How can forensic entomology help clinical wound healing? Xenobiotic effect on Lucilia sericata (Diptera: Calliphoridae) in the context of forensic entomology and larvatherapy. Part I: Tramadol

How can forensic entomology help clinical wound healing? Xenobiotic effect on Lucilia sericata (Diptera: Calliphoridae) in the context of forensic entomology and larvatherapy. 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Xenobiotic effect on Lucilia sericata (Diptera: Calliphoridae) in the context of forensic entomology and larvatherapy. Part I: Tramadol Jiri Hodecek, Damien Portevin, Magali Dovat-Sabatella, Frank Sporkert, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7638292/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Entomotoxicology can inform both forensic reconstructions and clinical larvatherapy, yet the consequences of patient-level drug exposure on therapeutic flies remain unexplored. We reared Lucilia sericata on bovine mince fortified with tramadol at therapeutic (0.05 mg/100 g) and lethal (2.0 mg/100 g) levels, alongside blank controls, and assessed development time, wing morphometrics, MALDI-TOF protein profiles, and targeted toxicology of adult tissues. Development time did not differ significantly among treatments, whereas morphometrics showed strong effects of treatment, sex, and their interaction: lethal exposure produced smaller wings, therapeutic exposure larger wings relative to controls, and females exceeded males across treatments. MALDI-TOF PCA primarily separated samples by tissue and experiment (batch), but adult leg spectra consistently distinguished lethal exposure from blank/therapeutic groups, indicating subtle treatment-linked molecular variation. LC–MS/MS detected tramadol in adults exposed to lethal tramadol concentrations in two of three experiments while O-desmethyltramadol was not detected; both analytes were undetectable at therapeutic levels. Collectively, tramadol induced pronounced morphological shifts without measurable developmental delay, a combination that could bias PMI min estimates in forensic entomology and, in clinical settings, possibly influence maggot therapy performance. These findings support integrating morphometrics with targeted proteomics and toxicology in entomotoxicological evaluations. Health sciences/Medical research Biological sciences/Zoology Forensic entomotoxicology Lucilia sericata Tramadol MALDI-TOF mass spectrometry Wing morphometrics Post-mortem interval Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In forensic entomotoxicology we can use the bodies of necrophagous insects to detect xenobiotics previously present in the body of the deceased. This is particularly helpful in cases where the body is in such a state, that it is no longer possible to obtain samples for a toxicological analysis (Introna et al. 2001 ; da Silva et al. 2017 ; Hodecek 2020 ). However, different xenobiotics can also affect the developmental rate and behaviour of the immature insect stages, which can cause a severe bias to the minimal Post-mortem interval (PMI min ) estimation by a forensic entomologist (Introna et al. 2001 ; Magni et al. 2014 ; da Silva et al. 2017 ). Unfortunately, there are too many xenobiotics (and their mixtures) and too many species of necrophagous flies to study and although the number of manuscripts analysing different types of xenobiotics and their effect on the development of the necrophagous flies is rising, the field is still significantly understudied (Gosselin et al. 2011 ). Interestingly, the scope of entomotoxicology extends beyond forensic applications and has growing relevance in clinical contexts — particularly in larvatherapy, also known as maggot debridement therapy (MDT) or biosurgery (Hodecek 2020 ). This therapeutic approach uses sterile larvae of facultative calliphorids, with Lucilia sericata being the most widely used species to treat chronic wounds by debriding necrotic tissue, reducing bacterial load, and promoting healing. Larvatherapy has seen renewed clinical interest due to its simplicity, cost-effectiveness, and efficacy against antibiotic-resistant infections (Courtenay et al., 2000; Sherman, 2003 ; Whitaker et al. 2007). However, like in forensic cases, larvae used in this medical procedure may be exposed to xenobiotics — such as analgesics — present in the patient’s body. This raises important questions about how drugs might influence larval development, behavior, and therapeutic efficacy. Among these xenobiotics, tramadol is particularly noteworthy. Tramadol is a commonly prescribed opioid analgesic and serotonin–norepinephrine reuptake inhibitor used to manage moderate to severe pain (Musshoff & Madea, 2001 ). It is frequently encountered not only in clinical pain management but also in suicide and overdose cases (Michaud et al. 1999 ; Musshoff & Madea 2001 ; Barbera et al. 2013 ; Niznansky et al. 2024). Therapeutic blood-plasma concentrations of tramadol generally range between 0.1 and 1.0 mg/L. In cases, where tramadol had been taken therapeutically, tramadol concentrations until 30 mg/L in urine, 0.3 mg/L in bile, 0.3 mg/kg in liver and 0.4 mg/kg in kidney can be measured post-mortem. Toxic plasma levels start from 1 mg/L and comatose-fatal concentrations from 1.3–2.0 mg/L (up to 20 mg/L), often in mixtures with other xenobiotics (Musshoff & Madea 2001 ; Barbera et al. 2013 ). In lethal cases, the concentration of tramadol can range between 46 and 110 mg/L in urine, 46 mg/L in bile, from 6.2 to 69 mg/kg in liver and 3.1 to 37 mg/kg in kidney (Molina, 2009 ; Michaud et al. 1999 ; Schulz et al. 2012 ). In well-perfused skeletal muscle tissue, slightly higher concentrations can generally be expected for basic drugs compared to their corresponding peripheral blood concentrations (Rees et al. 2013 , Øiestad et al. 2018 ). A recent publication has allowed to put in evidence a transfer of tramadol from human skeletonized remains (concentrations in bone marrow of 4.5 µg/g and in muscle of 4.02 µg/g) to fly larvae found in the direct vicinity of the remains (concentration in larvae of 0.28 µg/g) (Niznansky et al. 2024). Given tramadol’s dual relevance – both as a commonly prescribed painkiller during larvatherapy and as a frequent toxicant in forensic cases – understanding its impact on Lucilia sericata development is of high clinical and forensic importance. In this study, we investigate how both therapeutic and lethal levels of tramadol in body fluids and tissues affect the growth and development of Lucilia sericata , the primary species used in larvatherapy (Jones & Wall, 2008 ), and a widely distributed necrophagous fly in European forensic entomology (Lutz et al., 2021 ; Hodecek & Jakubec, 2022 ; Hodecek et al. 2024 ). By bridging forensic entomotoxicology with clinical larvatherapy, this research offers new insights into the broader implications of xenobiotic exposure in medically and legally significant insect species. Results The developmental duration of Lucilia sericata varied slightly across the treatment groups. Mean emergence times were longest in the lethal tramadol group, followed by the therapeutic dose, and shortest in the control group (Fig. 1 ). However, statistical analysis using one-way analysis of variance (ANOVA) indicated no significant difference among the groups (p = 0.950). This finding was corroborated by a non-parametric Kruskal-Wallis test (H = 0.800, p = 0.670), suggesting that the observed variation in developmental time was not statistically significant. It is likely that the lack of statistical significance is due to the limited number of repeated experiments (n = 3 per group). On the contrary, wing morphometric analysis revealed significant effects of both treatment and sex on wing size in Lucilia sericata. A multivariate analysis of variance (MANOVA) showed that treatment, sex, and their interaction significantly influenced wing length and width (p < 0.001 for all effects). Post-hoc comparisons indicated that flies exposed to the lethal tramadol concentration had significantly smaller wings than those in both the blank (control) and therapeutic groups (p < 0.001). Additionally, flies in the therapeutic group exhibited significantly larger wings than those in the control group (p < 0.001). Across all treatments, females