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Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major , is a neglected tropical disease affecting impoverished populations. Current treatments are limited by cost, resistance, and side effects, highlighting the need for affordable, sustainable interventions. Lucilia sericata larvae, used in maggot therapy, effectively treat chronic wounds through debridement, antimicrobial activity, and healing promotion. This study explores how L. sericata processes L. major and proposes its potential application in ZCL treatment. The life cycles of L. sericata and L. major were maintained in laboratory conditions. Larval-parasite interactions were tested across substrates [hen liver, rat spleen, Roswell Park Memorial Institute (RPMI) 1640 cell culture medium] and time intervals (30–240 minutes). Extracorporeal effects were evaluated using trypan blue exclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays; intracorporeal interactions via microscopy and nested-PCR targeting L. major rRNA genes. L. sericata excretion/secretion products and microbiota exhibited strong anti-leishmanial activity. Promastigotes were deformed within 1 hour post-exposure (hpe), fully inactivated at 4 hpe, and lysed by 6 hpe. In RPMI medium, the treatment group ( L. sericata  +  L. major ) showed significant reductions in active parasites and viable cells compared to controls after 4 hours. Microscopy revealed no parasites in larval guts, but PCR detected L. major DNA in all specimens, suggesting partial digestion. This study demonstrates that L. sericata can eliminate L. major through intra- and extra-oral digestion, supporting its potential as a biotherapeutic agent for ZCL-associated wounds. These findings offer a foundation for developing larval therapy protocols in dermatology. Further studies in animal models and clinical trials are required to validate this approach for managing ZCL.

Flies, especially those from the Calliphoridae family, play a crucial role in decomposition and are the first to colonize a cadaver. Firstly, accurate species identification is a prerequisite for entomological evidence-based calculation of postmortem interval (PMI). While morphological criteria for identifying the species of adult blow flies exist, there are either absent or inadequate keys for younger stages. In all phases of blow fly development, molecular identification offers a quick and accurate procedure. It is widely known that mitochondrial cytochrome oxidase subunit I has the capacity for molecular identification but is ineffective in certain species. This study was conducted to assess the effectiveness of the cytochrome oxidase 1 gene in the identification of seventeen different species of calliphorid flies involving four genera, Calliphora , Chrysomya , Lucilia , and Hemipyrellia . In West Bengal, 2,977 blow fly specimens were gathered from four distinct geo-climatic zones. COI barcodes were able to confirm morphological identification through low K2P intraspecific genetic divergences (0% to 1%) and moderate to high K2P interspecific genetic divergences (0.39% to 12.29%). The Neighbour-Joining (NJ) analysis demonstrated well-supported reciprocal monophyly among the species. The species grouping was in agreement with morphological and molecular identifications. The four delimitation methods, BIN, ASAP, PTP, and GMYC, used for species identification produced similar results and facilitated the proper identification of species. Therefore, it can be concluded that COI barcodes are a highly successful alternative for the molecular identification of blow flies, facilitating forensic cases and biodiversity research in India.

Phormia regina (Meigen, 1826; Diptera: Calliphoridae) is a Holarctic species that rapidly colonizes carcasses and has been used as an indicator for determining the minimum postmortem interval. However, studies using morphological methods to estimate the intrapuparial age of P. regina are lacking. In this study, morphological changes within the puparium were observed under a stereomicroscope at 7 constant temperatures ranging from 16 °C to 34 °C.The intrapuparial period was categorized into 12 substages. Morphological indicators, including compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, were recorded in detail. The observed morphological changes were divided into 6–10 substages, and the duration of each substage was also recorded in detail.The results of this study provide primary data for using the intrapuparial morphology of P. regina when pupae are collected at a crime scene and estimating the minimum postmortem interval.

