Temperature effects on development and cuticular hydrocarbons in forensically relevant Dermestes maculatus

Forensic entomology is a specialized discipline within forensic science that employs the developmental stages and succession patterns of insects to aid in criminal investigations, particularly in estimating the postmortem interval (PMI), detecting neglect or abuse, and understanding cadaver relocation^1,2. Among the most forensically significant insect species is the hide beetle, Dermestes maculatus (Coleoptera: Dermestidae), which is frequently found on decomposing remains during the later stages of decay³. This beetle plays a pivotal role in estimating PMI, particularly under arid or enclosed conditions where dipteran colonization is limited⁴. Dermestes maculatus is widely distributed across North America, Europe, Africa, Asia, and Australia, and has been recorded in various regions of Saudi Arabia, including Riyadh, Jazan, Jeddah, and Al-Baha^5,6,7,8. Both adults and larvae are capable of consuming dry animal tissues and are known to infest dried fish, hides, furs, and even museum specimens^5,9. These traits highlight their dual significance: not only in forensic investigations but also as pests of economic concern. Recent research has provided new developmental data for D. maculatus from different geographical regions, including China’s Yangtze River Delta region, where developmental duration of the immature stage ranged from 66.13 ± 8.58 days at 19 °C to 21.9 ± 2.01 days at 34 °C, with survival rates varying significantly across temperatures¹⁰. Similarly, comprehensive studies have examined the effects of temperature and laboratory rearing conditions on D. maculatus development, documenting survivorship exceeding 50% across seven larval instars, though morphometric parameters showed no consistent relationship with temperature, limiting the utility of isomegalen diagrams for PMI calculation¹¹.

In forensic investigations, accurate estimation of larval age is critical. Traditionally, morphological parameters such as larval length, weight, and the number of molts have been used to determine age¹². However, these methods can be affected by environmental conditions, food quality, and other factors, thus requiring supplementary techniques for more precise age estimation. Recent advancements have emphasized the utility of cuticular hydrocarbon (CHC) profiling as a non-invasive and species-specific method for determining insect age^13,14. The insect cuticle is covered with hydrocarbons that vary with age, sex, and environmental conditions, making CHCs a reliable biomarker^15,16. Alotaibi et al.¹⁷ demonstrates that CHC of Chrysomya albiceps components differed significantly between larval instars and across rearing temperatures (30–40 °C), with highest CHC abundance at 35 °C and lowest at 40 °C. Studies on Sarcophaga peregrina have documented comprehensive CHC profiles across all life stages, identifying 37 compounds ranging from C11 to C35 carbon chain lengths¹⁸.These temperature-dependent variations in CHC profiles parallel the morphological changes observed during development, suggesting that CHCs provide complementary chemical evidence for age estimation.

Given that temperature significantly influences both developmental rate and hydrocarbon composition, studying larval development under controlled thermal conditions becomes essential. The optimal temperature range for D. maculatus development is reported to be between 25 and 30 °C⁴. Therefore, integrating morphological and chemical analyses under varying temperatures can enhance the reliability of larval age estimation. Temperature is the most critical environmental factor influencing insect development rates. Insects are poikilothermic organisms whose metabolic rates and developmental processes are directly governed by ambient temperature within species-specific thermal tolerance ranges. Understanding temperature-development relationships is fundamental to forensic entomology, as miscalculation of thermal effects can lead to substantial errors in PMI estimates. As there are limited studies on the effect of temperature on cuticular hydrocarbon on insects of forensic importance, the present study aims to assess the effectiveness of larval morphological traits and cuticular hydrocarbon profiles for estimating the age of D. maculatus larvae under controlled temperature conditions of 20 °C, 30 °C, and 40 °C. By integrating morphometric and chemical markers, the study seeks to strengthen age estimation accuracy for forensic post-mortem interval analysis, particularly under conditions where conventional indicators may be unreliable.

Experimental insects

In May 2024, a pre-killed rabbit (Oryctolagus cuniculus domesticus), purchased from pet shop, was placed outdoor in a ventilated cage (60 × 50 × 42 cm) in northern Riyadh, Saudi Arabia after having the ethical approval from King Saud University ethical committee (KSU-SE-21–65). Rabbit carcass was used only as a collection substrate and not as a model for forensic succession or ecological inference. Larval specimens of D. maculatus were collected one-week post-placement and transferred to the laboratory at King Abdulaziz City for Science and Technology (KACST) for colony establishment. The larvae reared under controlled conditions (30 °C, 60% RH, 12:12 h L: D photoperiod) in a metal cage (30 × 30 × 30 cm³) till adults emerged.

