Forensic identification of Chrysomya megacephala from a hanging cadaver using mitochondrial COI gene sequencing

Introduction

Forensic entomology applies entomological evidence to medico-legal investigations, particularly in estimating the post-mortem interval (PMI), reconstructing events surrounding death, and providing corroborative evidence in criminal cases. Traditionally, insect species identification has relied on morphological characteristics, but this approach can be limited when specimens are immature, damaged, or morphologically ambiguous. To address these challenges, molecular methods have increasingly been incorporated into forensic workflows. Sperling, Anderson, and Hickey (1994) [5] pioneered the application of DNA-based identification for insects in PMI estimation, demonstrating that genetic tools could improve accuracy when morphological traits are inadequate. Among molecular markers, the mitochondrial cytochrome c oxidase subunit I (COI) gene has emerged as a gold standard for DNA barcoding due to its high interspecific variation and low intraspecific divergence, facilitating reliable discrimination among closely related taxa (Hebert et al., 2003) [3] . This COI barcode system has been widely adopted across animal groups and has become central to forensic entomology, enabling accurate species identification even from partial, immature, or degraded specimens (Wells & Stevens, 2008) [7] . Recent research continues to reinforce COI's value: Wang and colleagues (2025) [6] highlighted its ongoing utility in forensic insect identification and explored optimizations that enhance rapid and cost-effective species detection. Necrophagous blow flies, particularly those of the family Calliphoridae, are among the first colonizers of decomposing remains and have well-studied developmental patterns that make them especially useful in PMI estimation. Chrysomya megacephala (Fabricius), the oriental latrine fly, is frequently reported in forensic contexts and often dominates carrion and cadaver insect assemblages across tropical and subtropical regions (Ye et al., 2024) [8] .

Its forensic relevance has been underscored by both morphological and molecular studies, and rapid detection methods for this species continue to be developed to enhance on-site identification capabilities. Molecular identification methods not only confirm species identity through sequence similarity but also through phylogenetic context, allowing forensic investigators to place unknown samples within a broader evolutionary framework. Concordance between BLAST database matches, sequence alignments, and phylogenetic clustering significantly strengthens species-level assignments, as emphasized in recent reviews of molecular applications in forensic entomology (Scieuzo et al., 2025) [4] . Collectively, these advances underscore the importance of integrating COI barcoding with robust analytical frameworks to improve the precision and reliability of forensic insect identifications.

In this study, we applied an integrated molecular approach to identify an insect specimen collected from a hanging human body (F1-Hanging). By evaluating DNA quality, amplifying and sequencing the mitochondrial COI gene, and confirming species identity through BLAST similarity and phylogenetic clustering, we demonstrate the applicability of COI-based barcoding for forensic entomological investigations under real-world conditions.

Materials and Methods

Study Area

The present study was conducted at Jagdalpur District, under the Department of Biosciences, MATS University. Jagdalpur is located in the Bastar region of Chhattisgarh and is characterized by a tropical climate with distinct seasonal variations, which support a rich diversity of necrophagous insect fauna. Laboratory-based molecular work, including DNA extraction, PCR amplification, sequencing, and bioinformatic analyses, was carried out in the molecular biology facilities and information were provided of Department of Biosciences, MATS University, Raipur.

Sample Collection and Preservation

Cadaver-associated insect specimens were collected from a hanging human body (designated as Sample F1-Hanging) following standard forensic entomology collection protocols. The specimens were handled using sterile forceps to avoid cross-contamination. Collected samples were preserved immediately in 95-100% ethanol and stored at -20 °C until further molecular analysis. Proper labeling and documentation were maintained throughout the sampling and storage process.

DNA Extraction

Genomic DNA was extracted from individual insect specimens using a standard insect DNA extraction protocol. A small portion of tissue was homogenized, and DNA was isolated following cell lysis, protein digestion, and purification steps. The extracted DNA was finally eluted in nuclease-free water and stored at -20 °C for downstream applications.

DNA Quantification

The concentration and quality of the extracted DNA were assessed using spectrophotometric analysis. DNA concentration was recorded in ng/µl, and samples showing sufficient yield and purity were selected for PCR amplification. Only DNA extracts free from visible degradation and with adequate concentration were used for molecular identification.

