Wound debridement potential of glycosidases of the wound-healing maggot, Lucilia sericata

The wound-healing maggot, Lucilia sericata Meigen (Diptera: Calliphoridae), degrades extracellular matrix components by releasing enzymes. The purpose of this study was to investigate the glycosylation profiles of wound slough/eschar from chronic venous leg ulcers and the complementary presence of glycosidase activities in first-instar excretions/secretions (ES1) and to define their specificities. The predominant carbohydrate moieties present in wound slough/eschar were determined by probing one-dimensional Western blots with conjugated lectins of known specificities. The presence of specific glycosidase activities in ES1 was determined using chromogenic and fluorogenic substrates. The removal of carbohydrate moieties from slough/eschar proteins by glycosidases in ES1 was determined by two-dimensional electrophoresis and Emerald 300 glycoprotein staining. α-d-glucosyl, α-d-mannosyl and N-acetlyglucosamine residues were detected on slough/eschar-derived proteins. Furthermore, it was demonstrated that the treatment of slough/eschar with ES1 significantly reduced uptake of the carbohydrate-specific stain. Subsequently, α-d-glucosidase, α-d-mannosidase and N-acetylglucosaminidase activities were identified in ES1. Specific chromogenic and fluorogenic substrates and gel filtration chromatography showed that these activities result from distinct enzymes. These activities were mirrored in the removal of α-d-glucosyl, α-d-mannosyl and N-acetylglucosamine residues from proteins of slough/eschar from maggot-treated wounds. These data suggest that maggot glycosidases remove sugars from slough/eschar proteins. This may contribute to debridement, which is ultimately accomplished by a suite of biochemically distinct enzymes present in ES1.     

Influence of parasitism and soil compaction on pupation of the green bottle fly,Lucilia sericata

The influence of parasitoids and soil compaction on pupation behavior of blow flies was examined in a host–parasitoid system involving Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae). Larvae of L. sericata were introduced to containers with soil of different compaction levels, with or without parasitoids. Although females of N. vitripennis did not significantly affect the pupation depth of L. sericata, they increased the rate of pupal development by 15.0–23.7 h at 28.4 ± 1.2 °C, and increased the clumping of puparia. Pupation depth of L. sericata was negatively related to soil compaction; mean depth of pupation was 4.4 cm in uncompacted soil and 0.5 cm in high‐compaction soil. In high‐compaction soil, pupal development increased by 10.5–18.8 h at 25.2 ± 0.3 °C, and puparia were clumped. These results provide a framework for locating puparia in forensic investigations and releasing appropriate parasitoids for biological control of blow flies.     

Growth and survival of blowfly Lucilia sericata larvae under simulated wound conditions: implications for maggot debridement therapy

Maggot debridement therapy has become a well‐established method of wound debridement. Despite its success, little information is available about the optimum duration of the treatment cycle and larval growth in wounds. This study examines the development of Lucilia sericata (Diptera: Calliphoridae) larvae under two containment conditions (bagged and free range) under simulated wound conditions and assesses the impact of transport and further storage of larvae on their survival and growth. There was no significant difference in size between bagged and free‐range larvae over the 72‐h experimental period. Larvae grew fastest 8–24 h after inoculation and completed their growth at 40–48 h. Mortality rates were similar (0.12–0.23% per hour) in both containment conditions and did not differ significantly (P = 0.3212). Survival of free‐range larvae was on average 16% lower than survival of bagged larvae. Refrigeration of larvae upon simulated delivery for > 1 day reduced their survival to < 50% and caused a reduction in growth of up to 30% at 12 h, but not at 48 h, of incubation. Therefore, it is recommended that free‐range larvae are left in the wound for a maximum of 40–48 h, and bagged larvae for 48–72 h. Larvae should be used within 24 h of delivery to avoid high mortality caused by prolonged refrigeration.     

A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation

Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonah_m) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonah_m reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.     

