Green Bottle Fly Lucilia Sericata Research Articles

Morphological studies of fly puparia (Diptera: Calliphoridae) in the soil from a Joseon Dynasty grave in Korea.

Archaeoentomological investigations were conducted on soil contents from a grave belonging to the Joseon Dynasty as part of the Urban Environment Maintenance Project (UEMP) in Cheongjin 12-16 dong (districts), Jongno-gu, Seoul, Korea, from December 01, 2008 to February 19, 2011. A total of 28 insect puparia with hard shells of the common green bottle fly Lucilia sericata were identified in the soil. Evidence suggested that the corpse was placed outside for some days instead of being buried immediately after death. This is the first report of fly puparia in soil samples from a tomb of the Joseon Dynasty during 16-17 AD in Korea. Our findings may help determine the timeframe of burial and offer archaeological insights into the funerary customs of the period.

Vagococcus luciliae sp. nov., isolated from the common green bottle fly Lucilia sericata.

The genus Vagococcus belongs to the family Enterococcaceae (order Lactobacillales) and is closely related to the genus Enterococcus. Currently, 19 species of the genus have been validly named. In this study, we isolated strain G314FT from the common green bottle fly Lucilia sericata collected in Germany. Sequencing of its almost-complete 16S rRNA gene revealed that the isolate belongs to the genus Vagococcus, being closely related to Vagococcus bubulae SS1994T with high sequence identity (99.50 %), followed by Vagococcus martis D7T301T (98.86 %), Vagococcus vulneris SS1995T (98.71 %), Vagococcus teuberi DSM 21459T (98.64 %), Vagococcus silagei 2B-2T (98.64 %) and Vagococcus penaei CD276T (98.64 %). Genome sequencing of strain G314FT was performed by a combination of Illumina and Oxford Nanopore technology, yielding a circular genome with a size of 2 139 468 bp and an 11 kb plasmid. Average nucleotide identity and digital DNA-DNA hybridization values were calculated between G314FT and its closest-related taxa, and found to be <91 % and <40 %, respectively, thus strongly supporting that strain G314FT represents a novel species of the genus Vagococcus. Phylogenetic and core protein-based phylogenomic trees revealed that G314FT was closely related to a group of three species, V. bubulae SS1994T, V. martis D7T301T and V. teuberi DSM 21459T. Comparatively, the genome of G314FT is the smallest in the group of the four related species, and the biochemical pathway comparison using BlastKOALA revealed that G314FT has lost some amino acid biosynthetic proteins; however, it has gained enzymes for carbohydrate metabolism. Phenotypically, G314FT was consistent with other species of the genus Vagococcus including a negative catalase reaction and non-motility. Using the polyphasic approach, our data supports that the isolate represents a new species, for which we propose the name Vagococcus luciliae G314FT (=DSM 112651T= CCM 9164T).

Treatment of a Chronical Infected Wound in a Cat with Sterile Lucilia Sericata Larvae

The larvae of the green bottle fly Lucilia Sericata have been successfully used throughout history as a debridement method for chronic and infected wounds. In 1929, Alexander Fleming's discovery of penicillin dramatically reduced the use of surgical larvae, but soon after the emergence of antibiotic resistance, sterile larvae re-emerged as an alternative in treatment. For a long time, sterile larvae applications were neglected in veterinary medicine. However, recently, both in the world and in Turkey, MDT is seen as an effective treatment method for chronic wounds of animals. The case of this case report is a 1-yearold male stray cat brought to a private clinic by a benefactor with a chronic infectious wound of 2.5x 2.5 cm and 0.5 cm in depth extending from the distal right hind leg cruris to the region of the ossa tarsi bones. It was decided to apply MDT to the wound using sterile 2nd instar Lucilia Sericata larvae. Sterile 2nd instar larvae were placed in the wound area daily and after 24 hours the larvae were removed, the area was cleaned and then new larvae were placed. Larval treatment was applied 7 times and until the last larva application, the large and infected wound gradually shrank and 100% healing was achieved. In this study, it was reported that Lucilia Sericata larvae showed high recovery in the treatment of chronic wounds of animals and the importance of evaluating them among treatment alternatives was emphasized.

