Developmental differences in spatially distinct populations of the forensically relevant blow fly Lucilia sericata - About the comparability of developmental studies (and case work application).

The cosmopolitan blow fly Lucilia sericata is often used in forensic case work for estimating the minimum postmortem interval (PMImin). For this, the age of immature specimens developing on the dead body is calculated by measuring the time taken to reach the sampled developmental stage at a given temperature. To test whether regional developmental data of L. sericata is valid on a global scale, the time taken to reach different developmental stages was compared between a population from Mexico and one from Germany at two different constant temperatures. The German population of L. sericata was collected in Frankfurt/Main, while the Mexican population originated near Oaxaca de Juarez and was transported to Germany in the larval stage. Only the F1 generation was used to avoid adaption of the Mexican flies. Eggs were immediately placed at 20 °C and 30 °C. Five times 30 freshly eclosed larvae per replicate (n = 5) were then transferred to a cup of minced meat in separate containers. The larvae were checked every 8 h for migration, pupariation or emergence of adult flies. The time at which the first individual and 50 % of the specimens per container entered each of these stages, was recorded. Significant differences in the time of development between the two populations were observed at both temperatures. At 20 °C, the first specimens of the Mexican population reached all developmental stages a little (< 1 day to < 2 days) earlier than the German L. sericata. At 30 °C, the Mexican flies also reached the post-feeding stage slightly earlier (0.2 days). However, at 30 °C, the German flies started pupariation significantly earlier (after 5 days) than the Mexican flies (6.9 days) and the adults from Germany also emerged earlier (10.5 days compared to 13.1 days). The same pattern was observed when looking at 50 % of the total number of specimens per container. A comparison with previously published developmental studies was difficult as the experimental design varied widely between studies. However, the results were within the range of most studies. Our study has shown that age estimation can vary widely depending on the population on which the reference data used for the calculations are based. This highlights the importance of using local and population-specific developmental data for estimating the age of blow flies in case work.

The role of medical-grade maggots in facilitating healing of diabetic foot ulcers in Kano, northern Nigeria: a case series.

OBJECTIVE: Approximately 13% of people living with diabetes develop one or more ulcers during the course of the disease, and diabetic foot ulcer (DFU) is responsible for >60% of lower limb amputations worldwide. This case series aimed to demonstrate the effectiveness of medical-grade maggots on DFUs in promoting wound healing and reducing related hospital stays in northern Nigeria. METHOD: Maggot debridement therapy (MDT) was applied to the DFUs of patients who consented to this treatment between January-August 2021 at the Orthopaedic Unit of the Aminu Kano Teaching Hospital (AKTH), Kano, Nigeria. Sterile first instar larvae of Lucilia sericata were obtained indigenously and applied using the confinement (free-range) method under aseptic procedure. RESULTS: A total of 15 patients with DFUs of Wagner classification grades III (33.3%) and IV (66.7%), were seen and documented. The patients included 10 (66.7%) females and five (33.3%) males, giving a female:male ratio of 2:1. The mean age (±standard deviation) of the respondents was 51.6±10.8 years. The surface area of the wounds ranged from 24-140cm2, with a median value of 75cm2. Among the patients, 60% had two cycles of MDT, with a median duration of five days. Most of the wounds (53.3%) were debrided within five days; 13.3% (two wounds) took seven days to be fully debrided. A paired sample t-test showed a statistically significant association between wound surface area and therapy duration (t=8.0; p=0.000) and between wound surface area and cycles of therapy (t=8.3; p=0.000). Before maggot application, 14 (93.3%) DFUs were completely (100%) covered with slough and only one (6.7%) was 95% covered with slough. After maggot application, 10 (66.7%) wounds were completely debrided while five (33.3%) wounds required bedside surgical debridement to achieve complete debridement. A paired sample t-test showed statistically significant difference between the pre- and post-MDT slough covering the wounds (t=45.1; p=0.000). CONCLUSION: In this case series, MDT was successfully used in the debridement of DFUs, which facilitated the healing process with an encouraging clinical outcome.

Eye and hair color prediction of human DNA recovered from Lucilia sericata larvae.

