FlyTracks and SmellPrints: A Multiscale Forensic Blueprint Linking Necrophagous Insect Behavior, Environmental Decay, and Molecular Affinity for Cadaveric VOCs

Abstract

Forensic entomology has long been employed to estimate the post-mortem interval (PMI) by studying the ecological succession of necrophagous insects on decomposing remains. However, the mechanisms underlying species-specific attraction to decaying tissues, particularly in the early stages of decomposition, remain poorly understood. This study presents the first integrative investigation that bridges ecological, developmental, and molecular frameworks to explain dipteran attraction to decomposing remains and their potential application in biomedical detection. Over a 12-day semifield trial, we systematically documented the colonization dynamics and developmental timelines of three forensic indicator speciesLucilia sericata, Calliphora vomitoria, and Phormia reginausing euthanized albino rats as standardized vertebrate models. Statistical analyses, including one-way ANOVA, PCA, CVA, CCA, and path analysis, revealed distinct interspecies differences in larval development, pupation rate, adult emergence, and longevity. L. sericata emerged as the primary colonizer with the fastest development and highest early stage abundance, while P. regina exhibited delayed emergence and extended longevity patterns. In a novel molecular dimension, we identified conserved odorant receptor (OR) proteins across the three species using TBLASTN alignment and structural modeling via AlphaFold2. Molecular docking simulations revealed that L. sericata’s ORs showed the highest binding affinities to cadaverine and putrescinekey biogenic amines emitted during early decompositionsuggesting a biochemical basis for its rapid attraction. This is the first study to link behavioral ecology with protein-level chemosensory mechanisms and computational predictions. It opens promising translational avenues for using synthetic ORs in biomedical diagnostics, particularly for the early detection of necrotic and potentially cancerous tissues based on shared volatile organic compounds. This work thus redefines the forensic entomological framework and paves the way for biologically inspired biosensor technologies.

1. Introduction

Forensic entomology has firmly established itself as a pivotal discipline within modern forensic science, offering unparalleled insights into the post-mortem interval (PMI), the circumstances surrounding death, and, in certain instances, the location and cause of death. , At its core, this field integrates entomology, pathology, ecology, and statistical modeling to leverage the predictable patterns of insect colonization on decomposing remains. Necrophagous Dipteraprimarily blow flies (Calliphoridae), supplemented by flesh flies (Sarcophagidae) and house flies (Muscidae)colonize carcasses in successive waves, each species and developmental stage presenting distinct ecological and temporal signatures. These successional stages provide an invaluable forensic clock, enabling reconstruction of death timelines with increasing precision.

Indeed, forensic entomology’s lineage extends back to medieval China, where legal authorities allegedly inferred blood-stained knives by observing fly attraction. Since then, the discipline has evolved exponentially. Advances in molecular diagnostics, climate-based population modeling, ecological niche modeling, thermal ecology, geographic information systems (GIS), 3D morphometric imaging, and robust statistical frameworkssuch as principal component analysis (PCA), canonical variate analysis (CVA), and path modelinghave collectively refined our capacity to interpret insect-related evidence. In situations where traditional physiological markers of death (rigor mortis, livor mortis, algor mortis) fail, from advanced decomposition to mummification or desiccation, entomological evidence remains both consistent and informative. Consequently, it is now widely admissible as crucial temporal and ecological testimony in courts. ,

Necrophagous flies are often the first visible agents to colonize remains, and their developmentfrom egg through larval instars, pupation, and adult emergenceoccurs at species-specific rates under given environmental conditions. , By integrating species identity with ambient temperature, humidity, and microhabitat variables, forensic entomologists can derive a reliable minimum PMI (minPMI). However, these timelines are influenced by contextual factors such as geographic location, seasonality, substrate type (soil, water, clothing), and chemical constituentsincluding drugs or poisonspresent within the decedent. , As a result, deviations from expected colonization profiles can hint at corpse movement, concealment, veterinary or neglect-related scenarios, or chemical interference.

In complex forensic scenariossuch as mass disasters, clandestine burials, or illicit body concealmententomological evidence extends beyond PMI estimation: it may reveal spatial orientation, site disturbances, and microhabitat conditions. Institutional cases involving myiasis have likewise highlighted entomology’s forensic and humanitarian relevance, guiding legal investigations and improving welfare protocols. Similarly, veterinary forensic cases involving animal abuse or neglect rely on insect evidence to infer timelines of injury or deprivation. Collectively, these developments illustrate that insect-based evidence is increasingly recognized as an indispensable adjunct to both human and animal forensic investigations, as well as public health monitoring.