Addressing global food waste requires innovative and sustainable solutions. This study investigates the potential of Chrysomya megacephala (Diptera: Calliphoridae) larvae to convert food waste into valuable byproducts, while evaluating the antimicrobial properties of larval extracts. Under controlled laboratory conditions, the larvae reduced the weight of food waste by 21.87%, demonstrating their effectiveness in waste degradation. The optimal food waste-to-sawdust ratio was identified as 10:1. The bioconversion process resulted in 111.60-fold increase in larval biomass when reared on food waste and a 153.20-fold increase on fresh pork liver, highlighting their efficiency in converting protein-rich substrates. Larval extracts demonstrated significant antimicrobial activity against Bacillus subtilis and Pseudomonas aeruginosa , with minimum inhibitory concentrations (MICs) of 100 µg/ml. Proteomic analysis revealed proteins with potential antimicrobial and antioxidative properties. Furthermore, the extracts promoted cell growth in vitro without showing cytotoxic effects on HaCaT cell lines, suggesting potential applications in wound healing and infection control. These findings highlight the capacity of C. megacephala larvae to reduce food waste while generating antimicrobial agents, offering a sustainable approach to waste management with promising implications in medical applications.

The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (β-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.

In the medico-legal application of forensic entomology, estimating the time of death is critical and traditionally relies on changes in observable traits of carrion feeding insect larvae. Traits such as size, weight, and morphology can be used to predict the insect specimen age and help define the minimum time since death. The blowfly Phormia regina Meigen (Diptera: Calliphoridae) is a key forensic insect, yet age estimation for older maggots in this and other carrion-feeding species is particularly challenging due to the limited morphological changes in the late-stage larvae. To enhance age-estimation precision, we employed transcriptomic profiling on blowfly maggots, aiming to identify genes as markers for time of death estimation. Our study characterized maggot development, reinforcing that weight and behavior cannot precisely determine age between 100 and 130 hours at 27.5 °C. We built a chromosomal scale annotated genome, establishing a reliable database for uncovering transcriptomic signatures during larval development. Applying differential gene expression analyses, weighted gene co-expression network analysis, and the generalized linear model, we identified nine candidate genes (y5078, y5076, agt2, ech1, dhb4, asm, gabd, acohc, ivd) that delineate the age of otherwise indeterminate maggots. This research introduces a molecular approach to address a longstanding problem in forensic entomology and promises to increase precision in determining the time of death at a crime scene.

The oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae), is a medically important synanthropic blow fly species characterized by its necrophagy and coprophagy, often observed near carrion and animal feces. Notably, C. megacephala always arrives at carcass earlier than other species. To elucidate the underlying mechanisms behind the host choice in C. megacephala , we present the chromosome-scale genome assembly for this species. The genome size is 816.79 Mb, with a contig N50 of 1.60 Mb. The Hi-C data were anchored to six chromosomes, accounting for 99.93% of the draft assembled genome. Comparative genomic analysis revealed significant expansions in pathways of ligand-gated ion channel activity, passive transmembrane transporter activity, and protein methyltransferase activity, which may be closely associated with host localization and oviposition. After identifying 69 odor-binding proteins (OBPs) in the assembled genome, phylogenetic analysis showed that DmelOBP99b and CmegOBP99b exhibited high homology. Transcriptome analysis demonstrated that the relative expression of CmegOBP99b was consistently the highest during the metamorphosis, and RT-qPCR further confirmed the similar results. Additionally, CmegOBP99b exhibited a strong binding affinity to DMDS (dimethyl disulfide) as determined by molecular docking. To determine the protein expression level of CmegOBP99b in various body parts, we prepared recombinant CmegOBP99b protein and anti- CmegOBP99b polyclonal antibodies. Western blot analysis showed that CmegOBP99b was significantly expressed in the female’s head compared to other parts, which is consistent with RT-qPCR results. Therefore, CmegOBP99b may be the primary odor-binding protein responsible for olfactory recognition and the behavioral coordination of C. megacephala . This study not only provides valuable insights into the molecular mechanisms of oviposition localization in C. megacephala but also facilitates further research into the genetic diversity and phylogeny of the Calliphoridae family.