Forensic entomological evidence is employed to estimate minimum postmortem interval (PMImin), location, and identification of fly samples or human remains. Traditional forensic DNA analysis (i.e., STR, mitochondrial DNA) has been used for human identification from the larval gut contents. Forensic DNA phenotyping (FDP), predicting human appearance from DNA-based crime scene evidence, has become an established approach in forensic genetics in the past years. In this study, we aimed to recover human DNA from Lucilia sericata (Meigen 1826) (Diptera: Calliphoridae) gut contents and predict the eye and hair color of individuals using the HIrisPlex system. Lucilia sericata larvae and reference blood samples were collected from 30 human volunteers who were under maggot debridement therapy. The human DNA was extracted from the crop contents and quantified. HIrisPlex multiplex analysis was performed using the SNaPshot minisequencing procedure. The HIrisPlex online tool was used to assess the prediction of the eye and hair color of the larval and reference samples. We successfully genotyped 25 out of 30 larval samples, and the most SNP genotypes (87.13%) matched those of reference samples, though some alleles were dropped out, producing partial profiles. The prediction of the eye colors was accurate in 17 out of 25 larval samples, and only one sample was misclassified. Fourteen out of 25 larval samples were correctly predicted for hair color, and eight were misclassified. This study shows that SNP analysis of L. sericata gut contents can be used to predict eye and hair color of a corpse.

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.

Variables Associated with Attitudes toward Biodebridement Using Lucilia sericata Larvae in a Group of Nurses.

Despite numerous studies and recommendations, the acceptance of treatments involving medicinal maggots in many clinics has been slow. Several factors may account for this, including the gender of nurses administering the treatment, their level of work experience, and their perceived level of personal stress. The aim of the study was to assess the impact of selected variables (gender, work experience, stress level) on the readiness of nurses to administer maggot debridement therapy (MDT), which is a form of biodebridement. The study population was a cohort of 290 wound care nurses providing specialist care for patients with chronic wounds. It was assumed that the identified variables may determine the implementation of larval therapy in everyday professional practice. A subsample of 35 men and 35 women was further analyzed to determine if gender, work experience, and/or personal stress levels were correlated with attitudes towards the utilization of maggots in biodebridement. Assessment tools included the Perceived Stress Scale (PSS-10) and the MDT 10 Perception Assessment Questionnaire, a protocol by which the subject ranked six wound photographs in order of repulsiveness and responded to questions regarding demographic variables, which include education and work experience. The visual perception of pictures of a wound with larvae is indirectly an indicator of the attitude towards larval therapy. Selection of the photograph with maggots on the wound as the most repulsive image was associated with a personal appraisal of not being ready to implement maggot therapy (chi-square = 8.430, p = 0.015). Low work experience (chi-square = 14.039, df = 4, p = 0.007), and low readiness for MDT (chi-square = 8.430, df = 2, p = 0.015) were also associated with unpreparedness to administer maggot therapy. Neither gender nor perceived stress level were exclusively associated with disgust for maggots or lack of readiness to implement MDT. Low professional experience and a deficit of knowledge in maggot therapy may negatively affect the readiness of nurses to administer biodebridement. Gender and personal stress levels do not affect nurses' readiness to utilize larval therapy.

From open wound treatment to primary soft-tissue non-Hodgkin lymphoma diagnosis; a challenging case report.

Primary soft-tissue extranodal lymphomas are rare clinical entities. By their natures, they can cause significant swelling around the affected extremities, and they can be easily misdiagnosed with other conditions like sarcomas. We share an unfortunate experience of a young male patient who was admitted to another clinic with complaints of a large mass in his right thigh, the patient has been diagnosed with anaplastic pleomorphic sarcoma, and he was scheduled for surgery. The patient refused the operation and was admitted to our emergency clinic with an open wound on his right thigh. Successful open wound management was achieved with antibiotic therapy, tissue debridement, larvae therapy, and Vacuum-assisted closure. Meanwhile, the pathologic re-examination revealed diffuse large B-cell lymphoma. After an uneventful follow-up, the patient was referred to the hematology clinic. This case highlights the importance of considering alternative diagnoses before making surgical intervention decisions that may result in unpleasing consequences.

Mitochondrial genomic investigation reveals a clear association between species and genotypes of Lucilia and geographic origin in Australia.

