Determination of time since death is a cornerstone of medico‑legal investigation, directly influencing reconstruction of events, verification of alibis, and administration of justice. Conventional methods of PMI estimation—such as algor mortis, livor mortis, rigor mortis, and putrefactive changes—are time‑dependent and become increasingly unreliable as decomposition progresses.¹ Environmental variables, body habitus, cause of death, and post‑mortem handling further compromise their accuracy.²

Forensic entomology is defined as the application of insect biology to legal investigations.³Necrophagous insects colonise decomposing remains in a predictable manner, and their development is primarily governed by temperature and ecological conditions. By identifying insect species and determining their developmental stages, a minimum PMI can be estimated with considerable precision.⁴

The scientific foundation of forensic entomology dates back to the nineteenth century, with Bergeret’s landmark application in 1855 and Mégnin’s formulation of insect succession waves in 1894.^5,6 Subsequent experimental and case‑based studies across Europe, North America, and Australia validated insect succession and developmental data as reliable indicators of PMI.^7,8,9

However, insect fauna, succession patterns, and developmental rates vary with geography and climate. Data generated in one bio‑geoclimatic zone cannot be directly extrapolated to another.¹⁰ India, with its vast climatic diversity, lacks region‑specific human‑based forensic entomology studies. Punjab, despite its extreme seasonal variations, remains under‑represented in entomological literature.

Morphological identification of insects is often hindered by damaged specimens, immature stages, or close inter‑species similarity. Molecular identification using mitochondrial DNA markers, particularly the cytochrome c oxidase subunit I (COI) gene, has emerged as a robust adjunct for accurate species identification.^11,12

The present study was undertaken to generate region‑specific forensic entomological data from south‑west Punjab using human autopsy material, to validate molecular identification of necrophagous insects, and to assess the reliability of entomological PMI in medico‑legal practice.

Review of Literature

Forensic entomology has evolved from anecdotal observations to a well‑established forensic discipline. Sung Tz’u’sWashing Away of Wrongs in the 13th century represents the earliest documented use of insects in death investigation.¹³Redi’s experiments in 1668 disproved spontaneous generation, establishing that maggots arise from fly eggs.¹⁴

Bergeret’s application of insect development to estimate PMI in an infanticide case in 1855 marked the first medico‑legal use of forensic entomology.⁵Mégnin later proposed eight successive waves of insects colonising exposed corpses, forming the conceptual basis of insect succession.⁶

Twentieth‑century research expanded empirical evidence. Motter analysed insect fauna from 150 exhumations, highlighting the relationship between burial conditions and insect colonisation.¹⁵ Reed and Payne conducted controlled carcass studies demonstrating predictable succession patterns and the accelerating effect of insect activity on decomposition.^16,17

Rodriguez’s human cadaver studies established a direct relationship between decomposition stages and insect succession.¹⁸O’Flynn provided region‑specific developmental data for blowflies in Australia, emphasising climatic influence on PMI estimation.⁹

In India, Aggarwal studied 54 cases in Punjab and demonstrated improved PMI accuracy when entomological evidence was combined with conventional findings.¹⁹ Subsequent studies confirmed seasonal variation in insect activity and accelerated larval development during summer months.²⁰

Recent advances focus on molecular identification. COI gene sequencing has proven effective in distinguishing morphologically similar species and damaged specimens.^21,22,23However, genetic databases remain incomplete for Indian insect populations, underscoring the need for regional molecular data.

Aims and Objectives

1. To establish molecular identification of necrophagous insects infesting human corpses in south‑west Punjab.
2. To estimate minimum PMI using insect succession and developmental stages.
3. To compare entomological PMI with PMI derived from conventional methods.
4. To assess the applicability of forensic entomology in routine medico‑legal practice in India.

Study Design and Setting

A descriptive observational study was conducted at the Mortuary, Department of Forensic Medicine and Toxicology, and the Molecular Research Unit (MRU), Guru Gobind Singh Medical College & Hospital, Faridkot, Punjab.

Study Period and Sample Size

The study spanned 18 months (January 2023–June 2024). Twenty‑six medico‑legal autopsy cases fulfilling inclusion criteria were analysed.

Inclusion and Exclusion Criteria

Cases showing insect infestation with reasonably confirmed PMI were included. Bodies without insect evidence or unreliable PMI were excluded.

Collection of Entomological Evidence

Eggs, larvae, pupae, and adult insects were collected from natural orifices, wounds, and maggot masses using sterile forceps. Two sets were prepared: (1) live specimens for rearing and (2) preserved specimens for molecular analysis. Larvae were killed by hot‑water immersion before preservation in 70% ethanol.

Estimation of PMI

Minimum PMI was calculated based on the most advanced developmental stage, rearing observations, and ambient temperature data. Entomological PMI was compared with PMI estimated from police records and decomposition changes.

Molecular Identification

DNA extraction was performed using the NucleoSpin® Tissue Kit. PCR amplification targeted the mitochondrial COI gene. Sequencing results were analysed using MEGA‑X software and compared with reference databases.

Ethical Considerations

The study was approved by the Institutional Ethics Committee. Consent was obtained from relatives through standard medico‑legal procedures.

Demographic and Environmental Profile

Out of 26 cases, 23 (88.46%) were males. The predominant age group was 20–50 years, with 61.54% identified. Most cases occurred during summer and monsoon months (88.46%). Outdoor recoveries constituted 61.54% and where partially appeals worn in 80.77%.

Table1. Age &Sexdistributionofcasesobserved

Table 2.Distribution ofcasesacrossseasons

Table 3. Distribution of cases according to the state of Apparels over the bodies

Decomposition Stage

Nineteen bodies (73.08%) were in the bloat stage, and seven (26.92%) in active decay. 13 (50.00%)had larvae, 7 (26.92%) had eggs, and 6 (23.08%) had adult flies.

Table4.Distributionofcasesaccordingtotheirdecompositionstage

Table 5.Stageof insectfoundon bodies

Insect Species Identified morphologically and molecularly

The most common species was Chrysomyamegacephala (61.54%), followed by Muscadomestica (19.23%), Sarcophagacarnaria (15.38%), Luciliacuprina (3.85%), and Dermestesmaculatus (3.85%) which were confirmed by Molecular identification was done by amplifying COI region of these flies.

Table 6.Speciesofinsectfoundon bodies

Fig.1Sarcophagacarnaria

Fig.2DermestesMaculatus

Fig3:Luciliacuprina

Fig 4:Chrysomyamegacephala

Correlation of PMI

Entomological PMI matched known PMI in 25 cases (96.15%). One mismatch occurred in a drowning case.

The predominance of males and younger adults mirrors findings from Indian and international studies.^19,20 Seasonal clustering during warmer months reflects enhanced insect activity and accelerated larval development.²⁴

The dominance of Chrysomyamegacephala is consistent with tropical forensic entomology literature.²⁵ Molecular confirmation using COI gene region of these flies, which shows 98-99% similarity with the nucleotide sequences in GEN BANK submitted by former researchers after the Basic Local Alignmentsearch Tool (BLAST)

High concordance between entomological and known PMI confirms the reliability of forensic entomology. Discrepancies in aquatic deaths highlight delayed insect access as a limiting factor.²⁶

Forensic entomology supported by molecular identification is a reliable and practical method for estimating minimum PMI in decomposed bodies in south west Punjab. Development of regional entomological and genetic databases and integration of entomology into routine forensic practice in India are strongly advocated.

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