PCR Amplification of COI Gene

The mitochondrial cytochrome oxidase I (COI) gene was amplified using mitochondrial-specific primers (mtCOI-5′ and mtCOI-3′). PCR reactions were carried out in a total volume of 50 µl containing DNA template, primers, dNTPs, Taq DNA polymerase, assay buffer, and nuclease-free water. The PCR cycling conditions included an initial denaturation at 94 °C, followed by 35 cycles of denaturation, annealing at 40 °C, extension at 72 °C, and a final extension step. Amplification success was confirmed by agarose gel electrophoresis.

DNA Sequencing

Successfully amplified PCR products were purified and subjected to Sanger sequencing in both forward and reverse directions. High-quality consensus sequences were generated after trimming low-quality ends and resolving ambiguous bases.

Sequence Alignment and BLAST Analysis

The obtained COI sequences were aligned using standard alignment software to ensure correct reading frames and sequence integrity. Species identification was performed using the BLASTN tool against the NCBI nucleotide database. Sequence similarity, query coverage, alignment score, and E-values were considered for accurate specieslevel identification.

Phylogenetic Analysis

Phylogenetic analysis was conducted using reference COI sequences of closely related taxa retrieved from GenBank. Evolutionary distances were calculated using the Jukes-Cantor model, and phylogenetic trees were constructed using the Neighbor-Joining method. Bootstrap analysis was performed to assess the robustness of the tree topology and clustering patterns.

Data Interpretation

Species identification was confirmed based on concordance among BLAST similarity results, sequence alignment, and phylogenetic clustering. This integrated molecular approach ensured reliable identification of the cadaver-associated insect specimen for forensic entomological interpretation.

Results and Discussion

DNA Quantification

The extracted DNA concentration for the F1-Hanging sample was 116 ng/µl (Table 1). This concentration is well within the optimal range required for downstream molecular applications, including high-fidelity PCR amplification and Sanger sequencing. The yield indicates that the DNA extraction protocol was effective and produced DNA with minimal degradation or inhibitory contaminants. Adequate DNA quality and quantity are essential in forensic entomology for accurate species identification, particularly for mitochondrial COI gene amplification (Sperling, Anderson, & Hickey, 1994) [5] . The obtained concentration confirms the suitability of the F1-Hanging sample for reliable molecular identification and supports the effectiveness of the extraction methodology employed.

PCR Amplification and COI Sequencing

Both cadaver-associated insect samples successfully amplified the mitochondrial cytochrome oxidase I (COI) gene under the described PCR conditions (Table 4). The obtained sequence lengths were 656 bp for F1-Cow and 653 bp for F1-Hanging (Table 5). Successful amplification and sequencing indicate that the extracted DNA was sufficiently intact and free from major PCR inhibitors, thereby allowing reliable DNA barcoding. Amplification of the COI gene was achieved using mitochondrial-specific primers (mtCOI-5′ and mtCOI-3′), which generated the expected ~1.5 kb PCR product. The PCR cycling conditions-initial denaturation at 94 °C, followed by 35 cycles with annealing at 40 °C and extension at 72 °C-were effective for high-fidelity amplification. These optimized conditions ensured consistent and specific amplification of the target mitochondrial region. The successful PCR outcomes demonstrate that (i) the DNA quality was adequate with no significant degradation, (ii) the primers exhibited high specificity toward the mitochondrial COI region, and (iii) no PCR inhibition occurred, which is particularly important when working with forensic samples that may contain environmental contaminants. The presence of amplifiable mitochondrial DNA in both samples confirms their suitability for accurate molecular identification, supporting the reliability of DNA-based methods in forensic entomology (Wells & Stevens, 2008) [7] .

Sequence Alignment and Species

BLAST Analysis

BLASTN analysis of the mitochondrial COI sequence obtained from Sample F1-Hanging produced highly significant alignments with Chrysomya megacephala sequences available in the NCBI nucleotide database. The query sequence (653 bp) showed 98.93% sequence identity with multiple C. megacephala reference entries, accompanied by high alignment scores, 100% query coverage, and an E-value of 0.0, indicating an extremely reliable match. All top-ranked BLAST hits corresponded exclusively to C. megacephala, with no competing species appearing among the highest similarity results. The minimal number of nucleotide mismatches observed further supports the accuracy of the sequence and confirms the absence of contamination or sequencing artifacts. These BLAST parameters meet and exceed the accepted similarity thresholds for species-level identification of forensic insects using COI barcoding (Sperling et al., 1994) [5] .

Overall, the BLAST results conclusively identify the F1-Hanging specimen as Chrysomya megacephala, validating the molecular findings obtained through sequence alignment and phylogenetic analysis and confirming the reliability of COI-based DNA barcoding for forensic entomological investigations.