had significantly larger wings than males (p < 0.001). These trends were consistent for both wing length and wing width and are visually illustrated in Figs. 2 and 3 . Principal component analysis (PCA) analyses of matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) spectra revealed clear segregation among tissue types (larvae, legs, wings), indicating distinct metabolic or proteomic profiles (Fig. 4 A). Within tissue-specific subsets, variance was predominantly explained by experimental batches (MALDI plates), suggesting a significant batch effect (Fig. 4 B-D). However, within the leg dataset, signals consistently differentiated lethal tramadol samples from the blank and therapeutic groups, indicating possible treatment-specific molecular alterations detectable despite batch-related variance (Fig. 4 E-G). Toxicological target screening for tramadol showed increased presence of tramadol in the bodies of adult flies reared on lethal dosage in 2 out of the 3 experimental batches. The concentration of tramadol was ~ 23 pg/mg in the first experiment and ~ 81 pg/mg in the second experiment. The active metabolite (O-desmethyltramadol) was not detected as well as tramadol in the adult flies bred from meat fortified at therapeutic level (Table 1 ). Table 1 Detected concentration of tramadol and its metabolite O-desmethyltramadol in the adult fly tissues after exposure to therapeutic and lethal concentration (nd = not detected). Sample Experiment n. Dosage Drug concentration (pg/mg) tramadol 1 therapeutic nd tramadol 2 therapeutic nd tramadol 3 therapeutic nd tramadol 1 lethal 23.37 tramadol 2 lethal 80.55 tramadol 3 lethal nd O-desmethyltramadol 1 therapeutic nd O-desmethyltramadol 2 therapeutic nd O-desmethyltramadol 3 therapeutic nd O-desmethyltramadol 1 lethal nd O-desmethyltramadol 2 lethal nd O-desmethyltramadol 3 lethal nd Discussion This study evaluated the effects of tramadol on the development, morphology, and protein expression in Lucilia sericata , a fly species of major relevance in both forensic entomology and larvatherapy. Our results demonstrated a slight delay of the development under tramadol and although the results were not statistically significant, the observed trend towards prolonged emergence in the therapeutic and lethal concentration groups suggests that caution is needed when dealing with a tramadol overdose as the resulting PMI min can be underestimated. This pattern is consistent with previous findings from Abouzied ( 2016 ), who observed developmental delays in Sarcophaga argyrostoma reared on tramadol-treated rats and El-Samad et al. ( 2011 ), who saw similar effects on Lucilia sericata . Some other opioids also seem to prolong the developmental period. For example, morphine prolonged the development of Lucilia sericata , Chrysomya albiceps and Chrysomya megacephala (El-Samad et al. 2020 ). In contrast to the subtle effects in the developmental rate, wing morphometrics showed clear and significant differences across treatment groups for both – males and females. Flies from the lethal tramadol group developed smaller wings, while those exposed to therapeutic doses exhibited larger wings compared to controls. These morphometric effects suggest a non-linear or hormetic dose–response relationship, which has also been observed in other entomotoxicological studies (da Silva et al. 2017 ). Morphometric traits such as wing length and width are increasingly recognized as sensitive indicators of physiological stress in necrophagous flies, as they are often affected by different types of xenobiotics (Bhardwaj et al. 2020). Sexual dimorphism in wing size was also reaffirmed, as females consistently showed larger wings than males, in line with established trends for necrophagous blowflies (Baleba et al., 2019 ). This supports the importance of incorporating sex as a factor in morphometric analyses, particularly in experimental designs aiming to assess xenobiotic impact. From the toxicological measurements, it should be noted that tramadol was found in the adult flies bred from meat fortified at lethal levels. This result strengthens the fact that the xenobiotic was metabolized in some extent. The absence of detection of tramadol and active metabolite O-desmethyltramadol in the adult flies bred from meat fortified at therapeutic levels could indicate that the intake of tramadol was not sufficient to be accumulated in the flies during the breeding period or it was already metabolized and excreted. Indeed, as the developmental cycles were not statistically impacted, an increase of metabolization / elimination of tramadol cannot explain the absence of tramadol and its metabolite. In addition to noticeable phenotypic shifts, the MALDI-TOF analysis revealed consistent trends towards treatment-dependent proteomic patterns. However, the main variation was dominated by inter-experimental batch effects, potentially linked to plate preparation and acquisition conditions. Even within the leg tissue dataset – where the separation between treatment groups was most evident – batch variance remained the dominant source of clustering. This suggests that tramadol only slightly alter protein expression profiles in Lucilia sericata at the concentrations tested, to a degree barely resolvable via standard MALDI-TOF. It is also possible that more sensitive or targeted proteomic approaches, such as LC-MS/MS, would be required to detect subtle biochemical shifts. Moreover, the observed proteomic shifts may reflect quantitative rather than qualitative changes – not the induction of novel proteins, but rather upregulation or downregulation of existing ones. From a forensic perspective, the present findings reinforce that morphometric changes – in the absence of strong developmental delays – may indicate xenobiotic exposure and should not be overlooked in PMI min estimation. This is particularly relevant when chemical tissue analysis is no longer available. The use of morphometric proxies in entomotoxicology has been increasingly validated (Bhardwaj et al. 2020), and our results support expanding that toolkit to opioid-exposed cases. In the clinical domain, especially in maggot debridement therapy (MDT), our findings also raise interesting questions. Lucilia sericata is widely used in biosurgery for chronic wound care due to its debridement capacity, immune modulation, and antimicrobial effects (Sherman, 2003 ; van der Plas et al., 2007 ). However, many patients receiving MDT are concurrently administered analgesics such as tramadol. While our study does not suggest toxic or lethal outcomes for larvae at therapeutic levels, the observed morphological alterations indicate that physiological effects do occur and could theoretically influence maggot efficacy. Conclusion This study highlights that tramadol, particularly at lethal concentrations, induces significant morphometric changes in Lucilia sericata , even when developmental delays are not statistically noticeable. MALDI-TOF analysis revealed minimal protein expression shifts attributable to tramadol, suggesting limited proteomic disruption under the tested conditions. The integration of morphometrics and proteomics offers a nuanced lens for entomotoxicological analysis, useful in both forensic reconstruction and clinical evaluation of xenobiotic effects on maggot therapy agents. Future research should aim to: i) increase biological replication to resolve subtle trends with enhanced statistical power; ii) incorporate residue analysis from larval tissues to link physiological outcomes with internal drug burdens; iii) apply advanced proteomic and transcriptomic tools to explore drug-related molecular changes more sensitively. Methods Breeding protocol Before every experiment, ca. 500 eggs were ordered from Andermatt Biocontrol Suisse, which is also a supplier for larva-therapy at dermatology centres in Lausanne and Geneva (Grossdietwil, Switzerland). The package with the eggs was sent on Thursday afternoons packed with a small ice pack to prevent them from hatching earlier. On Friday mornings, a clump of Lucilia sericata eggs was placed on small plastic trays with meat. Each tray contained 100g of minced bovine meat. Three different trays with three different tramadol fortifications were prepared: 1) A lethal level tramadol