Larval rearing of fringe-lipped carp,Labeo fimbriatus (Bloch) with low cost diets including green bottle fly Lucilia sericata (Meigen) larvae meal incorporated diet

A study was conducted with non-conventional ingredients to test their efficacy as fishmeal (FM) replacers in the diet of fringe- lipped carp. Labeo fimbriatus first feeding larvae and fry were reared for 30 and 60 days in indoor, 50 L, aerated, circular plastic tanks at 100 and 30 numbers tank⁻¹, respectively. In the first feeding larvae to fry rearing experiment (Exp. 1), the fish were fed with either of the following isonitrogenous and isocaloric diets – live plankton, FM diet, green bottle fly (Lucilia sericata) larvae meal (GBFLM) diet and silkworm pupa (SWP) diet. The fry to fingerling rearing (Exp. 2), was also conducted using the same diets described above except live plankton. All compounded diets were formulated to contain 40% crude protein for the experiment 1 and 35% for experiment 2 and were fed ad libitum. Triplicate tanks were maintained for each treatment in both the experiments. In Exp. 1, the mean final weight of fry was higher with plankton and FM diets, while no difference (p > .05) was observed between FM and GBFLM diets. Weight of fish fed SWP diets was not statistically different from those fed GBFLM diet. No difference (p > .05) in final length, survival and condition factor was recorded. Analysis of digestive enzyme activity of whole fish revealed lower (p < .05) activity of amylase in fish fed plankton. In Exp. 2, no difference (p > .05) was observed between the different diet groups in terms of mean final weight, length, survival and condition factor. Analysis of digestive enzyme activity of whole fish revealed no difference (p > .05) in the activity of digestive enzymes between the treatments except a lower (p < .05) activity of trypsin in FM diet and lipase in FM and GBFLM diets. Since the survival and condition factors of animals is the most important aspect during nursery rearing, similar (p > .05) values recorded in different treatments indicate the possibility of incorporation of these non-conventional protein sources in the diet of L. fimbriatus during first feeding larvae to fry and fry to fingerling rearing.     

Long-term effects of diet on the neuroendocrine system of the sheep blowfly, Lucilia sericata

ABSTRACT. The medial neurosecretory cells (MNC), corpus allatum (CA) and ovaries of Lucilia sericata Mg. are quiescent in mature flies fed on a diet of sugar and water, but these tissues rapidly respond to the ingestion of a protein meal. The present study revealed that after oogenesis, virgin flies synthesized and stored neurosecretory material, but this activity declined as they grew older. At the same time, the CA, following a decrease in size during the latter stages of oogenesis, became enlarged again. This is interpreted as denoting synthesis but not release of CA hormone. In meat-fed mated flies, cycles of oviposition and oogenesis were reflected in the rise and fall in activity of the MNC and CA. The MNC and CA of male flies also responded to the ingestion of a protein meal, and the activity of these tissues persisted for several days before it declined. The similar response of the MNC and CA in both males and females demonstrates the importance of a meat meal to reproduction in this species: in females it is required for oogenesis, but its ingestion by both sexes is necessary for overall reproductive success.     

Cold shock and cold tolerance in larvae and pupae of the blow fly, Lucilia sericata

Abstract.  Understanding the effects of low winter temperatures on mortality is essential in the development of a full understanding of the long-term population dynamics of any insect. The present study aims to examine the survival of pupae and larvae of the blow fly, Lucilia sericata , at overwintering temperatures. Groups of pupae and diapausing and nondiapausing third-stage larvae of L. sericata are maintained in cooled incubators at either 3 °C and 6 °C. Groups are removed from the incubators at 3–4-day intervals and transferred either to−8 °C or to 25 °C. After 1 h in the freezer, the larvae and pupae exposed to this cold-shock are also transferred to 25 °C. Larvae and pupae are then allowed to continue development and the number of adults emerging from each group is counted. The results demonstrate that survival decreases linearly with the period of exposure at both 3 °C and 6 °C. Mortality is higher at 3 °C than at 6 °C and, in groups that receive the cold shock, cold-shock reduces emergence by over 50%. However, there is no consistent tendency for diapausing larvae to survive prolonged cold or cold shock better than other life-cycle stages. The results suggest that the facultative development of an overwintering diapause stage in L. sericata does not appear to be an adaptation to enhance cold tolerance or resistance to cold shock. It is concluded that the survival of overwintering L. sericata is likely to be relatively less affected by low temperatures than it is by, for example, biotic factors, particularly given the buffered soil environment and short time-scales over which periods of cold act.     