BACKGROUND: Lucilia cuprina and L. sericata (family Calliphoridae) are globally significant ectoparasites of sheep. Current literature suggests that only one of these blowfly subspecies, L. cuprina dorsalis, is a primary parasite causing myiasis (flystrike) in sheep in Australia. These species and subspecies are difficult to distinguish using morphological features. Hence, being able to accurately identify blowflies is critical for diagnosis and for understanding their relationships with their hosts and environment. METHODS: In this study, adult blowflies (5 pools of 17 flies; n = 85) were collected from five locations in different states [New South Wales (NSW), Queensland (QLD), Tasmania (TAS), Victoria (VIC) and Western Australia (WA)] of Australia and their mitochondrial (mt) genomes were assembled. RESULTS: Each mt genome assembled was ~ 15 kb in size and encoded 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a control region. The Lucilia species mt genomes were conserved in structure, and the genes retained the same order and direction. The overall nucleotide composition was heavily biased towards As and Ts-77.7% of the whole genomes. Pairwise nucleotide diversity suggested divergence between Lucilia cuprina cuprina, L. c. dorsalis and L. sericata. Comparative analyses of these mt genomes with published data demonstrated that the blowflies collected from sheep farm in TAS clustered within a clade with L. sericata. The flies collected from an urban location in QLD were more closely related to L. sericata and represented the subspecies L. c. cuprina, whereas the flies collected from sheep farms in NSW, VIC and WA represented the subspecies L. c. dorsalis. CONCLUSIONS: Phylogenetic analyses of the mt genomes representing Lucilia from the five geographic locations in Australia supported the previously demonstrated paraphyly of L. cuprina with respect to L. sericata and revealed that L. c. cuprina is distinct from L. c. dorsalis and that L. c. cuprina is more closely related to L. sericata than L. c. dorsalis. The mt genomes reported here provide an important molecular resource to develop tools for species- and subspecies-level identification of Lucilia from different geographical regions across Australia.

How to prove neglect in the context of the post-mortem examination.

Understanding the causes, extent, and period of neglect is not only a medical but also a forensic task when it comes to legal investigations. In this study, we evaluated 46 autopsied cases where there was clear evidence of physical neglect during the last period of the deceased's life. The age of the deceased ranged from 21 to 96 years; most of them were female (71.7%). The majority of cases (89.9%) took place in a domestic environment, with partners or relatives providing care. The most frequent post-mortem findings were pressure sores, followed by inflammatory skin changes, and signs of malnutrition and dehydration. Neglect was the cause or co-cause of death in 23% of the cases. More than half of the deceased showed severe contamination of the skin surface by excrement, and in almost 40% of the cases, fly infestation was found. The majority of insects belonged to the group of house flies (Diptera: Muscidae), mainly the common house fly, Musca domestica. By analyzing the entomological evidence, it was possible to prove an insect infestation period of at least several days ante-mortem. Since the period of neglect may be relevant in terms of legal proceedings, the present work demonstrates the particular importance of insect traces in providing this evidence. While prosecution and conviction of caregivers remain challenging, it is all the more essential that entomology and legal medicine collaborate on the analysis of findings of neglect.

[Diagnostic atypique d'une myiase]. / Atypical diagnosis of myiasis.

Muscoid larvae were observed on self-medicated dressing material loaded with purulent material taken from a 91-year-old hospital patient. These larvae were identified as Lucilia sericata. However, no larvae were found in the patient's tissues. The observation of larvae on dressings should not automatically lead to a diagnosis of cutaneous myiasis.

Detection of Three Opioids (Morphine, Codeine and Methadone) and Their Metabolites (6-Monoacetylmorphine and 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) in Larvae of Lucilia sericata Species by UHPLC-TF-MS and Validation.

Insects on corpses could be a useful tool for the detection of exogenous substances such as drugs of abuse. The identification of exogenous substances in carrion insects is critical for proper estimation of the postmortem interval. It also provides information about the deceased person that may prove useful for forensic purposes. High-performance liquid chromatography coupled with Fourier transform mass spectrometry is a highly sensitive analytical technique that can identify substances even at very low concentrations, such as in the case of searching for exogenous substances in larvae. In this paper, a method is proposed for the identification of morphine, codeine, methadone, 6-monoacetylmorphine (6-MAM) and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the larvae of Lucilia sericata, a common carrion fly widely distributed in temperate areas of the world. The larvae, which were reared on a pig meat substrate, were killed once they reached their third stage by immersion in hot water at 80 °C and aliquoted into 400 mg samples. The samples were fortified with 5 ng of morphine, methadone and codeine. After solid-phase extraction, the samples were processed with a liquid chromatograph coupled to a Fourier transform mass spectrometer. This qualitative method has been validated and tested on larvae from a real case. The results lead to the correct identification of morphine, codeine, methadone and their metabolites. This method could prove useful in cases where toxicological analysis must be conducted on highly decomposed human remains, where biological matrices are very limited. Furthermore, it could help the forensic pathologist to better estimate the time of death, as the growth cycle of carrion insects can undergo changes if exogenous substances are taken.