fortification in concentration of 2.0 mg/100g, which was demonstrating the level of tramadol usually found for an overdose (Molina 2009 ; Lewis et al. 2010 ); 2) A therapeutical level of tramadol in concentration of 0.05 mg/100g, which is a concentration present in human’s body during its therapeutical usage (Molina 2009 ; Lewis et al. 2010 ) and 3) A blank meat with no tramadol as a control. Tramadol used for the fortification was in liquid state (a syrup) and the homogenization of the mixture was ensured by a thorough mixing of the 100g minced meat with ca 10ml of the prepared liquid solution. The blank meat was mixed just with water to have the same resulted meat compound as with the fortified meat. The trays were put in polyacrylate boxes of 20x20x14 cm with 3 cm of sawdust on the bottom for the pupation of the larvae. The boxes were covered with a fabric mesh, and they were placed in an incubator (Lovibond TC 255S), where the flies were bred in darkness under 25°C. When the larvae reached 3rd instar, 5 specimens were removed from each box and put in the freezer (i.e. − 20°C) for the MALDI-TOF analysis. After all the adults emerged in all boxes, the boxes were put in the freezer, where all the flies died. 5 adults of each box were left in the freezer for the MALDI-TOF, and the rest was taken out to be dried out in room temperature. The experiment was repeated three times. The length of the developmental cycles was measured by active infrared cameras (AXIS M1065-LW), installed in the incubator, which were monitoring the first emergence in each box. Wing morphometrics In order to compare the sizes of the adults, the wing lengths and widths were measured. Random 100 adults of each box were selected for the wing measurement. After they were killed by freezing, they were kept in the room temperature to dry for approximately 2 months before the measurements were taken. The measurement technique was inspired by Smith & Wall ( 1997 ), Hwang and Tuner (2009) and Baleba et al. ( 2019 ). The dried right wings were cut off at the base of the stem-vein and glued to a white paper using small amount of transparent nail polish. The wings were then covered by a strip of clear tape. All wings were photographed under a stereoscope Leica S9i with 40x magnification, and the resulted pictures were saved as JPEG. For the wing length we chose two landmarks: costal vein intersection medial vein and medial vein intersection with distal median cubital. The resulted length of the wing is then the distance in between these two landmarks. For the wing width we chose costal vein intersection with radial vein 1 and basal median cubital vein and anterior cubital 2 (Baleba et al. 2019 ). The distance in between these landmarks was measured in ImageJ v.1.54g. Protein profiling MALDI-TOF was used to compare protein profiles of the differently treated batches. For this purpose, 3 different body parts of the studied specimens were utilized: 1) a head of 3rd instar larva; 2) a wing of an adult fly and 3) a hind leg of an adult fly. 5 + 5 specimens (3rd instar larvae + adults) of each batch were frozen and prepared for the MALDI-TOF analysis. The preparation of the plates was done in the laboratory of the Swiss Human Institute of Forensic Taphonomy (SHIFT) in Lausanne adapted from the protocol in Vogel et al. 2018 . The samples are prepared with a sinapinic acid matrix solution (40 mg sinapinic acid in 600 µL acetonitrile, 400 µL deuterated H 2 O and 3 µL TFA). All chemicals were obtained in Sigma Aldrich (Merck group, USA). The resulted plates were then sent to Mabritec AG (Riehen, Switzerland) for the spectrometry analysis. The measurements were done on a Shimadzu axima series MALDI-TOF MS machine (Shimadzu-Biotech, Kyoto, Japan) in linear positive mode, the mass range was set to 4’000–30’000 Da. The laser repetition rate was set at 50 Hz with a default acceleration voltage of 20 kV and an extraction delay time of 120 ns. The ion gate was fixed at 3’900 Da and the pulsed extraction optimized at 15’000 Da. A circular well raster with 91 positions with a spacing of 130 µm was used. MS profiles were taken at 50 positions with five laser shots totalling in 250 single raw spectra per sample. The samples plates were each externally calibrated from 4’365 to 20’171 Da using the ribosomal subunit masses in the reference spectra of Escherichia coli strain DH5α. Tramadol detection The concentration of tramadol was measured in 5 random bodies of adult flies from all treatment groups of all experiments in the adaption of an in-house validated method for the detection of drugs in hair samples. Approximately 20 mg of fly tissue were weighed into a screw-cap microcentrifuge tube. Two stainless-steel balls were added, the tube was sealed, and samples were homogenized in a Retsch MM400 mixer mill (Haan, Germany) for 15 min at 30 Hz. Subsequently, 0.4 mL deionized water and 0.4 mL methanol containing isotopically labeled tramadol-d3-^13C (125 ng.mL⁻¹) were added to the powdered material. Extraction was performed by micropulverized extraction for 40 min at 30 Hz according to a validated in-house procedure established for hair samples. After centrifugation performed twice, 500 µL of the supernatant were transferred to a 1.5 mL LC glass vial, and 10 µL were injected onto a Shimadzu Nexera LC-30 system coupled to a SCIEX 6500 + triple quadrupole mass spectrometer. Chromatographic separation was achieved on an Onyx Monolithic C18 column, 100 × 4.6 mm (Phenomenex), at 30°C with a flow rate of 0.6 mL.min⁻¹. Mobile phases were (A) water with 0.1% formic acid and (B) acetonitrile. The gradient was: 2% B at 0.00–0.02 min, 70% B at 8.00 min, 95% B at 10.00 min, then re-equilibration to 2% B at 14.00 min. Tramadol, O-desmethyltramadol, and tramadol-d3-^13C (Sigma-Aldrich, Buchs, Switzerland) were quantified in scheduled MRM mode (quantifier transition used for calibration and quantitation; qualifier monitored for confirmation) using SCIEX MultiQuant 3.2. Calibration ranged from 5–500 pg.mg⁻¹ for both analytes, with limits of quantification of approximately 5 pg.mg⁻¹. Morphometric statistical analyses Statistical analyses were performed using R software (version 4.3.2, R Foundation for Statistical Computing). Wing length and width measurements of Lucilia sericata were analyzed to assess the effects of treatment (blank, therapeutic, and lethal tramadol levels) according to the sex (male, female). Initially, a multivariate analysis of variance (MANOVA) was conducted to test the combined effects of treatment and sex on both wing length and width. Post-hoc comparisons were performed using Tukey's Honest Significant Difference (HSD) test to identify significant pairwise differences between treatment groups and sexes. Data were visually represented using mean plots with 95% confidence intervals to illustrate the trends in wing size across treatments and between sexes. Local Polynomial Regression Fitting (LOESS smoothing) was applied to the plots to enhance visualization of the trends, and the final figures were generated using the ggplot2 package. To evaluate the influence of tramadol exposure on the developmental duration of Lucilia sericata , emergence times (in hours) were compared among treatment groups (blank, therapeutic, and lethal) using a one-way analysis of variance (ANOVA). To verify robustness against non-normal data distribution, a non-parametric Kruskal-Wallis test was also conducted. Development time data were visualized using boxplots to display intergroup variation. MALDI statistical analyses For the statistical treatment of the MALDI signals, MALDIquant_1.22.3 and MALDIquantForeign_0.14.1 R packages were used. 1099 spectra from three independent experiments were imported of which one was removed following calculation and visual inspection of insufficient total ion currents (TIC). Spectra were then transformed (sqrt), smoothed (Savitzky-Golay) and baseline removed (SNIP) before new calculation of TIC and mass peak detection (MAD) at signal to noise ratio above 5. Values above Q3 + 1.5 IQR or below Q1–1.5 IQR for TIC (n = 5) and total mass peak per spectra (n = 12) outliers were considered as outliers (rstatix 0.7.2 R package). Spectra were then aligned to nine reference peaks detected in 20% of spectra after binning at 0.002 ppm. The warped spectra were calibrated (TIC) and peak intensities extracted to generate a matrix use for Principal Component Analysis (PCA) computation and visualization using FactoMineR 2.1 and factoextra 1.0.7 R packages. Discriminant analysis based on PCA was performed using adegenet 2.1.11 R package. Declarations Competing interests The authors declare no competing interests. Funding declaration The work presented in this research has received a financial support from Fondation BIOS pour la Recherche and the Fondation Rolf Gaillard pour la recherche en endocrinologie, diabétologie et métabolisme. Author Contribution JH: Conceptualization, Methodology, Investigation, Formal analysis, Resources, Writing—original draft, Writing—review & editing;DP: Methodology, Formal analysis, Writing—review & editing;MDS: Methodology, Formal analysis;FS: Methodology, Formal analysis, Writing—review & editing;SL: Methodology, Formal analysis;VP: Methodology, Formal analysis;GV: Methodology, Formal analysis;VV: Conceptualization, Methodology, Formal analysis, Resources, Writing—review & editing.All authors reviewed and approved the final manuscript. 