Mitochondrial genomes of the sheep blowfly, Lucilia sericata, and the secondary blowfly, Chrysomya megacephala

This paper presents complete mitochondrial genomes for the sheep blowfly, Lucilia sericata (Meigen), and the secondary blowfly, Chrysomya megacephala (Fabricius). Both L. sericata and C. megacephala had standard dipteran-type mitochondrial genome architectures and lengths of 15 945 bp and 15 831 bp, respectively. Additionally, C. megacephala possessed a tRNA duplication either side of the D-loop, as previously reported in another Chrysomya species, C. putoria; this duplication appears to be synapomorphic for the genus Chrysomya. As in other insect mitochondrial genomes, base compositions had a high AT content, with both genomes more than 76% AT-rich.     

Population suppression for control of the blowfly Lucilia sericata and sheep blowfly strike

Abstract.

• 1 The control of ovine myiasis by suppression of populations of the blowfly Lucifia sericata was investigated experimentally on three farms in the south-west of England in 1992 and 1993.
• 2 In blind trials, sheep on one farm (control) were given two doses of placebo, on a second two doses of the larvicide cyromazine (Vetrazin®, CibaGeigy), and on a third cyromazine and a subsequent dose of placebo.
• 3 The first treatment was given shortly before the predicted spring emergence of L.sericata and the second shortly before the predicted emergence of the second generation. Previous simulation analysis had identified strategic early-season treatment as the optimum for blowfly population suppression.
• 4 On both treatment farms significantly smaller L.sericata populations were recorded throughout 1992 and the incidence of strike was significantly lower than on the control farm. The results show that appropriate early-season timing of sheep treatment can suppress populations of L.sericata and could be used by farmers to reduce the incidence of blowfly strike.
• 5 The results suggest, however, that the effectiveness of population suppression and strike incidence may have been influenced by immigration into the control areas and by adverse weather, the latter changing the susceptibility of sheep to strike and resulting in rising strike incidence even when L.sericata population densities were low. In practice, therefore, blowfly population suppression should be employed as a component of an integrated strike management programme.

     

Geographic origin affects larval competitive ability in European populations of the blow fly, Lucilia sericata

The blow fly, Lucilia sericata (Meigen) (Diptera: Calliphoridae), is found throughout Europe. In northern areas, in addition to breeding in carrion, it can also commonly act as primary facultative ectoparasite of sheep, resulting in animal suffering and production losses. However, in the south of Europe, L. sericata is not usually implicated in myiasis and is found almost entirely in carrion. The causes of this behavioural difference are unclear. To begin to address the possible reasons for this difference, the present study examined the effects of competition on the survival and development of L. sericata populations derived from southern Spain, England, and a hybrid derived from a cross between Spanish females and UK males. Larvae were reared at a range of different initial densities and the mortality, development rate, and size of the resultant adults were measured. Mortality increased significantly with density, but the Spanish population had a significantly higher mortality rate than the UK population at all densities. Similarly, adult size declined with increased larval density, but individuals from the Spanish population were consistently larger than those from the UK population at all densities. The mortality and size of the hybrids was consistently intermediate between the Spanish and UK populations. No consistent effects on development rate were observed. This study demonstrates that insects from northwestern and southern European population have qualitatively different life history traits and competitive abilities and may contribute towards explaining the different behaviour of populations of this species in different part of its distribution range.     

Interaction of the neuroendocrine system and ovaries in sheep blowflies, Lucilia sericata, sterilized with tepa

ABSTRACT. Female Lucilia sericata Mg. fed a reproductive diet of meat were sterilized by injection of 10μg of tepa. Light microscopy revealed that the medial neurosecretory cells of the brain of sterilized flies were highly active compared with the same cells in untreated flies of similar age and nutritional status. The corpora allata of sterilized flies were also greatly enlarged. The evidence suggests that developing oocytes secrete a substance that inhibits the corpora allata hormone, thereby suppressing brain neurosecretory cell activity and preventing oogenesis in the subsequent oocytes.     