Discussion

The present study successfully demonstrated the utility of mitochondrial cytochrome oxidase I (COI) gene sequencing for the accurate species-level identification of a cadaverassociated insect from a hanging body (Sample F1-Hanging). The extracted DNA concentration (116 ng/µL) was adequate for downstream molecular analysis, indicating that the extraction protocol preserved DNA integrity and effectively minimized potential inhibitors. High-quality DNA is fundamental for reliable polymerase chain reaction (PCR) amplification and sequencing, particularly in forensic entomology where precise species identification is critical (Sperling, Anderson, & Hickey, 1994) [5] .

The PCR amplification and sequencing of the COI region yielded high quality sequences (~653 bp) that could be reliably compared with reference databases. Mitochondrial COI has been widely accepted as the standard marker for DNA barcoding of animal taxa due to its high interspecific divergence and low intraspecific variation, making it particularly effective in distinguishing closely related species in forensic contexts (Wells & Stevens, 2008; Hebert et al., 2003) [7,3] . BLAST analysis of the F1-Hanging COI sequence showed 98.93% identity with Chrysomya megacephala reference sequences, with high query coverage and zero E-value, confirming the accuracy of the match. The absence of top hits from alternative taxa and the minimal nucleotide mismatches observed support the conclusion that the sequence represents C. megacephala without evidence of contamination or sequencing errors. Such high levels of COI similarity are consistent with accepted thresholds for species-level identification in forensic entomology (Yusseff-Vanegas & Agnarsson, 2017) [9] . Phylogenetic analyses further corroborated the BLAST and alignment results. The F1-Hanging sequence clustered strongly within a clade of authenticated C. megacephala reference sequences with robust bootstrap support, reflecting low evolutionary divergence typical of conspecific samples. Congruence among BLAST similarity, phylogenetic clustering, and sequence alignment enhances confidence in the molecular identification, which is essential given that morphological identification can be challenging when specimens are immature, damaged, or lacking diagnostic features. Indeed, molecular approaches such as COI barcoding provide a standardized and reproducible mechanism for species determination in forensic casework (Scieuzo et al., 2025) [4] . The identification of C. megacephala has significant forensic implications. Members of the family Calliphoridae, including C. megacephala, are among the primary colonizers of decomposing remains and are extensively used in estimating the post-mortem interval (PMI) because their predictable colonization patterns and life history traits can serve as a biological clock in decomposition studies. This species' widespread distribution, rapid colonization behavior, and well-characterized developmental stages make it a valuable indicator in medico-legal investigations (Chrysomya megacephala, 2025) [1] . Molecular identification through COI barcoding also overcomes limitations of traditional morphological identification, which can be hindered by cryptic species, phenotypic variation, and the absence of key morphological characters in immature life stages. Employing standardized DNA barcoding protocols enhances the precision and reliability of taxonomic assessment, which is crucial for accurate PMI estimation and forensic interpretation (Grundy, 2025) [2] . Overall, the integration of DNA extraction, PCR amplification, sequencing, BLAST comparison, and phylogenetic confirmation demonstrates a robust molecular workflow that strengthens the evidential value of insect specimens in forensic cases. The results emphasize the importance of molecular tools in forensic entomology, particularly when traditional methods are insufficient or ambiguous.

Conclusion

The study confirms that mitochondrial COI gene-based DNA barcoding is a robust and reliable approach for the species-level identification of cadaver-associated insects in forensic investigations. The F1-Hanging sample yielded high-quality DNA in sufficient quantity, allowing successful PCR amplification and sequencing without evidence of degradation or inhibition. Sequence alignment, BLAST analysis, and phylogenetic clustering consistently identified the specimen as Chrysomya megacephala, with high sequence similarity and strong phylogenetic support, thereby validating the accuracy of the molecular workflow employed. The confirmed presence of this forensically important blowfly species, known for its early colonization behavior, underscores the relevance of molecular identification in improving the accuracy of post-mortem interval estimation, particularly in cases where morphological identification is limited or unreliable.

Acknowledgement

The authors sincerely thank MATS University, Raipur (C.G.), for providing laboratory facilities and academic support throughout this study. We are grateful to the Department of Bioscience for technical assistance during sample processing and molecular work. Special thanks to the forensic authorities who facilitated sample access and field support. The first author, Anupama Meshram, expresses deep appreciation to Dr. Aparna Jha for her