Maggot therapy (MT) is the most common medical use of sterile fly larvae of Lucilia sericata and other species of the family Calliphoridae as an alternative to surgery and long-term antiseptic therapy in the treatment of deep and infected non-healing wounds. Effective and reliable MT requires an aseptic technique to prevent bacterial infection of the wound. However, due to the habitat of Calliphoridae flies, the outer surface of their eggs is often contaminated with bacteria. In this study, Lucilia sericata eggs were sterilized using two techniques. NaOCI (sodium hypochlorite), which is widely used as a sterilant (Sterilant 1-S1), and Lysol surface disinfectant, which is rarely used (Sterilant 2-S2), and a control (unsterilized) were chosen. Sterilization efficacy, egg survival, and mortality were evaluated comparatively at 0.05, 1, 2, and 3 (%) concentrations and 1-, 2-, 3-, and 4-min application times at each concentration, and bacterial growth in the post-sterilization environment was controlled. In the in vitro sterility efficiency and reliability test, no bacterial colonies were found in sterilized eggs transferred to tryptic soy agar (TSA) culture. In contrast, bacterial and fungal growth was detected in unsterilized eggs. Protocol S1 provided effective egg surface sterilization at a concentration of 0.05% with a 1-min exposure time. This treatment resulted in 85.12% survival rate and 14.88% egg mortality. Based on its optimal balance of low mortality, high viability, and effective sterilization, we recommend a 1-min immersion in 0.05% NaOCl solution for sterilization of Lucilia sericata eggs.

The genetic structure of forensically important blow fly (Brauer & Bergenstamm) (Diptera: Calliphoridae) populations has remained elusive despite high relatedness within wild-caught samples. This research aimed to determine if the implementation of a high-resolution spatiotemporal sampling design would reveal latent genetic structure among blow fly populations and to elucidate any environmental impacts on observed patterns of genetic structure. Adult females of the black blow fly, Phormia regina (Meigen) (Diptera: Calliphoridae), were collected from 9 urban parks in Indiana, USA over 3 yr and genotyped at 6 polymorphic microsatellite loci. The data analysis involved 3 clustering methods: principal coordinate analysis (PCoA), discriminant analysis of principal components (DAPC), and STRUCTURE. While the PCoA did not uncover any discernible clustering patterns, the DAPC and STRUCTURE analyses yielded significant results, with 9 and 4 genetic clusters, respectively. Visualization of the STRUCTURE bar plot revealed N = 11 temporal demarcations indicating barriers to gene flow. An analysis of molecular variance of these STRUCTURE-inferred populations supported strong temporally driven genetic differentiation (FST = 0.048, F'ST = 0.664) relative to geographic differentiation (FST = 0.009, F'ST = 0.241). Integrated Nested Laplace Approximation and Boosted Regression Tree analyses revealed that collection timepoint and 4 main abiotic factors (temperature, humidity, precipitation, and wind speed) were associated with the genetic subdivisions observed for P. regina. A complex interplay between environmental conditions, the unique reproductive strategies of the blow fly, and the extensive dispersal abilities of these organisms likely drives the strong genetic structure of P. regina in the Midwestern US.

Tetracyclines are broad-spectrum antibiotics widely used in agriculture, medicine, and research. However, they are associated with harmful side effects. In arthropods, parental exposure to tetracyclines has been linked to reduced health and fitness in untreated offspring. These transgenerational effects of tetracyclines could jeopardize the success of pest control programs that use tetracyclines to control gene expression. In this study, we investigated the transgenerational effects of 2 tetracyclines, doxycycline (DOX) and anhydrotetracycline (ATC), in the blowfly Lucilia cuprina, a significant pest of sheep. To simulate the rearing conditions of a transgenic male-only release program, blowflies were reared on standard diet alone, or standard diet plus DOX or ATC, for 3 generations, and then reared for an additional fourth generation on standard diet alone. We used behavioral assays, 16S amplicon sequencing, and mRNA sequencing to determine how DOX and ATC influenced male sexual competitiveness, microbiome composition, and gene expression in the third and fourth generations. We found that 3 generations of DOX treatment led to lower sexual competitiveness in both third- and fourth-generation males. In addition, DOX and ATC shifted the composition of the blowfly microbiome and altered the expression of numerous mitochondria- and immunity-related genes in both generations. Our study supports an emerging body of evidence that tetracyclines exert not only direct but also transgenerational effects, and sheds light on the transcriptional and microbial responses to antibiotic exposure and removal. Our findings emphasize the need for pest control programs that use tetracyclines to evaluate the long-term effects of these antibiotics.