Effect of diet on the neuroendocrine system and egg development in the sheep blowfly, Lucilia sericata

ABSTRACT. A light microscope study of the endocrine and ovarian systems of Lucilia sericata under two diets revealed that in young females fed on sugar and water, medial neurosecretory cells (MNC) synthesized and stored neurosecretory material (NSM) as the flies matured. The MNC remained filled with NSM as long as the diet was maintained. Following a small increase immediately after emergence, the size of the corpora allata (CA) showed little further change, and the nuclei of nurse cells remained small. However, rapid changes occurred in these tissues soon after a meat meal: NSM was discharged from the MNC, and the CA increased in size. These changes were at a maximum 20 h after a meat meal. 4h later, vitellogenesis was well established and the nurse cell nuclei had increased in size 20-fold. Growth of the nurse cell nuclei continued until approximately 6 h before the completion of vitellogenesis when they are resorbed. Oögenesis took about 48 h at 25°C. When 100 μg of each of three different juvenoids were applied topically to different sugar-fed flies, the nuclei of both MNC and nurse cells became enlarged, whereas the CA were somewhat reduced in size. The relationship between protein ingestion and oogenesis is discussed, and the results obtained with L. sericata are compared with those of other species, especially the blowfly Calliphora erythrocephala.     

Responses of the sheep blowflies Lucilia sericata and Lcuprina to odour and the development of semiochemical baits

Abstract. The literature relating to the attraction of the sheep blowflies Lucilia sericata and Lucilia cuprina to their ovine hosts is reviewed. The responses of the two species are similar and different components of the behaviour leading to host location and oviposition appear to involve at least two distinct sets of semiochemical cues. Activation, upwind orientation and landing appear to occur in response to putrefactive sulphur-rich volatiles, originating from bacterial decomposition products. Oviposition is elicited primarily by the presence of ammonia-rich compounds; moisture, pheromones and tactile stimuli may also act as oviposition stimuli. There is a pronounced sex difference in the response of Lucilia to semiochemicals with a higher proportion of females attracted than males and a higher proportion of gravid than non-gravid females. While the mechanisms of host location by Lucilia are of intrinsic interest, understanding the responses to semiochemicals is important in the attempt to develop powerful synthetic baits for deployment with the traps or targets used for population sampling or suppression. The literature is discussed with respect to the development of synthetic semiochemical baits.     

Detection and partial characterisation of two antibacterial factors from the excretions/secretions of the medicinal maggot Lucilia sericata and their activity against methicillin-resistant Staphylococcus aureus (MRSA)

Maggot therapy is a simple and highly successful method for cleansing infected and necrotic wounds. The use of maggots has become increasingly important in the treatment of non-healing wounds, particularly those infected with the multidrug-resistant pathogen, methicillin-resistant Staphylococcus aureus (MRSA). The increasing challenge concerning the treatment of MRSA infections and the recent finding of vancomycin-resistant strains of MRSA have elicited the search for novel antibacterial compounds and, in particular, investigations into the potent antibacterial mechanism(s) behind maggot therapy. In this study, we report that excretions/secretions (ES) from the blowfly, Lucilia sericata, exhibit potent, thermally stable, protease resistant antibacterial activity against MRSA in vitro. We describe the initial characterisation of two antibacterial factors from native ES of L. sericata. A small, <500 Da factor with significant antibacterial activity against MRSA was partially isolated using ultrafiltration techniques. The potent activity of this factor was comparable to that of native excretions/secretions. A larger, 0.5-3-kDa factor with significant activity against S. aureus was also partially characterised.     