Yet, despite this progress, significant methodological and ecological challenges remain. Species misidentificationespecially in immature life stagesregion-specific developmental variation, and environmental fluctuations can introduce considerable uncertainty. Recent studies, such as Gbenonsi and Higley, have documented geographic variation in developmental rates for Lucilia sericata, undermining global PMI calibration frameworks. Additionally, substrate heterogeneity and temperature instability can delay or accelerate development. Therefore, there remains an urgent need for standardized, regionally tailored developmental baselines, combined with enhanced species identification toolssuch as DNA barcoding, gene expression assays, and larval morphometricsto improve reproducibility and forensic accuracy. ,

Beyond ecological and environmental factors, recent research has begun to reveal the molecular mechanisms underpinning insect colonization. , Key among these are the odorant-binding proteins (OBPs) of necrophagous flies, which mediate olfactory detection of volatile cues emitted by decomposing tissues. Two diaminescadaverine and putrescineare consistently released early in the decomposition process, generated through microbial decarboxylation of lysine and ornithine in necrotic cells. Their potent odor and ecological relevance have led to investigations into interspecies differences in olfactory sensitivity. ,

Comparative analyses of OBP sequences from L. sericata, Calliphora vomitoria, and Phormia regina have identified conserved ligand-interaction domains shared across species, with subtle amino acid variations that may influence binding specificity. These molecular differences provide a plausible mechanistic basis for the observed sequence of arrival: species with OBP variants that more readily bind cadaverine and putrescine may detect decomposition earlier and thus colonize remains sooner. While ecological field studies have long demonstrated consistent successional patterns, the molecular component offers a fundamental explanation rooted in sensory biology.

The current study highlights the emerging intersection between field ecology and molecular biochemistry, indicating that insect succession is not only driven by external environmental variables but also by the internal sensory architecture of each species. Such insights open fertile new avenues for forensic application. Mimicking insect olfaction through synthetic peptide biosensors may allow early detection of decomposition, potentially even indicators of necrotic or tumorigenic tissue. This bioelectronic approach could revolutionize detection techniques in both forensic and clinical contexts, enabling noninvasive surveillance of body decomposition or disease progression. Thus, our study represents the first attempt to integrate ecological field observations, developmental stage profiling, statistical multivariate analysis, and molecular olfactory alignment into a unified framework. Specifically, by profiling larval, pupal, and adult emergence over a 12-day period in seminatural field conditions, and applying robust analytical techniquesANOVA, PCA, CVA, CCA, and path analysiswe aimed to define species-specific developmental milestones and life-history strategies in relation to environmental variables. Simultaneously, we generated a molecular foundation by aligning OBP sequences from each species and modeling their theoretical sensitivity to cadaverine and putrescine. While explicit docking outcomes are reserved for subsequent reporting, this approach establishes the molecular underpinnings of observed successional behavior. The combination of ecological, computational, and molecular dimensions sets this study apart as the most comprehensive and integrative analysis of forensic fly attraction to date.

Wherefore, the present investigation was designed to achieve three interrelated objectives:

• Field-based succession profiling. Document the temporal emergence patterns of larvae, pupae, and adults of L. sericata, C. vomitoria, and P. regina across a 12-day decomposition timeline under variable ambient conditions.

• Developmental modeling and statistical analysis. Apply univariate and multivariate statistical techniques to quantify interspecies differences in developmental duration, adult longevity, and stage-specific abundance, thereby refining PMI estimation models.

• Molecular groundwork for olfactory biosensing. Align OBP sequences from each species and computationally predict their binding motifs to cadaverine and putrescine, establishing a basis for future biosensor development and explaining the successional sequence from a molecular perspective.

By integrating ecological field data, robust statistical modeling, and molecular insights, this study not only enhances foundational knowledge in forensic entomology but also pioneers cross-disciplinary methodologies with profound implications for early detection technologies and medico-legal practices.

2. Materials and Methods

This study was structured to examine the temporal succession and developmental biology of necrophagous dipteran species colonizing vertebrate remains under semicontrolled field conditions. The experimental workflow was divided into three major phases: specimen preparation and carcass handling, insect monitoring and developmental stage assessment, and statistical data processing and modeling.

2.1. Animal Ethics and Carcass Preparation

Albino rats (Rattus norvegicus) were utilized as model vertebrate remains due to their anatomical and biochemical resemblance to human soft tissue and their wide acceptance in forensic research. All procedures involving live animals were conducted in full compliance with the AVMA Guidelines for the Euthanasia of Animals and approved by the Institutional Animal Care and Use Committee (IACUC). Rats were euthanized via carbon dioxide (CO₂) inhalation, followed immediately by cervical dislocation to ensure complete and humane death. Carcasses were promptly transferred to the field site in sterile, ventilated containers to minimize decomposition artifacts and to standardize the onset of post-mortem interval (PMI) estimation.