Morphological and molecular identification

Morphological identification of adults was conducted using taxonomic keys^19,20 under a Leica M80 stereo microscope. Five random selected morphologically identified adults were preserved in ethanol and stored at −20 °C for molecular confirmation via mitochondrial cytochrome oxidase I (mt COI) gene sequencing.

DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen) and then quantified using a NanoDrop 2000 UV-VIS Spectrophotometer (Thermo Fisher Scientific Inc., USA). PCR amplification was performed using Folmer primers with annealing temperature adjusted after multiple optimization trials to achieve optimal amplification (Folmer 1994)²¹. The PCR reaction (20 µL) consisted of 2 µL of genomic DNA, 0.6 µL of forward and reverse primers, FIREPol 4 µLMaster Mix, and 14.8 µL distilled water. Amplification conditions were initial denaturation at 95 °C (5 min), 35 cycles of denaturation (95 °C, 30 s), annealing (43 °C, 30 s), extension (72 °C, 1 min), and final extension (72 °C, 5 min). PCR Amplicons were run on a 1.5% agarose gel stained with ethidium bromide and visualized under UV. Sequencing was performed using an Applied Biosystems 3130xl analyzer, and results were validated via BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Experimental design for temperature influence

Following morpho-molecular identification, D. maculatus adults were transferred into a rearing metal cage (30 × 30 × 30 cm³) (BioQuip Products Inc., USA) and provided with a piece of chicken as an oviposition medium and larval food, which was renewed every week.

Eggs from the second generation were reared at three temperatures (n = 300 eggs at each temperature) (20 °C, 30 °C, and 40 °C) with 60% RH and 12:12 h L: D photoperiod in incubator (MLR-351 H, Sanyo, Japan). From the hatched larvae, fifteen larvae from each instar per group reared at different temperature were periodically sampled (randomly) for morphometric analysis (length, width of head capsul, and weight). Selected larvae were killed in hot water (70 80 °C) for 3–5 min to prevent shrinkage when placed in ethanol²². Larvae were then placed in 75% ethanol in a 1.5-ml Eppendorf tube, labelled, and stored at − 20 °C for further use. Later, the body size, in terms of length and width of head capsul, of each larval instar of each experimental group was measured under the microscope (Leica M80 Microscope by Prescotts Inc.), provided with a digital camera (Qlmaging MicroPublisher 5.0 RTV, Biocompare). Larval weight was measured using an analytical balance (AG245 Analytical Balance, Mettler Toledo) according to the previously established procedure¹⁷.

Cuticular hydrocarbon extraction and analysis

Pools of larvae from selected instars (2nd, 4th, 6th) with 5 random replicates for each instar (Table 1) were used to measure their cuticular hydrocarbon according to²³. Living larvae were immersed in hexane for 10–15 min (Table 1). The volume of hexane used was sufficient to fully immerse all larvae. Extracts were purified on a silica gel column (60–120 mesh, 2 cm × 0.5 cm) using hexane as eluent. GC injection was 1 µL on a column of 0.25 mm ID, 0.25 μm film thickness. The extracts were purified using silica gel column chromatography (60–120 mesh, 2 cm × 0.5 cm) using hexane as the mobile phase to selectively elute nonpolar hydrocarbons while retaining polar compounds on the silica. The purified fractions were then evaporated and stored at 4 °C until analysis. The extracts of dried larvae were re-dissolved in 20 µl before injection into GC-Ms manually. All extracts (3 µl) were carried out on an Agilent Technologies 7890 A GC system using split/splitless injector operated in split mode with a split ratio of 10:1 at 260 °C, and capillary column (DB-5MS, Agilent, US) (30 m × 0.25 mm ID, 0.25 μm film thickness), and coupled to an Agilent mass 5975 C with triple-axis Detector (Agilent Technologies, US). The GC was coupled to a computer where data was processed with Agilent Chemstation software. Elution was carried out at a helium at 1.4 ml/min. The oven temperature was programmed to be held at 50 °C for 2 min, then ramped to 200 °C at 25 °C/min for 3 min, then from 200 to 260 °C at 3 °C/min and was held for 10 min. The solvent and column blank every 5 sample injections. Hydrocarbons were identified using a library search (NIST08).