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Care . 16 , R136. 10.1186/cc11441 (2012). Sherman, R. A. Maggot therapy for treating diabetic foot ulcers unresponsive to conventional therapy. Diabetes Care . 26 (2), 446–451. 10.2337/diacare.26.2.446 (2003). Smith, K. E. & Wall, R. The use of carrion as breeding sites by the blowfly Lucilia sericata and other Calliphoridae. Med. Vet. Entomol. 11 , 38–44. 10.1111/j.1365-2915.1997.tb00287.x (1997). van der Plas, M. J. A. et al. Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses. Microbes Infect. 9 (4), 507–514. 10.1016/j.micinf.2007.01.008 (2007). Vogel, G. et al. Functional characterization and phenotypic monitoring of human hematopoietic stem cell expansion and differentiation of monocytes and macrophages by whole-cell mass spectrometry. Stem Cell. Res. 26 , 47–54. 10.1016/j.scr.2017.11.013 (2018). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Nov, 2025 Reviews received at journal 31 Oct, 2025 Reviews received at journal 28 Oct, 2025 Reviewers agreed at journal 09 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers invited by journal 06 Oct, 2025 Editor assigned by journal 06 Oct, 2025 Editor invited by journal 06 Oct, 2025 Submission checks completed at journal 03 Oct, 2025 First submitted to journal 03 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7638292","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":530800208,"identity":"4e2cfe11-40cc-4e2a-b111-bf10f8dea2e7","order_by":0,"name":"Jiri Hodecek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACxmYGhgOMDQxAxMDM8IHhAEiQjXgtjDOI0QLRB9XCzEOMFuZ23ocHGHfYyPazHz5sbFNzJ3HDAeZnD/A7jN3gAOOZNOOZPWnJyTnHngG1sJkb4NfCBvRL2+HEDTd4jA/nNhxOnNnAwyZBlJb9N/g/H7YkScsGCR7mZEagln4GYrQktqUZzwD6x7Dn2GHjfmY2M7xaDPuPMX/42AYMsfbDjyV+1ByWbWNvfoZfSwOQSEARYsanHgjkCciPglEwCkbBKGBgAADxUUt6/50iUwAAAABJRU5ErkJggg==","orcid":"","institution":"University Centre of Legal Medicine, Lausanne University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jiri","middleName":"","lastName":"Hodecek","suffix":""},{"id":530800210,"identity":"bf1cf05a-a3f1-4e43-9253-5a3393ab8c2a","order_by":1,"name":"Damien Portevin","email":"","orcid":"","institution":"Swiss Tropical and Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Damien","middleName":"","lastName":"Portevin","suffix":""},{"id":530800213,"identity":"2cb70429-0be4-4832-85da-020cd675804b","order_by":2,"name":"Magali Dovat-Sabatella","email":"","orcid":"","institution":"University Centre of Legal Medicine, Lausanne University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Magali","middleName":"","lastName":"Dovat-Sabatella","suffix":""},{"id":530800215,"identity":"6144e917-7c6a-4baf-bb2d-b80aafbbf136","order_by":3,"name":"Frank Sporkert","email":"","orcid":"","institution":"University Centre of Legal Medicine, Lausanne University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Frank","middleName":"","lastName":"Sporkert","suffix":""},{"id":530800216,"identity":"a4b98f1f-a2d1-40b3-89d9-131700bc94eb","order_by":4,"name":"Samuel Ludin","email":"","orcid":"","institution":"Mabritec 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12:50:37","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81082,"visible":true,"origin":"","legend":"","description":"","filename":"662b1314874f48298eb3462aac976e9f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7638292/v1/1f2610650a60dd10d769d444.xml"},{"id":93778996,"identity":"558f5976-9f8e-4842-a97e-f79e0d42a2d6","added_by":"auto","created_at":"2025-10-17 12:50:37","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":88761,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7638292/v1/033a1509ffa5b8c006bce05a.html"},{"id":93780088,"identity":"ca95133e-12ac-414d-861e-8cc48f7561de","added_by":"auto","created_at":"2025-10-17 12:58:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63458,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment time (in hours) of \u003cem\u003eLucilia sericata\u003c/em\u003eunder different tramadol treatments. The boxplot shows the distribution of emergence times for three treatment groups: blank (B), therapeutic (T), and lethal (L), across three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7638292/v1/4ab51f6322cc9d98c9b3ecdc.png"},{"id":93778984,"identity":"517eda11-0e08-4600-8819-32473e692693","added_by":"auto","created_at":"2025-10-17 12:50:37","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114596,"visible":true,"origin":"","legend":"\u003cp\u003eThe mean wing length (mm) of \u003cem\u003eLucilia sericata\u003c/em\u003eacross three treatment groups (blank, therapeutic, and lethal levels of tramadol) for males (orange) and females (green). Error bars represent the 95% confidence intervals for the means.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7638292/v1/92ea42e68bd4f4dc04e19c29.jpeg"},{"id":93778987,"identity":"dfccbf57-51c8-44ac-8013-fd860f4ac646","added_by":"auto","created_at":"2025-10-17 12:50:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116112,"visible":true,"origin":"","legend":"\u003cp\u003eThe mean wing width (mm) of \u003cem\u003eLucilia sericata\u003c/em\u003eacross three treatment groups (blank, therapeutic, and lethal levels of tramadol) for males (orange) and females (green). Error bars represent the 95% confidence intervals for the means.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7638292/v1/1dae063af0f1c42b111df28c.jpeg"},{"id":93778989,"identity":"06a44522-0ffe-454f-a9d2-9289610bdc1f","added_by":"auto","created_at":"2025-10-17 12:50:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":246979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. PCA of MALDI-TOF spectra across tissue types (larva, leg, wing). \u003cstrong\u003eB-D\u003c/strong\u003e: PCA of MALDI-TOF spectra by tissue type (larvae, wings and legs) for each experiment. \u003cstrong\u003eE-G\u003c/strong\u003e: \u0026nbsp;PCA of MALDI-TOF spectra by treatment (blank, therapeutic and lethal levels) for each experiment. Data preprocessing included square-root transformation, Moving Average smoothing, SNIP baseline correction, and strict peak binning (tolerance = 0.002). \u003cstrong\u003eH\u003c/strong\u003e: Venn diagram of the mass peaks loadings driving PC1 across individual experiments.