Effect of increased ambient temperature on seasonal generation number in Lucilia sericata (Diptera, Calliphoridae)

Abstract Global climate change and, specifically, rising temperatures, may increase the number of generations of necrophagous insects. The common green bottle fly Lucilia sericata (Meigen, 1826) (Diptera, Calliphoridae) ranks among the most important cosmopolitan necrophagous insects that utilize corpses and cause myiasis in farm animals and humans. Based on the data simulations, the use of accumulated degree-hours enables to calculate the number of generations of this forensically important species of blowfly with a greater accuracy than before, considering short-term increases of temperature at the boundary of the cold and warm seasons. The number of generations of L. sericata has increased from 7.65 to 8.46 in the Ukrainian steppe zone over the last 15 years, while the active developmental period of this species has increased by 25 days due to earlier start in spring. The average temperature increase of 1 °C increased the number of generations of L. sericata by 0.85. With a global climate change following the Representative Concentration Pathway (RCP) 4.5 scenario (average temperature increase of 2.4 °C), adopted by the Intergovernmental Panel on Climate Change, by 2100 the number of generations of L. sericata in a simulated ecosystem will increase by 2.0 to 9.0 generations per year.

Tribolium castaneum defensin 1 kills Moraxella catarrhalis in an in vitro infection model but does not harm commensal bacteria

ABSTRACT Moraxella catarrhalis is a bacterial pathogen that causes respiratory tract infections in humans. The increasing prevalence of antibiotic-resistant M. catarrhalis strains has created a demand for alternative treatment options. We therefore tested 23 insect antimicrobial peptides (AMPs) for their activity against M. catarrhalis in a human in vitro infection model with primary macrophages, and against commensal bacteria. Effects on bacterial growth were determined by colony counting and growth curve analysis. The inflammatory macrophage response was characterized by qPCR and multiplex ELISA. Eleven of the AMPs were active against M. catarrhalis. Defensin 1 from the red flour beetle Tribolium castaneum significantly inhibited bacterial growth and reduced the number of colony forming units. This AMP also showed antibacterial activity in the in vitro infection model, reducing cytokine expression and release by macrophages. Defensin 1 had no effect on the commensal bacteria Escherichia coli and Enterococcus faecalis. However, sarcotoxin 1 C from the green bottle fly Lucilia sericata was active against M. catarrhalis and E. coli, but not against E. faecalis. The ability of T. castaneum defensin 1 to inhibit M. catarrhalis but not selected commensal bacteria, and the absence of cytotoxic or inflammatory effects against human blood-derived macrophages, suggests this AMP may be suitable for development as a new therapeutic lead against antibiotic-resistant M. catarrhalis.

Proline-Rich Antimicrobial Peptides in Medicinal Maggots of Lucilia sericata Interact With Bacterial DnaK But Do Not Inhibit Protein Synthesis.

In the search for new antibiotics to combat multidrug-resistant microbes, insects offer a rich source of novel anti-infectives, including a remarkably diverse array of antimicrobial peptides (AMPs) with broad activity against a wide range of species. Larvae of the common green bottle fly Lucilia sericata are used for maggot debridement therapy, and their effectiveness in part reflects the large panel of AMPs they secrete into the wound. To investigate the activity of these peptides in more detail, we selected two structurally different proline rich peptides (Lser-PRP2 and Lser-PRP3) in addition to the α-helical peptide Lser-stomoxyn. We investigated the mechanism of anti-Escherichia coli action of the PRPs in vitro and found that neither of them interfered with protein synthesis but both were able to bind the bacterial chaperone DnaK and are therefore likely to inhibit protein folding. However, unlike Lser-stomoxyn that permeabilized the bacterial membrane by 1% at the low concentration (0.25 µM) neither of the PRPs alone was able to permeabilize E. coli membrane. In the presence of this Lser-stomoxyn concentration significant increase in anti-E. coli activity of Lser-PRP2 was observed, indicating that this peptide needs specific membrane permeabilizing agents to exert its antibacterial activity. We then examined the AMPs-treated bacterial surface and observed detrimental structural changes in the bacterial cell envelope in response to combined AMPs. The functional analysis of insect AMPs will help select optimal combinations for targeted antimicrobial therapy.

