied. This effect resulted from the improved ATP-binding site affinity in the Na,K-ATPase molecule, as implicated by the reduction in K(M). Increasing the amount of SGE to 6.5 µg resulted in inhibition of the enzyme, which was characterized by reduction in V(max) and also K(M). This suggests that in the presence of relatively high Hybomitra bimaculata SGE concentration some SGE components affect Na,K-ATPase, when ATP is already bound to the enzyme.Our results indicate that SGE from the horse fly Hybomitra bimaculata contain at least two different biologically active compounds modifying the acute recovery and maintenance of excitability during contractile activity in the host tissue by affecting Na,K-ATPase with opposite effects, depending on the ratio of SGE-proteins to proteins of the host tissue.

Effect of the extract from salivary glands of Lucilia sericata on human neutrophils.

OBJECTIVES: There is incomplete information about host immune response to maggot therapy, nowadays increasingly used to clean chronic wounds from necrotic debris and infection. Maggots are applied to the wound during the inflammatory phase. At the same time neutrophils infiltrate the inflammatory site as the first defense line of the organism. Myeloperoxidase (MPO) and reactive oxygen species, generated during the respiratory burst by neutrophils, are the key players participating in microbial killing as well as in signalling pathways. AIM: We studied the effect of an extract from salivary glands (SGE) of Lucilia sericata (L. sericata) on opsonized zymosan stimulated whole blood chemiluminescence (CL), superoxide generation and MPO release from human neutrophils. METHODS: Formation of reactive oxygen species in whole blood was determined by luminol-enhanced CL. superoxide generation was measured as superoxide dismutase inhibitable reduction of cytochrome c, MPO activity as the oxidation of o-dianisidine in the presence of hydrogen peroxide. RESULTS: Crude SG extract of L. sericata had no significant effect either on superoxide generation and MPO release from isolated unstimulated human neutrophils or on activity of isolated enzymes. Crude SG extract of L. sericata in the highest concentration used significantly decreased opsonized zymosan (0.5 mg/ml) stimulated blood CL, superoxide generation and MPO release. CONCLUSION: On the basis of our results as well as from the literature we suggest that the beneficial effects of maggot therapy might involve the decrease of generation and release of proinflammatory factors, while neither phagocytosis nor subsequent apoptosis is disturbed.

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

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.