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7638292/v1/e84213f3e759b338685d16e3.png"},{"id":107350818,"identity":"e18e9567-9a27-4244-8d8f-107734345bf0","added_by":"auto","created_at":"2026-04-20 16:05:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":827322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7638292/v1/35131457-2832-45b3-a757-e4a5b3f48b9b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"How can forensic entomology help clinical wound healing? Xenobiotic effect on Lucilia sericata (Diptera: Calliphoridae) in the context of forensic entomology and larvatherapy. Part I: Tramadol","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn forensic entomotoxicology we can use the bodies of necrophagous insects to detect xenobiotics previously present in the body of the deceased. This is particularly helpful in cases where the body is in such a state, that it is no longer possible to obtain samples for a toxicological analysis (Introna et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; da Silva et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hodecek \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, different xenobiotics can also affect the developmental rate and behaviour of the immature insect stages, which can cause a severe bias to the minimal Post-mortem interval (PMI\u003csub\u003emin\u003c/sub\u003e) estimation by a forensic entomologist (Introna et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Magni et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; da Silva et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Unfortunately, there are too many xenobiotics (and their mixtures) and too many species of necrophagous flies to study and although the number of manuscripts analysing different types of xenobiotics and their effect on the development of the necrophagous flies is rising, the field is still significantly understudied (Gosselin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, the scope of entomotoxicology extends beyond forensic applications and has growing relevance in clinical contexts \u0026mdash; particularly in larvatherapy, also known as maggot debridement therapy (MDT) or biosurgery (Hodecek \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This therapeutic approach uses sterile larvae of facultative calliphorids, with \u003cem\u003eLucilia sericata\u003c/em\u003e being the most widely used species to treat chronic wounds by debriding necrotic tissue, reducing bacterial load, and promoting healing. Larvatherapy has seen renewed clinical interest due to its simplicity, cost-effectiveness, and efficacy against antibiotic-resistant infections (Courtenay et al., 2000; Sherman, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Whitaker et al. 2007). However, like in forensic cases, larvae used in this medical procedure may be exposed to xenobiotics \u0026mdash; such as analgesics \u0026mdash; present in the patient\u0026rsquo;s body. This raises important questions about how drugs might influence larval development, behavior, and therapeutic efficacy.\u003c/p\u003e\u003cp\u003eAmong these xenobiotics, tramadol is particularly noteworthy. Tramadol is a commonly prescribed opioid analgesic and serotonin\u0026ndash;norepinephrine reuptake inhibitor used to manage moderate to severe pain (Musshoff \u0026amp; Madea, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). It is frequently encountered not only in clinical pain management but also in suicide and overdose cases (Michaud et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Musshoff \u0026amp; Madea \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Barbera et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Niznansky et al. 2024). Therapeutic blood-plasma concentrations of tramadol generally range between 0.1 and 1.0 mg/L. In cases, where tramadol had been taken therapeutically, tramadol concentrations until 30 mg/L in urine, 0.3 mg/L in bile, 0.3 mg/kg in liver and 0.4 mg/kg in kidney can be measured post-mortem. Toxic plasma levels start from 1 mg/L and comatose-fatal concentrations from 1.3\u0026ndash;2.0 mg/L (up to 20 mg/L), often in mixtures with other xenobiotics (Musshoff \u0026amp; Madea \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Barbera et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In lethal cases, the concentration of tramadol can range between 46 and 110 mg/L in urine, 46 mg/L in bile, from 6.2 to 69 mg/kg in liver and 3.1 to 37 mg/kg in kidney (Molina, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Michaud et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Schulz et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In well-perfused skeletal muscle tissue, slightly higher concentrations can generally be expected for basic drugs compared to their corresponding peripheral blood concentrations (Rees et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u0026Oslash;iestad et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A recent publication has allowed to put in evidence a transfer of tramadol from human skeletonized remains (concentrations in bone marrow of 4.5 \u0026micro;g/g and in muscle of 4.02 \u0026micro;g/g) to fly larvae found in the direct vicinity of the remains (concentration in larvae of 0.28 \u0026micro;g/g) (Niznansky et al. 2024).\u003c/p\u003e\u003cp\u003eGiven tramadol\u0026rsquo;s dual relevance \u0026ndash; both as a commonly prescribed painkiller during larvatherapy and as a frequent toxicant in forensic cases \u0026ndash; understanding its impact on \u003cem\u003eLucilia sericata\u003c/em\u003e development is of high clinical and forensic importance. In this study, we investigate how both therapeutic and lethal levels of tramadol in body fluids and tissues affect the growth and development of \u003cem\u003eLucilia sericata\u003c/em\u003e, the primary species used in larvatherapy (Jones \u0026amp; Wall, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and a widely distributed necrophagous fly in European forensic entomology (Lutz et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hodecek \u0026amp; Jakubec, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hodecek et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). By bridging forensic entomotoxicology with clinical larvatherapy, this research offers new insights into the broader implications of xenobiotic exposure in medically and legally significant insect species.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe developmental duration of \u003cem\u003eLucilia sericata\u003c/em\u003e varied slightly across the treatment groups. Mean emergence times were longest in the lethal tramadol group, followed by the therapeutic dose, and shortest in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, statistical analysis using one-way analysis of variance (ANOVA) indicated no significant difference among the groups (p\u0026thinsp;=\u0026thinsp;0.950). This finding was corroborated by a non-parametric Kruskal-Wallis test (H\u0026thinsp;=\u0026thinsp;0.800, p\u0026thinsp;=\u0026thinsp;0.670), suggesting that the observed variation in developmental time was not statistically significant. It is likely that the lack of statistical significance is due to the limited number of repeated experiments (n\u0026thinsp;=\u0026thinsp;3 per group).\u003c/p\u003e\u003cp\u003eOn the contrary, wing morphometric analysis revealed significant effects of both treatment and sex on wing size in \u003cem\u003eLucilia sericata.\u003c/em\u003e A multivariate analysis of variance (MANOVA) showed that treatment, sex, and their interaction significantly influenced wing length and width (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all effects). Post-hoc comparisons indicated that flies exposed to the lethal tramadol concentration had significantly smaller wings than those in both the blank (control) and therapeutic groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, flies in the therapeutic group exhibited significantly larger wings than those in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Across all treatments, females had significantly larger wings than males (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These trends were consistent for both wing length and wing width and are visually illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003ePrincipal component analysis (PCA) analyses of matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) spectra revealed clear segregation among tissue types (larvae, legs, wings), indicating distinct metabolic or proteomic profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Within tissue-specific subsets, variance was predominantly explained by experimental batches (MALDI plates), suggesting a significant batch effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). However, within the leg dataset, signals consistently differentiated lethal tramadol samples from the blank and therapeutic groups, indicating possible treatment-specific molecular alterations detectable despite batch-related variance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eToxicological target screening for tramadol showed increased presence of tramadol in the bodies of adult flies reared on lethal dosage in 2 out of the 3 experimental batches. The concentration of tramadol was ~\u0026thinsp;23 pg/mg in the first experiment and ~\u0026thinsp;81 pg/mg in the second experiment. The active metabolite (O-desmethyltramadol) was not detected as well as tramadol in the adult flies bred from meat fortified at therapeutic level (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetected concentration of tramadol and its metabolite O-desmethyltramadol in the adult fly tissues after exposure to therapeutic and lethal concentration (nd\u0026thinsp;=\u0026thinsp;not detected).