Antibacterial Properties of Larval Secretions of the Blowfly, Lucilia sericata (Diptera: Calliphoridae)

Secretions aseptically collected from larvae or maggots of the green bottle fly Lucilia sericata (Meigen) (Diptera: Calliphoridae) exhibit antimicrobial activity along with other activities beneficial for wound healing. With the rise of multidrug-resistant bacteria. New approaches for identifying the active compounds responsible for the antimicrobial activity were examined. Five different pathogenic bacterial strains were used in this study, Three of them are gram positive bacteria, namely: Staphylococcus aureus, streptococcus sp, and proteus sp and two of them are gram negative bacteria, namely: E. coli. and Salmonella typhi. Considering the activity against organisms typically associated with clinical infection, may be a source of novel antibiotic-like compounds that may be used for infection control. Therefore, the aim of this study was to use a novel approach to investigate the output of secreted proteins from the maggots under conditions mimicking clinical treatments.Results revealed that the secretions aseptically markedly inhibit the growth of both gram-positive and gram-negative bacteria.

Effects of Floral Scent, Color and Pollen on Foraging Decisions and Oocyte Development of Common Green Bottle Flies.

The common green bottle fly Lucilia sericata (Meigen) and other filth flies frequently visit pollen-rich composite flowers such as the Oxeye daisy, Leucanthemum vulgare Lam. In laboratory experiments with L. sericata, we investigated the effect of generic floral scent and color cues, and of Oxeye daisy-specific cues, on foraging decisions by recently eclosed flies. We also tested the effect of a floral pollen diet with 0–35% moisture content on the ability of females to mature their oocytes. Our data indicate that (1) young flies in the presence of generic floral scent respond more strongly to a uniformly yellow cue than to any other uniform color cue (green, white, black, blue, red) except for ultraviolet (UV); (2) the floral scent of Oxeye daisies enhances the attractiveness of a yellow cue; and (3) moisture-rich pollen provides nutrients that facilitate ovary maturation of flies. With evidence that L. sericata exploits floral cues during foraging, and that pollen can be an alternate protein source to animal feces and carrion, Pollen apparently plays a major role in the foraging ecology of L. sericata and possibly other filth flies. These flies, in turn, may play a significant role as pollinators, as supported by a recently published study.

Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge.

The larvae of the common green bottle fly Lucilia sericata (Diptera: Calliphoridae) have been used for centuries to promote wound healing, but the molecular basis of their antimicrobial, debridement and healing functions remains largely unknown. The analysis of differential gene expression in specific larval tissues before and after immune challenge could be used to identify key molecular factors, but the most sensitive and reproducible method qRT-PCR requires validated reference genes. We therefore selected 10 candidate reference genes encoding products from different functional classes (18S rRNA, 28S rRNA, actin, β-tubulin, RPS3, RPLP0, EF1α, PKA, GAPDH and GST1). Two widely applied algorithms (GeNorm and Normfinder) were used to analyze reference gene candidates in different larval tissues associated with secretion, digestion, and antimicrobial activity (midgut, hindgut, salivary glands, crop and fat body). The Gram-negative bacterium Pseudomonas aeruginosa was then used to boost the larval immune system and the stability of reference gene expression was tested in comparison to three immune genes (lucimycin, defensin-1 and attacin-2), which target different pathogen classes. We observed no differential expression of the antifungal peptide lucimycin, whereas the representative targeting Gram-positive bacteria (defensin-1) was upregulated in salivary glands, crop, nerve ganglion and reached its maximum in fat body (up to 300-fold). The strongest upregulation in all immune challenged tissues (over 50,000-fold induction in the fat body) was monitored for attacin-2, the representative targeting Gram-negative bacteria. Here we identified and validated a set of reference genes that allows the accurate normalization of gene expression in specific tissues of L. sericata after immune challenge.