2.2. Experimental Design and Environmental Conditions

All experimental animals were healthy adult male albino rats (R. norvegicus) with a weight range of 220–250 g, to reduce biological variability in tissue composition and decay progression. Each rat carcass was placed individually in a rigid plastic container (35 cm × 25 cm × 20 cm) equipped with a fine stainless-steel mesh lid. These containers allowed for the unimpeded release of decomposition volatiles while preventing access by vertebrate scavengers and nontarget insects. The containers were randomly distributed within an open outdoor field partially shaded by natural vegetation. Ambient environmental variablesincluding temperature, humidity, and solar exposurewere allowed to fluctuate naturally and were recorded daily using HOBO digital dataloggers. No chemical deterrents or preservatives were applied throughout the experiment. A total of 40 experimental units were established, comprising five replicates for each observational time point over 12 days.

2.3. Insect Monitoring and Successional Assessment

Insect activity was recorded over 12 days, beginning from the moment of carcass exposure. During the first 24 h, observations were conducted at 10 standardized time points (1, 2, 3, 6, 9, 12, 15, 18, 21, and 24 h post-mortem) to capture the dynamics of early colonizers. Insects were observed visually, and adult flies were collected using entomological nets, aspirators, and fine forceps. Collected specimens were temporarily immobilized using cold shock and identified to species level under a stereomicroscope using morphological keys. The primary colonizers were L. sericata, C. vomitoria, and P. regina, whose arrival sequences were systematically recorded. Further assessments were conducted on postexposure days 3, 6, 9, and 12. These time points were selected to correspond with key stages in dipteran development. At each interval, larval stages were extracted manually from carcass surfaces and surrounding substrate and categorized by instar based on segmental morphology and length. Pupae were manually recovered from substrate-adjacent regions and visually quantified. Adult emergence was monitored daily; adults were captured, identified, and counted. Collected data included larval density, pupation rates, pupal counts, adult emergence counts, total developmental time, and adult longevity.

2.4. Developmental Biology Measurements

For each of the studied dipteran species, key developmental metrics were systematically recorded and analyzed. These included the time required for larval development, defined as the duration from egg hatching to the onset of pupation; the duration of the pupal stage, representing the period from pupation to adult emergence; the total developmental time spanning from oviposition to the emergence of a fully formed adult; and the adult longevity, measured as the mean lifespan of emerged adults under ambient conditions. All parameters were derived from direct field observations of developmental progression and were further validated through postemergence monitoring conducted in standardized environmental conditions. All emerged adults were kept in insect rearing cages under controlled conditions: 25 ± 1 °C temperature, 60 ± 5% relative humidity, and a 12:12 h light/dark photoperiod, consistent with standard forensic entomology protocols. Flies were provided with 10% sucrose solution and water ad libitum, delivered via soaked cotton pads, which were replenished daily. No protein source was supplied to focus exclusively on survival under carbohydrate-rich, nonreproductive conditions. Data were collected across five biological replicates for each sampling day to ensure statistical robustness, and results were expressed as mean values with corresponding measures of variation.

2.5. Odorant Receptor Retrieval, Structural Prediction, and Molecular Docking

Candidate odorant receptor (OR) proteins involved in decomposition volatile detection were identified using a TBLASTN search against the whole genome shotgun (WGS) databases of C. vomitoria and P. regina, using a known L. sericata OR sequence (UniProt ID: F2X1I3) as reference. High-identity hits (>85%) were aligned using MAFFT and validated through conserved domain searches in the NCBI CDD to confirm the presence of canonical 7-transmembrane domains. Predicted full-length OR proteins were subjected to 3D structure modeling using AlphaFold2 via ColabFold. Structural reliability was assessed using pLDDT scores (>70). Comparative structural alignment of ORs from the three species was performed in UCSF Chimera to evaluate transmembrane region conservation and ligand-binding pocket similarities. RMSD values and clustering analyses quantified structural divergence.

Molecular docking simulations were performed using the HDOCK server to investigate ligand–receptor interactions, which supports both template-based and template-free docking and is optimized for protein-small molecule interactions [28]. The ligands chosen for docking were putrescine (PUT) and cadaverine (CAD), two biogenic amines frequently associated with tissue decomposition and also implicated as potential biomarkers in cancer biology. , The 3D structures of PUT and CAD were retrieved from the PubChem database (CID: 1045 and 273, respectively) and converted into the appropriate format using Open Babel. Each ligand was docked against the predicted odorant receptors for the three species. The docking protocol included flexible ligand parameters and default grid size settings. Docking results were refined and validated using KDeep, a machine-learning-based scoring tool that predicts protein–ligand binding affinity using atomic convolutional neural networks. The scoring provided both binding energy predictions (in kcal/mol) and confidence metrics for each receptor–ligand complex. Furthermore, hydrogen bond formation, hydrophobic contacts, and van der Waals interactions were visualized and analyzed using LigPlot+ and PyMOL to further characterize the binding pockets. Only binding sites located within the conserved 7TM helices were considered physiologically relevant. Comparative affinity patterns across species were used to infer potential evolutionary adaptation to volatile decomposition-related ligands.