Nasopharyngeal Myiasis in Intensive Care Unit (ICU) Patients: Report of Two Cases.

Nosocomial myiasis is a rare event that has a higher incidence in the hospitals of poor and developing countries. The presence of nosocomial myiasis reflects the need for improved medical facilities and increased awareness among healthcare personnel. Severely ill patients are more susceptible, such as those with impaired consciousness, paralysis, and underlying diseases. The two cases here in described represent the first report of nosocomial myiasis in the Kurdistan Province, in Western Iran and one of them is the first report of myiasis involving a COVID-19-infected patient. The causal agent was Lucilia sericata. The taxonomical identification of the larvae of the second and third instar was based on the morphology of the cephaloskeleton, anterior spiracles, and peritreme plaques.

The in vitro and in vivo effects of Lucilia sericata larval secretions on Leishmania major.

The emerging of drug resistant against Leishmania parasites prompts scientists to seek for novel therapeutic strategies against theses infectious protozoan parasites. Among different strategies, the use of larvae secretions could be suggested as a possible therapy with low side effects. Accordingly, the current study evaluated the in vitro and in vivo effects of Lucilia sericata larval secretions on Leishmania major, the causative agent of cutaneous leishmaniasis (CL). After preparation of L. sericata larval stages (L2 and L3) secretions, the potential effects of secretions were evaluated against L. major promastigotes and amastigotes (in vitro) using MTT assay. The cytotoxicity effects of secretions were also checked on uninfected macrophages. In addition, in vivo experiments were also conducted to investigate the effects of larvae's secretions on the CL lesions induced in the BALB/c mice. Although the increased concentration of larvae secretions exhibited a direct effect on the promastigotes proliferation (viability), contrarily, L2 secretions at a concentration of 96 µg/ml represented the highest inhibitory effect on parasite (amastigotes) burden in infected macrophages. Interestingly, L3 secretions > 60 µg/ml induced inhibitory effects on amastigotes. The results relevant to the cytotoxicity effects of L2 and L3 secretions on uninfected-macrophages showed a dose dependent correlation. In vivo results were also significant, compared to the positive control group. This study suggested the plausible inhibitory effects of L. sericata larvae's secretions on the L. major amastigotes and CL lesions progression. It seems that the characterization of all effective components/proteins in the larvae secretions and their specific targets in parasite structure or in cell (macrophage) responses could further reveal more details regarding the anti-leishmanial properties of these compounds.

Defensins of Lucilia sericata Larvae and Their Influence on Wound Repair Processes in Practical Assessment-A Study of Three Cases.

Bacteria inhabiting chronic wounds form a biofilm that prolongs and slows down the healing process. Increasingly common antibiotic resistance requires clinicians to search for effective and alternative treatment methods. Defensins are the most common antimicrobial peptides capable of eradicating pathogens. Their discovery in maggot secretions allowed for a broader understanding of the healing mechanisms, and approving the use of Lucilia sericata fly larvae in the treatment of infected wounds resulted in an effective and safe procedure. The aim of the study was to present the possibility of biofilm elimination in a chronic wound by means of medical maggots (Lucilia sericata) with the example of three selected clinical cases. The observation included three women who met the inclusion criterion of having venous insufficiency ulcers with inhibited regeneration processes. Medical maggots were applied in a biobag for three days, and observation was conducted for 21 consecutive days. In 2 cases, a significant elimination of necrotic tissue from the wound bed with local granulation tissue was observed 72 h after application of a larvae colony on the wounds. In 1 case, the application of the larvae accelerated the repair process by reducing the wound area by approximately 40% at the time of observation. The formation of biofilm in a chronic wound is one of the main causes of disturbances in its effective healing. Combining procedures (scraping, antiseptic compresses, MDT, NPWT) related to wound debridement increases the effectiveness of biofilm elimination. The use of medical maggots is a safe and effective method of choice, and it enhances the processes of debridement. However, confirmed indisputable data on their effectiveness and frequency of use in the process of stimulating healing processes are still not available in the literature.