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExperiment n.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDosage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDrug concentration (pg/mg)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etherapeutic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etherapeutic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etherapeutic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003etramadol\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003elethal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e23.37\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003etramadol\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003elethal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e80.55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003elethal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eO-desmethyltramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etherapeutic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eO-desmethyltramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etherapeutic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eO-desmethyltramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etherapeutic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eO-desmethyltramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003elethal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eO-desmethyltramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003elethal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eO-desmethyltramadol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003elethal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study evaluated the effects of tramadol on the development, morphology, and protein expression in \u003cem\u003eLucilia sericata\u003c/em\u003e, a fly species of major relevance in both forensic entomology and larvatherapy. Our results demonstrated a slight delay of the development under tramadol and although the results were not statistically significant, the observed trend towards prolonged emergence in the therapeutic and lethal concentration groups suggests that caution is needed when dealing with a tramadol overdose as the resulting PMI\u003csub\u003emin\u003c/sub\u003e can be underestimated. This pattern is consistent with previous findings from Abouzied (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who observed developmental delays in \u003cem\u003eSarcophaga argyrostoma\u003c/em\u003e reared on tramadol-treated rats and El-Samad et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), who saw similar effects on \u003cem\u003eLucilia sericata\u003c/em\u003e. Some other opioids also seem to prolong the developmental period. For example, morphine prolonged the development of \u003cem\u003eLucilia sericata\u003c/em\u003e, \u003cem\u003eChrysomya albiceps\u003c/em\u003e and \u003cem\u003eChrysomya megacephala\u003c/em\u003e (El-Samad et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast to the subtle effects in the developmental rate, wing morphometrics showed clear and significant differences across treatment groups for both \u0026ndash; males and females. Flies from the lethal tramadol group developed smaller wings, while those exposed to therapeutic doses exhibited larger wings compared to controls. These morphometric effects suggest a non-linear or hormetic dose\u0026ndash;response relationship, which has also been observed in other entomotoxicological studies (da Silva et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Morphometric traits such as wing length and width are increasingly recognized as sensitive indicators of physiological stress in necrophagous flies, as they are often affected by different types of xenobiotics (Bhardwaj et al. 2020). Sexual dimorphism in wing size was also reaffirmed, as females consistently showed larger wings than males, in line with established trends for necrophagous blowflies (Baleba et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This supports the importance of incorporating sex as a factor in morphometric analyses, particularly in experimental designs aiming to assess xenobiotic impact.\u003c/p\u003e\u003cp\u003eFrom the toxicological measurements, it should be noted that tramadol was found in the adult flies bred from meat fortified at lethal levels. This result strengthens the fact that the xenobiotic was metabolized in some extent. The absence of detection of tramadol and active metabolite O-desmethyltramadol in the adult flies bred from meat fortified at therapeutic levels could indicate that the intake of tramadol was not sufficient to be accumulated in the flies during the breeding period or it was already metabolized and excreted. Indeed, as the developmental cycles were not statistically impacted, an increase of metabolization / elimination of tramadol cannot explain the absence of tramadol and its metabolite.\u003c/p\u003e\u003cp\u003eIn addition to noticeable phenotypic shifts, the MALDI-TOF analysis revealed consistent trends towards treatment-dependent proteomic patterns. However, the main variation was dominated by inter-experimental batch effects, potentially linked to plate preparation and acquisition conditions. Even within the leg tissue dataset \u0026ndash; where the separation between treatment groups was most evident \u0026ndash; batch variance remained the dominant source of clustering. This suggests that tramadol only slightly alter protein expression profiles in \u003cem\u003eLucilia sericata\u003c/em\u003e at the concentrations tested, to a degree barely resolvable via standard MALDI-TOF. It is also possible that more sensitive or targeted proteomic approaches, such as LC-MS/MS, would be required to detect subtle biochemical shifts. Moreover, the observed proteomic shifts may reflect quantitative rather than qualitative changes \u0026ndash; not the induction of novel proteins, but rather upregulation or downregulation of existing ones.\u003c/p\u003e\u003cp\u003eFrom a forensic perspective, the present findings reinforce that morphometric changes \u0026ndash; in the absence of strong developmental delays \u0026ndash; may indicate xenobiotic exposure and should not be overlooked in PMI\u003csub\u003emin\u003c/sub\u003e estimation. This is particularly relevant when chemical tissue analysis is no longer available. The use of morphometric proxies in entomotoxicology has been increasingly validated (Bhardwaj et al. 2020), and our results support expanding that toolkit to opioid-exposed cases.\u003c/p\u003e\u003cp\u003eIn the clinical domain, especially in maggot debridement therapy (MDT), our findings also raise interesting questions. \u003cem\u003eLucilia sericata\u003c/em\u003e is widely used in biosurgery for chronic wound care due to its debridement capacity, immune modulation, and antimicrobial effects (Sherman, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; van der Plas et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, many patients receiving MDT are concurrently administered analgesics such as tramadol. While our study does not suggest toxic or lethal outcomes for larvae at therapeutic levels, the observed morphological alterations indicate that physiological effects do occur and could theoretically influence maggot efficacy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights that tramadol, particularly at lethal concentrations, induces significant morphometric changes in \u003cem\u003eLucilia sericata\u003c/em\u003e, even when developmental delays are not statistically noticeable. MALDI-TOF analysis revealed minimal protein expression shifts attributable to tramadol, suggesting limited proteomic disruption under the tested conditions. The integration of morphometrics and proteomics offers a nuanced lens for entomotoxicological analysis, useful in both forensic reconstruction and clinical evaluation of xenobiotic effects on maggot therapy agents. Future research should aim to: i) increase biological replication to resolve subtle trends with enhanced statistical power; ii) incorporate residue analysis from larval tissues to link physiological outcomes with internal drug burdens; iii) apply advanced proteomic and transcriptomic tools to explore drug-related molecular changes more sensitively.