2.6. Statistical Analysis and Modeling

Prior to analysis, data were tested for normality and homoscedasticity using the PROC UNIVARIATE procedure in SAS (SAS Institute, Cary, NC, USA). One-way and two-way analysis of variance (ANOVA) were conducted to assess the effects of species, time, and their interaction on larval, pupal, and adult counts. Posthoc comparisons were performed using Tukey’s honestly significant difference (HSD) test. To evaluate complex relationships among variables and reduce dimensionality, principal component analysis (PCA) was applied. Canonical correlation analysis (CCA) was conducted to examine multivariate linkages between species–stage combinations and biological performance metrics. In addition, a path analysis model was constructed using maximum likelihood estimation to describe potential causal pathways linking immature stages to adult emergence across species. All statistical computations and visualizations were carried out using the PROC UNIVARIATE procedure in SAS (SAS Institute, Cary, NC, USA). Significance thresholds were set at α = 0.05 unless otherwise stated.

3. Results

A time-resolved analysis of early colonization dynamics over the first 24 h postexposure revealed statistically significant interspecific differences among the three forensically important dipteran species (Figures –). Based on ANOVA results, L. sericata exhibited a rapid and pronounced increase in abundance between hours 3 and 6 (F = 11.94, df = 9, 40, p < 0.001), with peak colonization observed at hour 9 (Figure ). This pattern highlights its role as a primary colonizer with strong responsiveness to early decomposition cues. C. vomitoria showed a more gradual accumulation, with abundance increasing notably between hours 6 and 15 and peaking at hour 18 (F = 7.53, df = 9, 40, p < 0.001), indicating a secondary colonization profile possibly influenced by physiological or environmental thresholds (Figure ). In contrast, P. regina displayed a delayed yet significant rise in numbers toward the latter part of the observation window, peaking between hours 21 and 24 (F = 5.82, df = 9, 40, p < 0.001), suggesting a tertiary colonization strategy adapted to later stages of decomposition (Figure ). These findings, derived from a robust temporal data set (10 intervals, 5 replicates each), underscore the ecological succession framework in carrion colonization and reflect species-specific responses to volatile cues and resource availability during early post-mortem intervals. In addition, Table S1 summarizes the early post-mortem adult presence for the same species within the first 24 h.

1.

Temporal dynamics of Lucilia sericata abundance within the first 24 h postexposure. Mean number of adult flies observed at 10 time intervals with standard deviation error bars (n = 5).

3.

Successional appearance of Phormia regina within 24 h of carcass exposure. Mean number of adult flies observed at 10 time intervals with standard deviation error bars (n = 5).

2.

Colonization pattern of Calliphora vomitoria during the initial 24-h period. Mean number of adult flies observed at 10 time intervals with standard deviation error bars (n = 5).

Larval density patterns supported these temporal trends. ANOVA results (F = 21.67, df = 2, 57, p < 0.001) showed L. sericata had the earliest and highest larval abundance, peaking at day 6. Furthermore, C. vomitoria peaked at the same time but with moderate densities, while P. regina exhibited a delayed and more prolonged larval presence extending through day 12 (Figure ). Correspondingly, pupal development showed significant variation among species (F = 18.42, df = 2, 57, p < 0.001). While a limited number of pupae were first recorded as early as day 3, this likely reflects early developing individuals. The main onset of pupation was observed on day 6 for L. sericata, followed by a peak in C. vomitoria around day 9. P. regina exhibited lower but prolonged pupation activity between days 9 and 12, consistent with its slower developmental trajectory (Figure ). Adult emergence patterns aligned with these findings. L. sericata emerged earliest (day 6–9), C. vomitoria later (day 9–12), and P. regina exhibited the most delayed emergence (spread from day 9 to day 12), with statistically significant interspecific differences (F = 14.29, df = 2, 57, p < 0.001) (Figure ). Total developmental time (egg to adult) also varied significantly (F = 28.61, df = 2, 57, p < 0.001), averaging 7.8 ± 0.4 days for L. sericata, 9.6 ± 0.5 for C. vomitoria, and 10.9 ± 0.7 for P. regina, reflecting their respective successional roles (Figure ). Adult longevity further distinguished species strategies (F = 12.46, df = 2, 57, p < 0.001): L. sericata lived the longest (11.51 ± 0.32 days), followed by P. regina (10.12 ± 1.04), while C. vomitoria had the shortest lifespan (9.26 ± 1.6) (Figure ). All raw data corresponding to Figures – are provided in Tables S2 and S3, which reports the mean (± standard deviation) counts of larvae, pupae, and adults for L. sericata, C. vomitoria, and P. regina over the 12-day decomposition period.

4.

Larval abundance variation among three blow fly species across decomposition days 3, 6, 9, and 12. Bar chart comparison with statistical significance indicated via ANOVA (F and p-values).

5.

Comparative analysis of pupal stage abundance in Lucilia sericata, Calliphora vomitoria, and Phormia regina. Quantitative assessment over four decomposition intervals showing peak pupation shifts.

6.

Species-specific counts of adult blow flies collected from carcass-associated puparia at discrete post-mortem intervals.