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).

Larval Therapy and Larval Excretions/Secretions: A Potential Treatment for Biofilm in Chronic Wounds? A Systematic Review.

Chronic wounds present a global healthcare challenge and are increasing in prevalence, with bacterial biofilms being the primary roadblock to healing in most cases. A systematic review of the to-date knowledge on larval therapy's interaction with chronic-wound biofilm is presented here. The findings detail how larval therapy-the controlled application of necrophagous blowfly larvae-acts on biofilms produced by chronic-wound-relevant bacteria through their principle pharmacological mode of action: the secretion and excretion of biologically active substances into the wound bed. A total of 12 inclusion-criteria-meeting publications were identified following the application of a PRISMA-guided methodology for a systematic review. The findings of these publications were qualitatively analyzed to provide a summary of the prevailing understanding of larval therapy's effects on bacterial biofilm. A further review assessed the quality of the existing evidence to identify knowledge gaps and suggest ways these may be bridged. In summary, larval therapy has a seemingly unarguable ability to inhibit and degrade bacterial biofilms associated with impaired wound healing. However, further research is needed to clarify and standardize the methodological approach in this area of investigation. Such research may lead to the clinical application of larval therapy or derivative treatments for the management of chronic-wound biofilms and improve patient healing outcomes at a time when alternative therapies are desperately needed.

The therapeutic effect of larval saliva and hemolymph of Lucilia sericata on the treatment of Leishmania major lesion in BALB/c mice946.

BACKGROUND: Treatment of cutaneous leishmaniasis (CL) remains a major challenge for the public health and medical community. It has been claimed that natural compounds derived from fly larvae have anti-leishmania properties against some species of Leishmania. The present study aimed at assessing the in vitro effects of larval products of Lucilia sericata against the promastigote and intracellular amastigote forms of Leishmania major. Also, the therapeutic effect of larval products on lesions induced by L. major infection was evaluated in BALB/c mice models. METHODS: Parasite specimens and macrophage cells were exposed to varying concentrations of larval products for 24-120 h. Lesion progression and parasite load were investigated in the models to assess the therapeutic effects of the products. RESULTS: The larval products displayed more potent cytotoxicity against L. major promastigotes. The IC50 values for larval saliva and hemolymph were 100.6 and 37.96 ug/ml, respectively. The IC50 of glucantime was 9.480 ug/ml. Also, the saliva and hemolymph of L. sericata exhibited higher cytotoxicity against the promastigotes of L. major but were less toxic to the macrophage cells. Treatment with leishmanicidal agents derived from larvae of L. sericata decreased the infection rate and the number of amastigotes per infected host cell at all concentrations. Lesion size was significantly (F (7, 38) = 8.54, P < 0.0001) smaller in the treated mice compared with the untreated control group. The average parasite burden in the treated mice groups (1.81 ± 0.74, 1.03 ± 0.45 and 3.37 ± 0.41) was similar to the group treated with a daily injection of glucantime (1.77 ± 0.99) and significantly lower (F (7, 16) = 66.39, P < 0.0001) than in the untreated control group (6.72 ± 2.37). CONCLUSIONS: The results suggest that the larval products of L. sericata were effective against L. major parasites both in vivo and in vitro. However, more clinical trial studies are recommended to evaluate the effects of these larval products on human subjects.

Maggot debridement therapy and complementary wound care: a case series from Nigeria.