Statistical analysis

All statistical analyses were conducted using IBM SPSS Statistics (Version 22.0, IBM Corp., Armonk, NY, USA) and R software (Version 4.3.1).

For morphometric data (length, head capsul width, and weight), fifteen independent larvae per instar per temperature (n = 15 biological replicates) were analyzed. Data were first assessed for normality using the Anderson–Darling test and for homogeneity of variances using Levene’s test. As assumptions were satisfied, differences between temperature groups within each instar were analyzed using one-way analysis of variance (ANOVA). When ANOVA results were significant (P < 0.05), Tukey’s Honestly Significant Difference (HSD) post-hoc test was applied for pairwise comparisons. Effect sizes were calculated using eta-squared (η²) to estimate the magnitude of temperature effects.

Developmental duration (egg, larval, and pupal stages) between 20 °C and 30 °C was compared using independent-samples t-tests. Kaplan–Meier survival analysis was additionally performed to assess differences in developmental progression across temperatures. Statistical significance was set at P < 0.05.

For cuticular hydrocarbon (CHC) analysis, five independent biological pools (n = 5) per instar per temperature were analyzed. CHC peak areas were normalized and expressed as relative percentage abundance prior to statistical analysis to minimize variation due to injection volume or detector sensitivity.

To evaluate differences in CHC profiles between instars and temperatures, multivariate statistical analyses were performed as described below. All multivariate analyses were conducted using standardized (z-score transformed) relative abundance data.

Morphological and molecular identification of adults D. maculatus

Morphological examination of adult specimens using standard taxonomic keys confirmed the species as Dermestes maculatus. Diagnostic features were consistent with published descriptions. In addition, Molecular confirmation using mitochondrial COI gene sequencing revealed high similarity with reference sequences in GenBank, validating species identity (one sequence deposited in GenBank under accession number PX705857).

Morphometric variation across instars and temperatures

Mean morphometric measurements (weight, head capsule width, and length) for each larval instar reared at 20 °C and 30 °C are presented in Table 2. Early instars (1st–3rd) showed no statistically significant differences in body weight, head capsule width, or length between the two temperature treatments (ANOVA, P > 0.05). Growth trajectories during early development were therefore comparable under both thermal regimes.

However, significant divergence emerged in mid-development. In the 4th instar, larvae reared at 30 °C exhibited significantly greater body weight (24.54 ± 0.99 mg) compared to those at 20 °C (8.81 ± 0.14 mg) (P < 0.001). Similarly, larval length was significantly higher at 30 °C (10.69 ± 0.04 mm) than at 20 °C (10.21 ± 0.04 mm) (P < 0.05).

In the 5th instar, significant differences were observed in all three morphometric parameters. Larvae reared at 30 °C showed markedly greater weight (56.73 ± 1.59 mg), head capsule width (2.09 ± 0.01 mm), and length (14.77 ± 0.04 mm) compared to those at 20 °C (P < 0.001).

By the 6th instar, although absolute values remained higher at 30 °C, differences were no longer statistically significant for all parameters (P > 0.05), indicating partial convergence of final instar morphology.

Developmental progression also differed qualitatively: larvae reared at 20 °C progressed to a 7th instar, whereas those at 30 °C completed development at the 6th instar. Overall, temperature significantly influenced morphometric development, particularly during mid-larval stages.

Temperature-dependent developmental duration

Developmental durations for each life stage are presented in Table 3. Total larval development was significantly shorter at 30 °C (15 days) compared to 20 °C (28 days) (independent t-test, P < 0.001). This represents approximately a 46% reduction in larval development time at the higher temperature.

Similarly, pupal duration was significantly reduced at 30 °C (5 days) relative to 20 °C (9 days) (P < 0.001). Egg incubation time was also shortened at elevated temperature.

At 40 °C, only approximately 20% of eggs hatched, and all larvae died before reaching the second instar, indicating that this temperature exceeds the species’ upper developmental threshold under the tested conditions.

These results confirm a strong inverse relationship between temperature and developmental duration within viable thermal limits.

Cuticular hydrocarbon (CHC) profiles in D. maculatus larval instars

GC–MS analysis identified approximately 40 hydrocarbon compounds across the 2nd, 4th, and 6th larval instars reared at 20 °C and 30 °C. Identified compounds included n-alkanes, alkenes, methyl-branched alkanes, and cyclic hydrocarbons with carbon chain lengths ranging from C16 to C34 (Table 4).