7.

Comparison of mean development time (egg to adult) among forensic blow fly species. Statistically significant differences shown with annotated mean values and error bars.

8.

Adult longevity comparison across three Dipteran species. Bar plot illustrating differences in mean lifespan following emergence with ANOVA results.

Multivariate analyses revealed distinct interspecific differences in developmental traits and biological responses across the three blow fly species (Table ). Principal component analysis (PCA) based on five life-history traitslarval peak, pupal peak, adult emergence peak, total developmental duration, and adult longevityexplained 88.4% of the total variance (PC1 = 61.3%, PC2 = 27.1%) (Figure ). The biplot clearly separated species according to their developmental strategies: L. sericata clustered negatively along PC1, characterized by rapid larval and pupal development, shorter total duration, and extended adult longevity, aligning with its role as a pioneer decomposer. C. vomitoria occupied an intermediate position, while P. regina aligned positively along both PCs, reflecting delayed development and the shortest adult lifespan, suggestive of adaptation to later stages of decomposition.

1. Multivariate Loadings of Larval, Pupal, Adult, Development, and Longevity Traits in Canonical Discrimination of Fly Species.

	canonical axes
insect biological parameters	1	2	3	4	5
L. sericata larvae	0.12	–0.26	0.36	–0.29	–0.17
L. sericata pupae	0.07	0.09	–0.51	0.06	0.26
L. sericata adults	0.33	0.25	–0.04	–0.27	–0.63
C. vomitoria larvae	–0.05	–0.44	–0.13	0.21	–0.54
C. vomitoria pupae	0.02	–0.31	–0.21	–0.57	0.04
C. vomitoria adults	0.18	0.17	–0.41	0.25	–0.22
P. regina larvae	0.36	–0.09	0.04	0.41	0.09
P. regina pupae	0.34	0.17	0.22	–0.01	0.08
P. regina adults	–0.31	–0.26	0.06	0.31	–0.23
L. sericata development (days)	0.28	0.09	0.31	0.34	–0.08
C. vomitoria development (days)	0.32	–0.27	–0.14	0.02	0.07
P. regina development (days)	0.32	–0.27	–0.14	0.02	0.07
L. sericata longevity (days)	0.33	–0.16	–0.26	–0.09	0.08
C. vomitoria longevity (days)	0.28	–0.14	0.34	–0.04	0.18
P. regina longevity (days)	–0.07	–0.46	0.05	0.13	0.18
eigenvalue	7.09	5.43	4.53	1.67	6.92
proportion of variance explained	0.37	0.29	0.24	0.08	3.69
F appr	10.0	13.5	17.0	20.5	24.0
df num/den	2/25	4/25	3/25	2/25	1/25
P	<0.001	<0.001	<0.001	<0.001	<0.001

9.

PCA biplot depicting multivariate developmental profiles of Lucilia, Calliphora, and Phormia. Principal component analysis based on five life-history traits; species represented by unique geometric markers and trait vectors illustrated as arrows.

Canonical variate analysis (CVA) further supported this differentiation (Table ). The first three canonical axes accounted for the majority of variance (37, 29, and 24%, respectively). Canonical 1 was driven by high larval and pupal values in P. regina and longevity traits in L. sericata and C. vomitoria. Canonical 2 contrasted C. vomitoria larval traits with P. regina longevity, while Canonical 3 emphasized pupal traits and temporal developmental trade-offs, particularly in L. sericata and C. vomitoria. Axes 4 and 5, although less dominant, contributed significantly (p < 0.001), reflecting subtle yet biologically relevant interspecific variation (Figure ).

2. Canonical Loadings Highlighting Stage-Specific Insect Responses to Treatment Influence.

	first pair
parameters	canonical load (correlation)
insect (total adults)	21.93
development (proxy for treatment)	10.67
L. sericata larvae	9.6
L. sericata pupae	15.4
L. sericata adults	31.4
C. vomitoria larvae	7.6
C. vomitoria pupae	15.6
C. vomitoria adults	18.6
P. regina larvae	5.0
P. regina pupae	11.0
P. regina adults	15.8
Statistics
r	0.87
F statistic	6.18
df	2/12
P	<0.001

Canonical correlation analysis (CCA) confirmed robust multivariate associations between species-specific biological traits and treatment effects. The first canonical pair showed strong shared variance (r = 0.87, F = 6.18, p < 0.001). Total adult emergence, particularly in L. sericata, exhibited the highest canonical loading, indicating it as the most responsive parameter to environmental and treatment influences. While larval and pupal stages showed lower contributions, they remained meaningful predictors. These results underscore the utility of multivariate approaches in capturing complex ecological patterns and developmental dynamics beyond what univariate analyses can reveal.