OBJECTIVE: Maggot debridement therapy (MDT) is an emerging procedure involving the application of sterile maggots of the Dipteran species (commonly Lucilia sericata) to effect debridement, disinfection and promote healing in wounds not responding to antimicrobial therapy. Data on MDT in sub-Saharan Africa (including Nigeria) are scarce. This study aimed to use medicinal grade maggots as a complementary method to debride hard-to-heal necrotic ulcers and thereby promote wound healing. METHOD: In this descriptive study, we reported on the first group of patients who had MDT at Aminu Kano Teaching Hospital (AKTH), a tertiary hospital in northern Nigeria. The first instar larvae of Lucilia sericata were applied using the confinement (free-range) maggot therapy dressing method under aseptic conditions. RESULTS: Diabetic foot ulcer (DFU) grade III-IV constituted more than half of the wounds (53.3%), followed by necrotising fasciitis (30%), and post-traumatic wound infection (10%). Others (6.7%, included pyomyositis, surgical site infection and post traumatic wound infection). The median surface area of the wounds was 56cm2. Of the 30 patients, half (50%) had two MDT cycles with a median time of four days. Of the wounds, 22 (73%) were completely debrided using maggots alone while eight (27%) achieved complete debridement together with surgical debridement. Wound culture pre-MDT yielded bacterial growth for all the patients and Staphylococcus aureus was the predominant isolate in 17 wounds (56.7%) while Pseudomonas aeruginosa and Streptococcus pyogenes were predominant in five wounds (16.7%) each. Only four (13.3%) wound cultures yielded bacterial growth after MDT, all Staphylococcus aureus. CONCLUSION: A good prognosis was achieved post-MDT for various wounds. MDT effectively debrides and significantly disinfects wounds involving different anatomical sites, thus enhancing wound healing and recovery. MDT is recommended in such wounds.

Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae).

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Anti-inflammatory and wound healing potential of medicinal maggot excretions/secretions at the ocular surface.

PURPOSE: In the skin, Lucilia sericata maggot excretions/secretions (ES) accelerate wound healing and limit inflammation. This study aimed to determine whether ES have similar beneficial effects at the ocular surface. METHODS: Human corneal epithelial cells (HCEC) were cultured with ES and cell viability was determined by the MTT assay. Additionally, mRNA expression of growth factors, antimicrobial peptides (AMPs) and cytokines was assessed by qPCR. ES ability to modulate TLR-induced IL-6 and IL-8 expression was determined by qPCR and ELISA. ES potential to promote corneal healing was evaluated in vitro by a migration assay in HCEC, and in vivo using a mouse model. RESULTS: ES did not impair HCEC viability up to 25 µg/ml. Among the factors evaluated, only hBD-2 was upregulated (2.5-fold) by 1.5 µg/ml ES after 6 hrs (P = 0.04). In HCEC, ES reduced Poly I:C-induced IL-6 and IL-8 mRNA (P ≤ 0.001) and protein (P ≤ 0.0001) expression. A similar effect was observed with Flagellin (TLR5 agonist) but it was less robust for FSL-1 (TLR2/6 agonist) and Pam3CSK4 (TLR1/2 agonist). The greatest in vitro migration effect was observed with 6.2 µg/ml ES after 44 hrs where gap area compared to vehicle was 53.3 ± 3.7% vs. 72.6 ± 5.4% (P = 0.001). In the mouse model, the maximum healing effect was present with 1.5 µg/ml ES after 12 hrs with a wound area of 19.0 ± 2.7% vs. 60.1 ± 21.6% (P = 0.003) or 77% reduction of the wound area compared to the negative control. CONCLUSIONS: ES significantly reduce in vitro TLR-induced production of inflammatory cytokines and promote corneal wound healing.

Carrion flies (Insecta: Diptera) found on human cadavers in Chiba prefecture, Honshu, Japan, with the first record of Fannia prisca from a human corpse.

Forensic entomology (FE) involves the collection and analysis of necrophagous insects and arthropods for postmortem interval estimation based on their growth and succession. In Japan, research on FE is limited, and the predominant species of necrophagous insects and their distribution have not been clarified. The present study aimed to clarify the actual situation of insects, targeting Diptera collected from human cadavers, in Chiba Prefecture. We targeted the cases for which specimens could be collected from forensic autopsies conducted at the Legal Medicine Department of Chiba University Graduate School of Medicine or from human bodies handled by the Chiba Police in 2019. We specifically chose cases from which adult insects could be bred. Fifty cases were applicable, and 47 cases were successful in rearing adult flies. Sixteen species of Diptera were identified: six species of Calliphoridae, four species of Sarcophagidae, and a few species of Fanniidae, Muscidae, Phoridae, Piophilidae, and Stratiomyidae. The most frequently observed species were Lucilia sericata (20 cases) and Chrysomya pinguis (18 cases). For the first time, globally, we recorded the presence of Fannia prisca in a human cadaver. In conclusion, this is the first report to reveal the presence of carrion flies in human death cases in Chiba Prefecture, Japan. Research in FE is important in the context of the Japanese natural and social environment since this can aid forensic investigations conducted by authorities.