Relative abundance data (mean ± SD of five biological pools per instar per temperature) revealed both stage-specific and temperature-specific variation in CHC composition (Figs. 1 and 2). Several hydrocarbons, including pentacosane, heptacosane, and tetracosane, were consistently detected across all instars and temperatures. In contrast, other compounds exhibited stage-dependent expression patterns. For example, certain iodinated hydrocarbons were predominantly detected in early instars at 20 °C but absent at later stages.

Temperature influenced both the diversity and abundance of CHCs. The 2nd instar reared at 30 °C displayed a greater number of detectable compounds compared to 20 °C. In later instars, several hydrocarbons were exclusive to one temperature treatment, indicating temperature-specific chemical signatures.

Multivariate analysis of CHC profiles

Principal Component Analysis (PCA) performed on standardized relative abundance data revealed clear clustering of samples according to both larval instar and rearing temperature. The first two principal components accounted for the majority of variance in CHC composition, demonstrating that developmental stage and temperature are major drivers of chemical differentiation.

Linear Discriminant Analysis (LDA) further confirmed separation among the six groups (2nd, 4th, and 6th instars at 20 °C and 30 °C). Complete group separation was observed in discriminant space (Table 5), indicating strong multivariate differentiation in hydrocarbon composition.

Although predictive cross-validation could not be performed due to the use of mean profiles rather than individual replicate-level data, the clear separation observed suggests that CHC composition encodes both developmental and thermal information.

Forensic entomology plays a vital role in criminal investigations by utilizing the presence and developmental stages of insects to estimate the postmortem interval (PMI)^24,25. During the late decay and skeletal stages of decomposition, beetles, particularly those from the Dermestidae family, become dominant, making Dermestes maculatus a species of notable forensic relevance²⁶.

Dermestes maculatus was selected for this study due to its wide distribution, frequent occurrence in forensic cases, and its economic and forensic significance. Previous studies have reported its presence across Saudi Arabia, including Riyadh, Jeddah, and the Southwestern Mountains^7,27,28. Additionally, the species is a known pest of stored animal products and silk⁵ and is increasingly recognized as an indicator species for PMI estimation²⁴.

Accurate species identification is essential in forensic entomology and often involves morphological examination. However, morphological similarities among closely related species can complicate accurate identification²⁹. In this study, adult morphological characteristics were consistent with earlier descriptions^19,30, and species identity was further confirmed through molecular analysis using the mitochondrial COI gene, a proven genetic marker for beetle identification^31,32. Combining morphological and molecular techniques increases confidence in taxonomic accuracy.

The most striking and forensically significant finding was the complete developmental failure at 40 °C, where 80% of eggs failed to hatch and all hatched larvae died before reaching the second instar. This upper thermal limit is consistent with the findings of Zhang et al.¹⁰, who identified an upper lethal threshold of 34.03 °C for D. maculatus. Similarly, Alotaibi et al.¹⁷ reported that Chrysomya albiceps development ceased at 45 °C. These findings have critical implications for forensic practice: in hot climates or during summer months when surface temperatures may exceed 40 °C, D. maculatus colonization may be delayed or prevented entirely, potentially leading to significant errors in PMI estimation if investigators assume continuous insect activity. Forensic entomologists must therefore carefully consider microhabitat temperatures and seasonal variations when interpreting Dermestes evidence from remains.

The temperature-dependent acceleration of development observed in our study has direct practical applications for PMI estimation. At 30 °C, the total larval development period was approximately 47% shorter than at 20 °C, emphasizing the necessity of accurate temperature data for forensic calculations. Wang et al.³³ reported similar temperature effects in the related species Dermestes tessellatocollis, reinforcing the importance of species-specific developmental databases across multiple temperatures. These findings corroborate those of Martín-Vega et al.³⁴ and Zanetti et al.¹¹, emphasizing the inverse relationship between temperature and development time. Larval instar counts also varied with temperature and were generally lower at higher temperatures, though variations across studies may reflect geographical, genetic, and environmental differences^22,35. For forensic practitioners, our data provide reference values for D. maculatus development at 20 °C and 30 °C that can be incorporated into thermal summation models and isomorphen diagrams for PMI estimation in cases involving mummified or skeletonized remains.