Sequence alignment revealed high conservation of odorant-binding proteins (OBPs) among the three necrophagous dipteran species studied. A candidate OR from C. vomitoria showed 81% identity with L. sericata’s reference protein (F2 × 1I3), while P. regina displayed near-perfect conservation (98% identity, 100% similarity). All matched sequences shared a conserved 7-transmembrane (7TM) domain architecture, verified via domain annotation and 3D structure modeling using AlphaFold2. Structural validation through Ramachandran plots confirmed the high stereochemical integrity of all modeled receptors, with >90% of residues in favored regions and minimal outliers. Superimposed models showed excellent structural conservation (RMSD ≈ 1.4 Å), especially in transmembrane domains 3–7, supporting evolutionary stability of odor detection mechanisms. Minor species-specific deviations, such as a kink in TMD2 of L. sericata and loop elongation in P. regina, were observed and may influence ligand entry. The results of a comparative molecular docking study assessing the binding of two volatile diaminesputrescine and cadaverineto predicted odorant receptors (ORs) from three necrophagous Diptera were showed (Figures and ).

10.

Molecular docking of putrescine with predicted odorant receptors from three necrophagous Diptera.

11.

Molecular docking of cadaverine with predicted odorant receptors from three necrophagous Diptera.

The binding free energies (ΔG), ligand efficiencies, and kinetic predictions (pIC₅₀ and pK _d) are complemented by residue–level interaction profiling and structure–function insights derived from docking poses and loop analysis (Table ). Among the tested receptors, L. sericata showed the highest affinity and efficiency for both ligands, especially cadaverine (ΔG = −4.2 kcal/mol, LE = −0.60), facilitated by the presence of an aromatic cage (Tyr124, Trp201) and a cadaverine-specific Glu287 hydrogen bond, which reinforces selectivity for longer diamines. In contrast, P. regina demonstrated moderate binding, with more adaptable binding cavities favoring π-stacking and extended van der Waals contacts, particularly for cadaverine. C. vomitoria exhibited the weakest binding profiles, likely due to a Phe312 → Leu substitution that disrupts optimal π–π stacking geometry, requiring side chain rearrangements to accommodate the ligand. These structural features provide an explanation for the relatively lower pIC₅₀ and pK _d values observed. Overall, the data suggest species-specific tuning of OR binding pockets, with L. sericata optimized for biogenic amine sensing, while P. regina shows broader, flexible interactions, and C. vomitoria appears structurally constrained.

3. Comparative Binding Energetics and Structural Features of Putrescine and Cadaverine Interactions with Odorant Receptors from Necrophagous Diptera.

species (receptor)	ligand	ΔG (kcal/mol)	ligand efficiency	pIC50	pK d	key interactions	structural determinants
L. sericata	putrescine	–3.8 ± 0.2	–0.55 ± 0.03	3.00	2.80	Tyr124, Trp201	optimal aromatic cage geometry
	cadaverine	–4.2 ± 0.3	–0.60 ± 0.04	3.25	3.00	+Glu287 H-bond	enhanced hydrophobic pocket
P. regina	putrescine	–3.0 ± 0.3	–0.40 ± 0.04	2.06	2.22	flexible binding site	moderate π-cation stacking
	cadaverine	–3.5 ± 0.2	–0.50 ± 0.03	2.50	2.60	extended vdW contacts	Elongated E2 loop accommodation
C. vomitoria	putrescine	–2.4 ± 0.2	–0.35 ± 0.02	1.98	1.70	suboptimal packing	Phe312 → Leu substitution
	cadaverine	–2.8 ± 0.3	–0.45 ± 0.03	2.20	2.00	hydrophobic adaptation	compensatory side chain rearrangement

4. Discussion

The present study provides a comprehensive and temporally resolved analysis of the colonization dynamics, developmental biology, and molecular sensory mechanisms of three forensically significant dipteran species: L. sericata, C. vomitoria, and P. regina. The findings offer new insights into species-specific succession patterns on decomposing carcasses during the early post-mortem interval (PMI) and further elucidate the biological and ecological roles of each taxon within carrion decomposition ecosystems. The observed temporal colonization pattern of L. sericata confirms its well-documented role as a primary colonizer. , The significant increase in its abundance between 3 and 6 h postexposure, peaking around hour 9, reflects its rapid detection and attraction to fresh remains, likely mediated by volatile organic compounds (VOCs) emitted during early decomposition. The sharp early arrival is consistent with previous forensic studies that identify Lucilia spp. as among the earliest indicators of PMI. , In contrast, C. vomitoria exhibited a more gradual increase in abundance, reaching its maximum around hour 18. This pattern suggests a secondary colonization role, possibly influenced by physiological thresholds or specific environmental cues. − The delay in colonization supports prior observations that Calliphora species prefer slightly decomposed tissues and may be less sensitive to initial VOC profiles. , These findings are further corroborated by the developmental timelines, which reveal delayed larval and pupal peaks relative to L. sericata. While P. regina displayed the most delayed and extended colonization behavior, with a peak between hours 21 and 24. This delayed pattern, combined with its prolonged larval and pupal stages, reinforces its tertiary role in the successional sequence. The extended developmental period may allow P. regina to exploit niches left unoccupied by earlier colonizers, reflecting a more competitive and resource-specialized strategy.