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eBreeding protocol\u003c/p\u003e\u003cp\u003eBefore every experiment, ca. 500 eggs were ordered from Andermatt Biocontrol Suisse, which is also a supplier for larva-therapy at dermatology centres in Lausanne and Geneva (Grossdietwil, Switzerland). The package with the eggs was sent on Thursday afternoons packed with a small ice pack to prevent them from hatching earlier. On Friday mornings, a clump of \u003cem\u003eLucilia sericata\u003c/em\u003e eggs was placed on small plastic trays with meat. Each tray contained 100g of minced bovine meat. Three different trays with three different tramadol fortifications were prepared: 1) A \u003cem\u003elethal level\u003c/em\u003e tramadol fortification in concentration of 2.0 mg/100g, which was demonstrating the level of tramadol usually found for an overdose (Molina \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lewis et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e); 2) A \u003cem\u003etherapeutical level\u003c/em\u003e of tramadol in concentration of 0.05 mg/100g, which is a concentration present in human\u0026rsquo;s body during its therapeutical usage (Molina \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lewis et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and 3) A \u003cem\u003eblank meat\u003c/em\u003e with no tramadol as a control. Tramadol used for the fortification was in liquid state (a syrup) and the homogenization of the mixture was ensured by a thorough mixing of the 100g minced meat with ca 10ml of the prepared liquid solution. The blank meat was mixed just with water to have the same resulted meat compound as with the fortified meat. The trays were put in polyacrylate boxes of 20x20x14 cm with 3 cm of sawdust on the bottom for the pupation of the larvae. The boxes were covered with a fabric mesh, and they were placed in an incubator (Lovibond TC 255S), where the flies were bred in darkness under 25\u0026deg;C. When the larvae reached 3rd instar, 5 specimens were removed from each box and put in the freezer (i.e. \u0026minus;\u0026thinsp;20\u0026deg;C) for the MALDI-TOF analysis. After all the adults emerged in all boxes, the boxes were put in the freezer, where all the flies died. 5 adults of each box were left in the freezer for the MALDI-TOF, and the rest was taken out to be dried out in room temperature. The experiment was repeated three times. The length of the developmental cycles was measured by active infrared cameras (AXIS M1065-LW), installed in the incubator, which were monitoring the first emergence in each box.\u003c/p\u003e\u003cp\u003eWing morphometrics\u003c/p\u003e\u003cp\u003eIn order to compare the sizes of the adults, the wing lengths and widths were measured. Random 100 adults of each box were selected for the wing measurement. After they were killed by freezing, they were kept in the room temperature to dry for approximately 2 months before the measurements were taken. The measurement technique was inspired by Smith \u0026amp; Wall (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), Hwang and Tuner (2009) and Baleba et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe dried right wings were cut off at the base of the stem-vein and glued to a white paper using small amount of transparent nail polish. The wings were then covered by a strip of clear tape. All wings were photographed under a stereoscope Leica S9i with 40x magnification, and the resulted pictures were saved as JPEG. For the \u003cem\u003ewing length\u003c/em\u003e we chose two landmarks: costal vein intersection medial vein and medial vein intersection with distal median cubital. The resulted length of the wing is then the distance in between these two landmarks. For the \u003cem\u003ewing width\u003c/em\u003e we chose costal vein intersection with radial vein 1 and basal median cubital vein and anterior cubital 2 (Baleba et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The distance in between these landmarks was measured in ImageJ v.1.54g.\u003c/p\u003e\u003cp\u003eProtein profiling\u003c/p\u003e\u003cp\u003eMALDI-TOF was used to compare protein profiles of the differently treated batches. For this purpose, 3 different body parts of the studied specimens were utilized: 1) a head of 3rd instar larva; 2) a wing of an adult fly and 3) a hind leg of an adult fly. 5\u0026thinsp;+\u0026thinsp;5 specimens (3rd instar larvae\u0026thinsp;+\u0026thinsp;adults) of each batch were frozen and prepared for the MALDI-TOF analysis. The preparation of the plates was done in the laboratory of the Swiss Human Institute of Forensic Taphonomy (SHIFT) in Lausanne adapted from the protocol in Vogel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e. The samples are prepared with a sinapinic acid matrix solution (40 mg sinapinic acid in 600 \u0026micro;L acetonitrile, 400 \u0026micro;L deuterated H\u003csub\u003e2\u003c/sub\u003eO and 3 \u0026micro;L TFA). All chemicals were obtained in Sigma Aldrich (Merck group, USA). The resulted plates were then sent to Mabritec AG (Riehen, Switzerland) for the spectrometry analysis.\u003c/p\u003e\u003cp\u003eThe measurements were done on a Shimadzu axima series MALDI-TOF MS machine (Shimadzu-Biotech, Kyoto, Japan) in linear positive mode, the mass range was set to 4\u0026rsquo;000\u0026ndash;30\u0026rsquo;000 Da. The laser repetition rate was set at 50 Hz with a default acceleration voltage of 20 kV and an extraction delay time of 120 ns. The ion gate was fixed at 3\u0026rsquo;900 Da and the pulsed extraction optimized at 15\u0026rsquo;000 Da. A circular well raster with 91 positions with a spacing of 130 \u0026micro;m was used. MS profiles were taken at 50 positions with five laser shots totalling in 250 single raw spectra per sample. The samples plates were each externally calibrated from 4\u0026rsquo;365 to 20\u0026rsquo;171 Da using the ribosomal subunit masses in the reference spectra of \u003cem\u003eEscherichia coli\u003c/em\u003e strain DH5α.\u003c/p\u003e\u003cp\u003eTramadol detection\u003c/p\u003e\u003cp\u003eThe concentration of tramadol was measured in 5 random bodies of adult flies from all treatment groups of all experiments in the adaption of an in-house validated method for the detection of drugs in hair samples. Approximately 20 mg of fly tissue were weighed into a screw-cap microcentrifuge tube. Two stainless-steel balls were added, the tube was sealed, and samples were homogenized in a Retsch MM400 mixer mill (Haan, Germany) for 15 min at 30 Hz. Subsequently, 0.4 mL deionized water and 0.4 mL methanol containing isotopically labeled tramadol-d3-^13C (125 ng.mL⁻\u0026sup1;) were added to the powdered material. Extraction was performed by micropulverized extraction for 40 min at 30 Hz according to a validated in-house procedure established for hair samples. After centrifugation performed twice, 500 \u0026micro;L of the supernatant were transferred to a 1.5 mL LC glass vial, and 10 \u0026micro;L were injected onto a Shimadzu Nexera LC-30 system coupled to a SCIEX 6500\u0026thinsp;+\u0026thinsp;triple quadrupole mass spectrometer. Chromatographic separation was achieved on an Onyx Monolithic C18 column, 100 \u0026times; 4.6 mm (Phenomenex), at 30\u0026deg;C with a flow rate of 0.6 mL.min⁻\u0026sup1;. Mobile phases were (A) water with 0.1% formic acid and (B) acetonitrile. The gradient was: 2% B at 0.00\u0026ndash;0.02 min, 70% B at 8.00 min, 95% B at 10.00 min, then re-equilibration to 2% B at 14.00 min. Tramadol, O-desmethyltramadol, and tramadol-d3-^13C (Sigma-Aldrich, Buchs, Switzerland) were quantified in scheduled MRM mode (quantifier transition used for calibration and quantitation; qualifier monitored for confirmation) using SCIEX MultiQuant 3.2. Calibration ranged from 5\u0026ndash;500 pg.mg⁻\u0026sup1; for both analytes, with limits of quantification of approximately 5 pg.mg⁻\u0026sup1;.