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

Lucimycin, an antifungal peptide from the therapeutic maggot of the common green bottle fly Lucilia sericata

We report the identification, cloning, heterologous expression and functional characterization of a novel antifungal peptide named lucimycin from the common green bottle fly Lucilia sericata. The lucimycin cDNA was isolated from a library of genes induced during the innate immune response in L. sericata larvae, which are used as therapeutic maggots. The peptide comprises 77 amino acid residues with a molecular mass of 8.2 kDa and a pI of 6.6. It is predicted to contain a zinc-binding motif and to form a random coil, lacking β-sheets or other secondary structures. Lucimycin was active against fungi from the phyla Ascomycota, Basidiomycota and Zygomycota, in addition to the oomycete Phytophtora parasitica, but it was inactive against bacteria. A mutant version of lucimycin, lacking the four C-terminal amino acid residues, displayed 40-fold lower activity. The activity of lucimycin against a number of highly-destructive plant pathogens could be exploited to produce transgenic crops that are resistant against fungal diseases.

Maggots reveal a case of antemortal insect infestation

Insects on corpses can give a wide range of information in forensic investigations. The most common application is calculating when insects oviposited on a dead person to give an estimation of the postmortem interval [1]. Since the infestation of a dead body may be delayed by certain circumstances such as indoor locations [2] or wrapping [3], it is more accurate to talk about the colonization time. Insects, being restricted to a certain area, can give further hints to the investigators such as if a body has been moved to a different location after death; also, body parts of insects can link a suspect to a crime scene [4]. Another application is the detection of toxic substances in insects feeding from dead persons' tissues [5]. There have been several studies done according to the biology of these animals' living associated with corpses as well as their application in legal investigations in the last years [6–12]. Many necrophagous flies are attracted by the odor of decomposition [13], but for some, other odors such as those from urine, feces, and the associated bacterial fauna are quite attractive and stimulate oviposition [14, 15]. The infestation of the living by dipterous fly larvae is known as myiasis. Wounds or anogenital regions are often found being fed by maggots in cases of neglect in humans or animals [16–19]. In certain cases, the chronologically nonmatching infestation of the anogenital area and favorable sites for oviposition of flies being attracted by the dead, such as the face, can give both the information of how long somebody has been neglected as well as how long a person has been dead [14, 15, 20]. Flies of the genus Lucilia belong to one of the first and most abundant summer species on corpses in Europe [21] and play an important role as facultative parasite causing myiasis [22]. It has to be emphasized that blowfly maggots will mostly feed from necrotic tissues [23] and will seldom eat the fresh and healthy skin or organs in humans or animals which has been reported for other fly species such as those from the family Oestridae under mostly tropical circumstances [24]. This unique property of fly maggots is commonly used in the maggot debridement therapy for treating nonhealing wounds [25]. Sterile fly larvae are applied to such resulting in a cleaning, disinfection, and increased healing-rate of the damaged tissue. The green bottle fly Lucilia sericata is the usual species used in this form of wound management [26]. The following case of an elderly dead woman reports the possibility to distinguish between maggot infestation before and after death, helping to reconstruct the formerly unknown circumstances.