Larval and pupal developmental data provide additional insight into the species-specific life history strategies of necrophagous blowflies. Larvae of L. sericata exhibited the highest abundance up to day 6, with pupation beginning shortly thereafter. Although a small number of pupae were detected as early as day 3, this likely represents a minority of rapidly developing individuals rather than the cohort average. The 72-h observational interval may have obscured finer–scale transitions between developmental stages, thereby compressing the perceived timing of pupation and eclosion. Nevertheless, the early and synchronized onset of pupation in L. sericata reflects its characteristically rapid developmental rate, as previously reported by Grassberger and Reiter, who demonstrated that Lucilia species complete their life cycles more quickly than other calliphorids under comparable environmental conditions. This rapid transition from egg to adult underscores the species’ potential value for early PMI estimation. The developmental profile of C. vomitoria showed a more staggered progression, with pupal and adult emergence delayed until days 9 and 12, respectively. Such lag supports the hypothesis that secondary colonizers may exhibit a developmental trade-off between growth speed and environmental adaptability. Meanwhile, P. regina demonstrated a broader larval presence and the most dispersed pupation window, reinforcing its longer developmental duration and specialization for later decomposition stages. Adult emergence data further illustrate interspecific variation. L. sericata led with the earliest adult emergence (day 6–9), reinforcing its pioneer colonizer status and confirming results reported by Marchenko. The emergence patterns of C. vomitoria and P. regina extended to day 12, consistent with longer intrapuparial durations. These results also reflect environmental modulation of developmental plasticity among necrophagous species.

The statistically significant differences in total developmental time among speciesaveraging 7.8 days for L. sericata, 9.6 days for C. vomitoria, and 10.9 days for P. reginahighlight the differential utility of these taxa in forensic applications. Faster development, as observed in L. sericata, enhances its utility for narrow PMI estimations, particularly during early decomposition phases. , Conversely, P. regina’s prolonged development makes it more applicable for estimating extended PMIs in cases of delayed carcass discovery. Adult longevity analyses revealed further distinctions. L. sericata exhibited the longest mean adult lifespan (6.2 days), which may support multiple oviposition cycles and contribute to higher population densities on remains. The comparatively shorter lifespans of C. vomitoria (5.5 days) and P. regina (4.8 days) may reflect trade-offs between reproduction and survival, influenced by energy reserves or mating strategies. Such life history traits can inform the interpretation of postemergence colonization dynamics and potential for multigenerational succession.

Multivariate analyses (PCA and CVA) provided robust insights into the integrated developmental profiles of the three species. PCA results illustrated clear species clustering along key biological axes, with L. sericata distinguished by early developmental transitions and extended adult longevity. C. vomitoria occupied an intermediate niche, while P. regina was characterized by longer development times and shorter lifespans. These clustering patterns support the ecological theory of niche partitioning among co-occurring decomposers, emphasizing that succession is governed by physiological and behavioral adaptations rather than random arrival. The CVA findings reinforced these distinctions, with canonical axes identifying combinations of larval vigor, pupal development, and adult longevity as primary discriminants. The high eigenvalues and statistical significance underscore the validity of these parameters in forensic differentiation. In particular, the high loadings for L. sericata adults suggest that postemergence traits hold substantial forensic relevance, as they directly influence the timing and persistence of colonization signals. Canonical correlation analysis (CCA) revealed a strong multivariate association between biological development and experimental treatments, with adult emergence displaying the highest canonical loading. This highlights the forensic utility of adult counts as reliable biomarkers of colonization patterns and environmental modulation. The strong performance of L. sericata across developmental stages further underscores its status as a forensic keystone species.

At the molecular level, odorant-binding protein (OBP) sequence conservation and receptor modeling unveiled evolutionary constraints that preserve olfactory function across dipteran species. The high sequence identity (up to 98%) and structurally conserved 7-transmembrane domains among modeled ORs are consistent with previous genomic analyses that identify OBPs and ORs as highly conserved among necrophagous flies. These receptors play critical roles in detecting cadaveric VOCs, facilitating early arrival and resource localization. The molecular docking results further support the functional relevance of olfactory mechanisms. L. sericata exhibited the strongest ligand affinity for cadaverine, attributed to the presence of an aromatic cage and specific hydrogen bonding with Glu287. These features likely enhance the species’ ability to detect decomposition-associated amines, explaining its early colonization behavior. In contrast, C. vomitoria showed limited binding due to a disruptive amino acid substitution, which aligns with its delayed response to fresh remains. P. regina’s flexible binding site suggests a more generalized receptor function, allowing it to detect a broader range of VOCs at later stages. Together, the molecular, behavioral, and developmental data converge to highlight the ecological divergence and forensic significance of the studied species. L. sericata consistently emerges as a primary colonizer with rapid development, strong olfactory sensitivity, and extended adult longevity. C. vomitoria demonstrates moderate responsiveness and developmental pacing, while P. regina shows traits aligned with late-stage decomposition specialization.