The impacts of larval density and protease inhibition on feeding in medicinal larvae of the greenbottle fly Lucilia sericata

Larval therapy, the therapeutic use of blowfly larvae to treat chronic wounds, is primarily used in debridement. There are, however, gaps in current knowledge of the optimal clinical application of the therapy and mechanisms of action in the debridement process. Using an artificial assay, two studies were undertaken to investigate these aspects of larval debridement by Lucilia sericata Meigen (Diptera: Calliphoridae); the first studied the effects of the density of larvae on tissue digestion and larval mass, and the second considered the effects on the same parameters of incorporating protease inhibitors into the feeding substrate. The total mass of tissue digested increased with larval density until saturation was observed at 5.0–7.5 larvae/cm². This range was considered optimal as lower doses resulted in the removal of less tissue and higher doses offered no additional tissue removal and appeared to exacerbate competition for feeding. In the second study, increased protease inhibitor concentration led to significant decreases in tissue digestion and larval mass, suggesting that serine proteases, particularly trypsin, may play major roles in larval digestion. Such information is important in elucidating the main constituents that make up larval digestive products and may be significant in the development of new therapies.     

Parental and direct effects of photoperiod and temperature on the induction of larval diapause in the blow fly Lucilia sericata

Abstract.  Lucilia sericata (Meigen) (Diptera: Calliphoridae) shows a facultative diapause in the third, and final larval, instar after the cessation of feeding. The effects of photoperiod and temperature on the induction and duration of diapause were examined in parental (G0) and current (G1) generations. Insects of the G0 generation were reared under four combinations of conditions, involving two photoperiods, LD 16 : 8 and LD 12 : 12 h, and two temperatures, 25 and 20 °C. The G1 generation, present in the eggs laid by these insects, were transferred to 10 combinations of conditions, involving the above two photoperiods and five temperatures, 25, 20, 17.5, 15 and 12.5 °C. In the G1 generation, the time from hatching to cessation of feeding was significantly affected by temperature only, whereas the induction of diapause was influenced by both photoperiod and temperature experienced by the G0 as well as the G1 generation. Short-day and low-temperature conditions in the G0 and in the G1 generation had diapause-inducing effects. In this species, it is likely that, for purposes of acquiring reliable seasonal information, induction of diapause is sensitive to environmental factors both in the G0 and G1 generations. The function of high-intensity diapause, induced by short-day conditions and high temperature in the parental generation, appeared to be the prevention of accidental pupariation in warm autumn weather.     

Antimicrobial factors of hemolymph and excretion of larvae Lucilia sericata (meigen) (diptera, calliphoridae)

The present study deals with molecular nature and peculiarities of functioning of two main protective systems of larvae Lucilia sericata--the antimicrobial compounds of hemolymph and of the excretion released by feeding larvae into environmental. There are identified a number of inducible antibacterial peptides including defensins (3844, 4062, and 4117 Da), P-peptide (3043 Da), and four new polypeptides (3235, 3702, 3746, and 3768 Da) In hemolymph of the larvae submitted to bacterial infestation, by the chromatomasspectrometry methods. The excretion of larvae Lucilia sericata contains peptides analogous or identical to hemolymph antibacterial peptides (diptericins: 8882 Da and 9025 Da), high molecular compounds of peptide nature (6466 Da, 6633 Da, 5772 Da, 8631 Da, etc.) differing from the known hemolymph components and low molecular compounds (130-700 Da). Spectrum of excretion bactericidal activity includes various groups of bacterial including the most actual pathogen from medical point of view--the meticillin-resistant Staphylococcus aureus, unlike the hemolymph that does not have antistaphylococcal activity. The excretion components suppressing growth and development of this staphylococcus are represented by substances of the low molecular nature (from 160 to 1020 Da). The performed studies characterize the strategies used by "surgical maggots" for protection from pathogens and for suppression of microbial competitors and allow better understanding of molecular mechanisms of larval therapy of purulent infectious diseases. These studies in perspective can serve the basis for creation of the principally new drugs for struggle with usual and antibiotics-resistant bacterial infections.     

Cloning,recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly,Lucilia cuprina

While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non‐specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC₅₀ values at µg levels. Trimethoprim and pyrimethamine were 5–30‐fold less active. Relative IC₅₀ values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active‐site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.