Morphometric data revealed that temperature effects become most pronounced during mid-larval development (4th–5th instars). This finding is forensically relevant because these instars are commonly encountered during advanced decomposition. While early instars showed limited morphometric divergence, later instars exhibited measurable temperature-induced differences in weight and body dimensions. Thus, morphometric analysis remains useful but may require temperature-specific reference datasets. This pattern contrasts with findings from Zanetti et al.¹¹, who reported no relationship between morphometric parameters and temperature in D. maculatus, concluding that isomegalen diagrams were unsuitable for PMI calculation. The discrepancy may reflect differences in experimental design, temperature ranges tested, or population-specific variations. Such data are essential in estimating larval age and, by extension, the PMI. However, post-feeding shrinkage and environmental variability can compromise morphological age estimation³⁶. Our data suggest that morphometric analysis can provide useful age estimation, particularly for later instars, but should be interpreted cautiously and ideally combined with other biomarkers.

Our GC-MS analysis identified approximately 40 unique hydrocarbon compounds (24 alkanes, 11 alkenes, 3 methyl-branched alkanes, and 2 cyclic hydrocarbons) with carbon chain lengths ranging from C16 to C34. This diversity is comparable to that reported in forensically important Diptera. For example, Sharma et al.³⁷ identified 23 compounds in Chrysomya rufifacies larvae, while Moore et al.1³⁸ detected compounds with varying proportions of n-alkanes (15–16%), alkenes (16–25%), and methyl-branched alkanes (52–59%) in Sarcophaga species. The presence of similar compound classes across taxonomically distinct necrophagous insects suggests that CHC profiling represents a broadly applicable approach in forensic entomology.

More importantly, CHC profiling demonstrated strong stage- and temperature-specific chemical signatures. The consistent presence of certain hydrocarbons across all conditions suggests potential baseline biomarkers for species confirmation, while temperature-exclusive compounds may function as indicators of environmental history. The observed multivariate separation in PCA and LDA analyses indicates that CHC profiles capture both intrinsic developmental information and extrinsic thermal exposure. Alkanes were consistently more abundant than alkenes across all stages and temperatures, consistent with previous research^17,39. Polar compounds were also detected, likely due to their partial solubility in the used non-polar solvent^23,40.

Greater variability in CHC profiles was observed among early instars, which may be due to, exposure to environmental conditions, or physiological differences in hydrocarbon biosynthesis⁴¹. Specific hydrocarbons such as pentacosane, heptacosane, and tetracosane were consistently detected, while others like octadecane and 1-octadecene decreased with larval maturity, indicating a shift in metabolic priorities⁴². Increased levels of heneicosane in later instars suggest a potential role in signaling or communication⁴³.

Temperature influenced not only developmental timing but also CHC composition. Several hydrocarbons were detected only at specific temperatures and instars. A key finding of our study was the temperature-specific variation in CHC profiles. At the 2nd instar, we detected 18 hydrocarbon compounds at 20 °C compared to 27 compounds at 30 °C, indicating that higher temperatures promote greater CHC diversity. Furthermore, specific compounds showed temperature-dependent abundance including 1-iodo-2-methylundecane and 1-iodohexadecane at 20 °C and docosene and cyclohexane at 30 °C (at early instars). These compounds may serve specialized functions in early development⁴⁴. This temperature-dependent CHC variation is consistent with findings from Alotaibi et al.¹⁷, who reported that CHC components in C. albiceps larvae differed significantly across temperatures, with the highest CHC concentrations at 35 °C and lowest at 40 °C. Similarly, Shang et al.¹⁸ demonstrated that CHC profiles of Sarcophaga peregrina pupae showed time-dependent variations at different constant temperatures (20 °C, 25 °C, 30 °C). Similar findings across other insect taxa suggest CHC changes can occur rapidly in response to environmental cues and may reflect critical physiological transitions. [43, 44]. Changes in the cuticular hydrocarbons of insects can occur over a timescale ranging from minutes to weeks, depending on the species and the insect’s survival rate^40,43.

The identification of temperature-exclusive compounds represents a particularly valuable forensic tool. In our 6th instar larvae, nine compounds were exclusive to 20 °C (including 1-nonadecene, heptadecane, 1-iodooctadecane, heneicosane, 5-eicosene (E), 1-iododocosane, 1-iodoeicosane, 9-tricosene (Z), and 13-methylheptacosane), while five compounds were exclusive to 30 °C (octadecane, 1-docosene, hentriacontane, 1-pentacosene, and 9-octylheptadecane). These temperature-specific signatures could potentially serve as “chemical thermometers,” providing retrospective information about the thermal conditions experienced by larvae during development. However, this application requires further validation under fluctuating temperature conditions that more closely approximate field scenarios.