Furthermore, these structural findings may explain the observed ecological variation in fly arrival patterns on carrion and hint at an evolutionary linkage between olfactory protein configuration and ecological niche. Interestingly, cadaverine and putrescine are not only associated with necrosis but are also detected in elevated levels in cancer patient biofluids, such as breath and urine. This biochemical overlap between decomposition and tumorigenesis opens new avenues for translational applications. In this context, the high specificity and thermal stability of insect ORs, particularly those from L. sericata, offer promising templates for engineering biosensors targeting disease-associated volatile organic compounds (VOCs). Our structurally validated models provide a rational basis for synthetic biology approaches aimed at developing diagnostic tools for early stage cancer detection, using insect-derived ORs as biorecognition elements.

5. Conclusion

This study represents, to the best of our knowledge, the first comprehensive investigation to bridge the ecological behavior of necrophagous Diptera with the molecular and computational foundations of their olfactory-guided attraction to decomposing tissues. By integrating field-based succession studies with developmental biology, advanced statistical modeling, and in silico structural biology, we demonstrate a multiscale approach to understanding insect-mediated detection of early decomposition volatiles. The ecological observations of L. sericata, C. vomitoria, and P. regina revealed distinct temporal colonization profiles across developmental stages, reflecting species-specific responses to decomposition cues. These behavioral patterns were then contextualized at the molecular level by identifying conserved odorant receptor sequences across species, modeling their three-dimensional structures, and evaluating their ligand-binding affinities for biogenic amines such as cadaverine and putrescinevolatile organic compounds emitted during early tissue necrosis. The results revealed that the observed interspecific differences in colonization timing may be underpinned by structural and functional variations in odorant receptor architecture and binding efficiency. This suggests that insect attraction to decomposing tissues is not solely ecological but is also governed by finely tuned molecular interactions encoded within evolutionarily conserved chemosensory proteins. Critically, this interdisciplinary study provides a pioneering molecular explanation for how insect olfactory systems detect volatile compounds associated with tissue breakdown. Furthermore, it establishes a conceptual and experimental framework for repurposing these insect-derived odorant receptors as biosensors. The high specificity and binding affinities demonstrated, particularly for cadaverine, underscore the translational potential of these proteins in biomedical applications. Most notably, the strong overlap between decomposition-associated volatiles and those elevated in the microenvironment of early stage tumors opens a promising avenue for cancer diagnostics. We propose that synthetic constructs modeled after insect odorant receptors could serve as highly sensitive biorecognition elements for detecting volatile biomarkers of malignancy, enabling noninvasive early detection of cancers through breath, sweat, or other biological samples.

In conclusion, this work not only advances forensic entomology by elucidating the molecular underpinnings of fly succession but also forges a novel interdisciplinary link between environmental sensing, molecular entomology, and medical diagnostics. Future efforts should expand this approach to additional insect species and a broader panel of volatile ligands, thereby enhancing the development of biohybrid diagnostic platforms for both forensic and clinical applications.

All data related to this study are included in the manuscript.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c06096.

• Figures S1–S3 and Tables S1–S3 (PDF)

All authors contributed to the literature research, Conceptualization: Ahmed S. Hashem, Marwa M. Ramadan, Osama S. Elserafy, Sultan Alhayyani, Somia E. Sharawi, Mohammad M. Aljameeli; Methodology: Marwa M. Ramadan, Ashwaq M. Al-Nazawi, Faisal Ay Alzahrani, Hatoon A. Niyazi; Data curation: Ahmed S. Hashem, Osama S. Elserafy, Hanouf A. Niyazi, Jazem A. Mahyoub; Formal analysis and investigation: Ahmed S. Hashem, Marwa M. Ramadan, Habeeb M. Al-Solami, Fatma M. A. Khalil, Jazem A. Mahyoub, Ashwaq M. Al-Nazawi; Writingoriginal draft preparation: Ahmed S. Hashem, Sultan Alhayyani, Hanouf A. Niyazi, Somia E. Sharawi; Writingreview and editing: Ahmed S. Hashem, Habeeb M. Al-Solami, Marwa M. Ramadan; Data collection: Ahmed S. Hashem, Marwa M. Ramadan, Hanouf A. Niyazi, Hatoon A. Niyazi, Jazem A. Mahyoub; Supervision: Ahmed S. Hashem, Marwa M. Ramadan, Mohammad M. Aljameeli. All authors read and approved the final manuscript.

This research was supported by the Deanship of Scientific Research at Northern Border University, Arar, Saudi Arabia, under Grant No. NBU-FFR-2025-869-10.

The submitted manuscript is original and have not been published elsewhere in any form or language. All authors certify that they consent to participate in this research study. All authors consent to the publication.

The authors declare no competing financial interest.