A novel approach to the antimicrobial activity of maggot debridement therapy

ObjectivesCommercially produced sterile green bottle fly Lucilia sericata maggots are successfully employed by practitioners worldwide to clean a multitude of chronic necrotic wounds and reduce wound bacterial burdens during maggot debridement therapy (MDT). Secretions from the maggots exhibit antimicrobial activity along with other activities beneficial for wound healing. With the rise of multidrug-resistant bacteria, new approaches to identifying the active compounds responsible for the antimicrobial activity within this treatment are imperative. Therefore, the aim of this study was to use a novel approach to investigate the output of secreted proteins from the maggots under conditions mimicking clinical treatments.MethodscDNA libraries constructed from microdissected salivary glands and whole maggots, respectively, were treated with transposon-assisted signal trapping (TAST), a technique selecting for the identification of secreted proteins. Several putative secreted components of insect immunity were identified, including a defensin named lucifensin, which was produced recombinantly as a Trx-fusion protein in Escherichia coli, purified using immobilized metal affinity chromatography and reverse-phase HPLC, and tested in vitro against Gram-positive and Gram-negative bacterial strains.ResultsLucifensin was active against Staphylococcus carnosus, Streptococcus pyogenes and Streptococcus pneumoniae (MIC 2 mg/L), as well as Staphylococcus aureus (MIC 16 mg/L). The peptide did not show antimicrobial activity towards Gram-negative bacteria. The MIC of lucifensin for the methicillin-resistant S. aureus and glycopeptide-intermediate S. aureus isolates tested ranged from 8 to >128 mg/L.ConclusionsThe TAST results did not reveal any highly secreted compounds with putative antimicrobial activity, implying an alternative antimicrobial activity of MDT. Lucifensin showed antimicrobial activities comparable to other defensins and could have potential as a future drug candidate scaffold, for redesign for other applications besides the topical treatment of infected wounds.

Formation of boldenone and boldenone-analogues by maggots of Lucilia sericata

Current evidence suggests that neo formation of the anabolic steroid boldenone (androsta-1,4-diene-17-ol-3-one) occurs in calves’ faecal material, making it difficult to distinguish between illegally administered boldenone and its potential endogenous presence. This strengthens the urgent need to elucidate the pathway leading to boldenone formation. In our laboratory, the invertebrate Neomysis integer (Crustacea, Mysidacea) was used since 2004 as an alternative model for the partial replacement of vertebrate animals in metabolisation studies with illegal growth promotors and veterinary drugs, e.g. boldenone. The present study evaluates the metabolic capacity of other invertebrates, the brine shrimp Artemia franciscana and maggots of the greenbottle fly Lucilia sericata. The first results indicate that maggots of L. sericata are able to convert phytosterols and –stanols, nowadays in substantial amounts added to animal feed, into androsta-1,4-diene-3,17-dione (ADD), the precursor of boldenone, at a yield of 0.10–0.14% (p<0.001, significance compared to endogenous excretion of maggots) but not to boldenone itself. Furthermore, β-testosterone, an endogenous hormone, was transformed into androst-4-ene-3,17-dione (AED), ADD and β-boldenone at a significant (p<0.001, significance compared to endogenous excretion of maggots) yield of circa 13%, 0.80% and 2.2%, respectively. In future studies these results are of value to further evaluate the use of maggots of L. sericata as an invertebrate model in metabolisation studies.

Antibacterial properties of larval secretions of the blowfly, Lucilia sericata

The antibacterial properties of secretions aseptically collected from larvae of the greenbottle fly Lucilia sericata (Meigen) (Diptera: Calliphoridae) were examined. These investigations revealed the presence of small (<1 kDa) antibacterial factor(s) within the larval secretions, active against a range of bacteria. These include the Gram-positive Staphylococcus aureus, both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA), Streptococcus pyogenes and to a lesser extent the Gram-negative Pseudomonas aeruginosa. These secretions were shown to be highly stable as a freeze-dried preparation and, considering the activity against organisms typically associated with clinical infection, may be a source of novel antibiotic-like compounds that may be used for infection control and in the fight against MRSA.

Lucilia sericata (Diptera: Calliphoridae) causing hospital-acquired myiasis of a traumatic wound

A case of traumatic nosocomial myiasis caused by the green bottle fly Lucilia sericata (Meigen, 1826) occurred in a patient hospitalized following a serious road traffic accident. The patient had suffered extensive polytrauma particularly in the facial area of the skull. A total of 50 larvae was discovered in the oral cavity, nose, paranasal sinuses and enucleated eye-socket. Projected timing indicated that the eggs were laid while the patient was hospitalized. The development of myiasis was facilitated by the mental and physical debility and dependency of the patient, numerous and deep facial necrotic wounds and a lengthy period of hot weather which led to prolonged open window ventilation of his room.