\u003c/p\u003e\u003cp\u003eMorphometric statistical analyses\u003c/p\u003e\u003cp\u003eStatistical analyses were performed using R software (version 4.3.2, R Foundation for Statistical Computing). Wing length and width measurements of \u003cem\u003eLucilia sericata\u003c/em\u003e were analyzed to assess the effects of treatment (blank, therapeutic, and lethal tramadol levels) according to the sex (male, female). Initially, a multivariate analysis of variance (MANOVA) was conducted to test the combined effects of treatment and sex on both wing length and width. Post-hoc comparisons were performed using Tukey's Honest Significant Difference (HSD) test to identify significant pairwise differences between treatment groups and sexes. Data were visually represented using mean plots with 95% confidence intervals to illustrate the trends in wing size across treatments and between sexes. Local Polynomial Regression Fitting (LOESS smoothing) was applied to the plots to enhance visualization of the trends, and the final figures were generated using the ggplot2 package.\u003c/p\u003e\u003cp\u003eTo evaluate the influence of tramadol exposure on the developmental duration of \u003cem\u003eLucilia sericata\u003c/em\u003e, emergence times (in hours) were compared among treatment groups (blank, therapeutic, and lethal) using a one-way analysis of variance (ANOVA). To verify robustness against non-normal data distribution, a non-parametric Kruskal-Wallis test was also conducted. Development time data were visualized using boxplots to display intergroup variation.\u003c/p\u003e\u003cp\u003eMALDI statistical analyses\u003c/p\u003e\u003cp\u003eFor the statistical treatment of the MALDI signals, MALDIquant_1.22.3 and MALDIquantForeign_0.14.1 R packages were used. 1099 spectra from three independent experiments were imported of which one was removed following calculation and visual inspection of insufficient total ion currents (TIC). Spectra were then transformed (sqrt), smoothed (Savitzky-Golay) and baseline removed (SNIP) before new calculation of TIC and mass peak detection (MAD) at signal to noise ratio above 5. Values above Q3\u0026thinsp;+\u0026thinsp;1.5 IQR or below Q1\u0026ndash;1.5 IQR for TIC (n\u0026thinsp;=\u0026thinsp;5) and total mass peak per spectra (n\u0026thinsp;=\u0026thinsp;12) outliers were considered as outliers (rstatix 0.7.2 R package). Spectra were then aligned to nine reference peaks detected in 20% of spectra after binning at 0.002 ppm. The warped spectra were calibrated (TIC) and peak intensities extracted to generate a matrix use for Principal Component Analysis (PCA) computation and visualization using FactoMineR 2.1 and factoextra 1.0.7 R packages. Discriminant analysis based on PCA was performed using adegenet 2.1.11 R package.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding declaration\u003c/h2\u003e\n\u003cp\u003eThe work presented in this research has received a financial support from Fondation BIOS pour la Recherche and the Fondation Rolf Gaillard pour la recherche en endocrinologie, diab\u0026eacute;tologie et m\u0026eacute;tabolisme.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eJH: Conceptualization, Methodology, Investigation, Formal analysis, Resources, Writing\u0026mdash;original draft, Writing\u0026mdash;review \u0026amp; editing;DP: Methodology, Formal analysis, Writing\u0026mdash;review \u0026amp; editing;MDS: Methodology, Formal analysis;FS: Methodology, Formal analysis, Writing\u0026mdash;review \u0026amp; editing;SL: Methodology, Formal analysis;VP: Methodology, Formal analysis;GV: Methodology, Formal analysis;VV: Conceptualization, Methodology, Formal analysis, Resources, Writing\u0026mdash;review \u0026amp; editing.All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eMost data supporting the findings of this study are available within the article. 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Res.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 47\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.scr.2017.11.013\u003c/span\u003e\u003cspan address=\"10.1016/j.scr.2017.11.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Forensic entomotoxicology, Lucilia sericata, Tramadol, MALDI-TOF mass spectrometry, Wing morphometrics, Post-mortem interval","lastPublishedDoi":"10.21203/rs.3.rs-7638292/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7638292/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEntomotoxicology can inform both forensic reconstructions and clinical larvatherapy, yet the consequences of patient-level drug exposure on therapeutic flies remain unexplored. We reared \u003cem\u003eLucilia sericata\u003c/em\u003e on bovine mince fortified with tramadol at therapeutic (0.05 mg/100 g) and lethal (2.0 mg/100 g) levels, alongside blank controls, and assessed development time, wing morphometrics, MALDI-TOF protein profiles, and targeted toxicology of adult tissues.\u003c/p\u003e\u003cp\u003eDevelopment time did not differ significantly among treatments, whereas morphometrics showed strong effects of treatment, sex, and their interaction: lethal exposure produced smaller wings, therapeutic exposure larger wings relative to controls, and females exceeded males across treatments.\u003c/p\u003e\u003cp\u003eMALDI-TOF PCA primarily separated samples by tissue and experiment (batch), but adult leg spectra consistently distinguished lethal exposure from blank/therapeutic groups, indicating subtle treatment-linked molecular variation.\u003c/p\u003e\u003cp\u003eLC\u0026ndash;MS/MS detected tramadol in adults exposed to lethal tramadol concentrations in two of three experiments while O-desmethyltramadol was not detected; both analytes were undetectable at therapeutic levels.\u003c/p\u003e\u003cp\u003eCollectively, tramadol induced pronounced morphological shifts without measurable developmental delay, a combination that could bias PMI\u003csub\u003emin\u003c/sub\u003e estimates in forensic entomology and, in clinical settings, possibly influence maggot therapy performance. These findings support integrating morphometrics with targeted proteomics and toxicology in entomotoxicological evaluations.\u003c/p\u003e","manuscriptTitle":"How can forensic entomology help clinical wound healing? Xenobiotic effect on Lucilia sericata (Diptera: Calliphoridae) in the context of forensic entomology and larvatherapy. Part I: Tramadol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 12:50:32","doi":"10.21203/rs.3.rs-7638292/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-03T08:02:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T13:50:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T10:28:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28265156031587354801854286674740383578","date":"2025-10-09T06:39:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288216809378977075934862308373977274483","date":"2025-10-08T17:35:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-06T17:28:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T17:23:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-06T16:06:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-03T14:12:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-03T14:09:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2abd6e1a-6599-4d2b-8755-325000d3d642","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56430092,"name":"Health sciences/Medical research"},{"id":56430093,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-04-20T16:02:21+00:00","versionOfRecord":{"articleIdentity":"rs-7638292","link":"https://doi.org/10.1038/s41598-026-46732-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-18 15:58:39","publishedOnDateReadable":"April 18th, 2026"},"versionCreatedAt":"2025-10-17 12:50:32","video":"","vorDoi":"10.1038/s41598-026-46732-2","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46732-2","workflowStages":[]},"version":"v1","identity":"rs-7638292","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7638292","identity":"rs-7638292","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}