Our observation that certain compounds (pentacosane, heptacosane, and tetracosane) were consistently present across all larval stages aligns with findings from Stewart-Yates et al.⁴⁶, who noted that while CHC profiles vary with insect development, some compounds remain stable and can serve as reliable markers. The progressive shift from shorter-chain to longer-chain hydrocarbons with increasing larval age mirrors the pattern reported by Sharma et al.³⁷ in C. rufifacies, where nonane (C9) was most abundant in 1 st instars, while hentriacontane (C31) and tritriacontane (C33) dominated post-feeding 3rd instars. This ontogenetic shift in CHC composition likely reflects changes in cuticular lipid biosynthesis associated with increasing body size and cuticle thickness, and represents a robust age-dependent biomarker.

Our finding that early instars (1st-3rd) showed similar morphometric measurements at 20 °C and 30 °C, but distinct CHC profiles, highlights the superior sensitivity of chemical markers for age estimation in early developmental stages. Conversely, the clear morphometric differences observed in later instars (4th-6th) demonstrate that traditional measurements remain valuable, particularly when CHC analysis is unavailable or impractical. This suggests a tiered approach to forensic age estimation: morphometric analysis as a rapid screening tool, with CHC profiling reserved for cases requiring higher precision or when morphometric data are ambiguous. The integration of multiple biomarkers also addresses the challenge of intraspecific variation. Paula et al.⁴⁷ demonstrated significant intraspecific variation in cuticular chemical profiles of Chrysomya megacephala between geographical populations, while Kula et al.⁴⁸ reported geographical variation in CHC profiles of Calliphora vicina across three populations. By combining morphometric and chemical data, forensic investigators can potentially distinguish between population-level variation and age-related changes, improving the reliability of PMI estimates.

While our study provides valuable data for forensic applications, several limitations must be acknowledged. First, our experiments were conducted under constant temperature and humidity conditions (60% RH, 12:12 h L: D photoperiod), which do not reflect the fluctuating environmental conditions encountered in forensic scenarios. Shang et al.¹⁸ demonstrated that fluctuating temperatures (18–36 °C; 22–30 °C) prolonged S. peregrina development and reduced pupariation/eclosion rates compared to constant temperatures, highlighting the potential for significant PMI estimation errors when laboratory-derived data are applied to field cases. Future research should prioritize validation of our findings under fluctuating temperature regimes that simulate realistic forensic scenarios.

Second, our CHC analysis was performed on living larvae immediately after extraction, whereas forensic specimens are typically preserved in ethanol or dried. Sharif et al.⁴⁹ investigated the impact of microenvironmental factors on CHC degradation in Lucilia sericata puparia, demonstrating that temperature, humidity, and UV light significantly affect CHC stability over time. Stewart-Yates et al.⁴⁶ noted that environmental factors can alter CHC composition and degradation, potentially compromising forensic interpretations. The stability of D. maculatus CHC profiles under various preservation and storage conditions requires systematic investigation before CHC profiling can be confidently applied to forensic casework.

Third, our study examined only two viable temperature conditions (20 °C and 30 °C), with complete developmental failure at 40 °C. Zhang et al.¹⁰ tested six temperatures (19 °C, 22 °C, 25 °C, 28 °C, 31 °C, 34 °C) and established comprehensive thermal summation models with a lower developmental threshold of 15.28 °C and an intrinsic optimum of 28.36 °C. To develop robust PMI estimation tools, additional temperature points between 20 °C and 30 °C, as well as temperatures below 20 °C, should be investigated to establish complete thermal development models for D. maculatus populations in Saudi Arabia.

In conclusion, the present study demonstrates that D. maculatus larval morphometric traits and cuticular hydrocarbon profiles are temperature-dependent biomarkers that can enhance PMI estimation accuracy in forensic investigations involving mummified or skeletonized remains. The integration of morphological and chemical markers provides complementary information across developmental stages and environmental conditions, offering a robust multi-modal approach to age estimation. Future research should prioritize field validation, expansion of thermal development databases, and application of chemometric modeling to enhance the precision and reliability of D. maculatus as a forensic indicator species. Further studies are needed to broaden CHC profiling to other forensically important insect species, assess how humidity and other climatic factors affect larval survival and CHC composition, confirm developmental patterns under open-field conditions, and develop new, cross-species methods for estimating larval age.