An innovative molecular approach towards the cost-effective entomological authentication of honey

The study addresses the growing need for reliable, practical, and economical methods to ensure food authenticity and traceability, with a focus on honey—a high-value product prone to fraud. Honey’s authenticity must consider both botanical and entomological origins, as different Apis species produce honeys with distinct composition, quality, and market value. Mislabeling undermines consumer rights and fair trade and influences beekeeping practices and species composition. Current standards and sensory attributes are insufficient to distinguish honeys from different bee species. Therefore, the research aims to develop a rapid, reliable, and cost-effective molecular method to determine the entomological origin of honey, enabling robust traceability and supporting sustainable industry development.

Prior work has explored multiple traceability approaches, with DNA-based methods standing out for stability, applicability, and specificity. Studies have used species-specific primers targeting genes such as tRNA-cox2 (Apis cerana), MRJP2 (A. cerana and A. mellifera), and ND2 (A. cerana, A. mellifera, A. dorsata). Real-time PCR with high-resolution melting (HRM) can differentiate A. cerana from A. mellifera and identify A. mellifera subspecies, but requires costly equipment and curated melting-curve databases. PCR-RFLP is a classical, low-cost, accurate, and easily testable approach widely used in food authenticity, offering intuitive results without complex analysis and minimal equipment requirements (PCR and gel electrophoresis). Despite its advantages and use in other food products, PCR-RFLP had not been applied to identify honey’s entomological origin. The authors identified a gap: no simple, feasible method existed to differentiate honeys from six Apis species simultaneously.

Sample collection: Worker bees (positive controls) from six Apis species were collected in China: A. mellifera (multiple locations including Hangzhou, Jinhua, Shaoxing, Beijing), A. cerana (Hangzhou, Jiaxing, Yichun, Nanchang), A. laboriosa (Honghe Hani and Yi Autonomous Prefecture, Lincang, Dehong Dai and Jingpo Autonomous Prefecture), A. dorsata (Xishuangbanna), A. florea (Xishuangbanna, Chongzuo), and A. andreniformis (Xishuangbanna). In total, 148 authentic honey samples with known entomological origin were collected during 2014–2022: A. mellifera (n=30), A. cerana (n=30), A. laboriosa (n=30), A. dorsata (n=30), A. florea (n=26), A. andreniformis (n=2). High-fructose corn syrup served as a negative control. Pre-treatment and DNA extraction: Bee thoraxes were homogenized in PBS with zirconia beads (1 min, 200 Hz), centrifuged, and pellets stored at −20 °C. For honey, 15 g was diluted in 30 mL water, heated to 45 °C for 10 min, homogenized, centrifuged at 10,000 rpm for 30 min; the pellet was resuspended in 1 mL water and centrifuged at 12,000 rpm for 15 min; pellets stored at −80 °C. DNA was extracted from bee and honey pellets using TIANamp Genomic DNA Kit. DNA concentration and purity (A260/A280) were measured by NanoDrop 2000. Primer design and PCR: Mitochondrial 16S rRNA gene sequences of the six target species (GenBank NC_051932.1, NC_014295.1, NC_036155.2, NC_037709.1, NC_021401.1, NC_039709.1) were aligned (MEGA7.0) to design conserved-region primers (Primer Premier 5). Universal primers: 16S rRNA-F 5′-TGACTTACGTCGATTTGAAC-3′ and 16S rRNA-R 5′-GACTGTACAAAGGTAGCATAAT-3′. PCR mix: 2 µL DNA template, 10 µL 2× Taq PCR StarMix, 0.5 µL each primer (10 µM), 7 µL water. Cycling: 94 °C 2 min; 30 cycles of 94 °C 30 s, 50 °C 30 s, 72 °C 30 s; final 72 °C 5 min. Products were run on 2% agarose gel with 100–2000 bp ladder. Sequencing and analysis: Amplicons (470–479 bp) were Sanger sequenced (BLAST identification). Sequences were deposited in GenBank (PP621844, PP621799, PP621846, PP621845, PP621848, PP621849). Pairwise genetic distances (Kimura 2-parameter) and a neighbor-joining phylogenetic tree with 1000 bootstraps were produced in MEGA7.0. Restriction enzyme selection and PCR-RFLP: Candidate restriction sites were analyzed with NEBcutter V2.0, identifying AseI (recognition site ATTAAT) as suitable to generate species-distinct profiles. Digestion: 25 µL reaction with 10 µL PCR product, 2.8 µL 10× NEBuffer r3.1, 1 µL (10 U) AseI, 11.2 µL water; 37 °C for 2 h. Digests were separated on 4% agarose gel; fragment sizes determined with 50 bp ladder. Suitability testing: Focusing on A. mellifera honey, PCR-RFLP was evaluated across processing/storage states: room temperature 1 year and 5 years; −20 °C for 6 and 8 years; water-bath heating at 60, 70, 80, 90, 100 °C for 1 h; crystallized; and commercial honey. Three samples per condition were analyzed.

- DNA extraction yielded high-quality DNA from both bees and honey despite potential inhibitors: bee DNA 19.5–556.6 ng/µL, purity 1.84–2.19; honey DNA 8.5–1585.6 ng/µL, purity 1.76–2.25. - A 470–479 bp mitochondrial 16S rRNA fragment was successfully amplified from all six Apis species and their honeys, indicating primer specificity and absence of PCR inhibitors. - Sequenced amplicons matched species identities in GenBank with 99–100% similarity; accession numbers: PP621844 (A. mellifera), PP621799 (A. cerana), PP621846 (A. laboriosa), PP621845 (A. dorsata), PP621848 (A. florea), PP621849 (A. andreniformis). Phylogenetic analysis clustered samples by species. - Pairwise genetic distances among the six species (16S rRNA) ranged from 3.5% to 12.1%. - Single-enzyme PCR-RFLP using AseI produced species-specific patterns for both bee tissue and honey: • A. mellifera: 414 bp, 60 bp. • A. cerana: 219 bp, 202 bp, 54 bp. • A. laboriosa: 217 bp, 125 bp, 60 bp (additional expected fragments <50 bp not clearly resolved). • A. dorsata: 175 bp, 128 bp, 60 bp (additional expected fragments <50 bp not clearly resolved). • A. florea: 249 bp, 165 bp, 60 bp. • A. andreniformis: 250 bp, 229 bp. - The method was robust across honey processing/storage conditions. Identical PCR-RFLP profiles were obtained from raw honey and honey stored at room temperature for up to 5 years, frozen at −20 °C for up to 8 years, heated up to 100 °C for 1 hour, crystallized, and commercial samples. - The approach requires only one primer pair and a single restriction enzyme to distinguish honeys from six closely related Apis species.

The study demonstrates that a simple, low-cost PCR-RFLP assay targeting a conserved mitochondrial 16S rRNA fragment and a single restriction enzyme (AseI) can unambiguously authenticate the entomological origin of honey from six Apis species. This addresses the need for practical tools that avoid the complexity, expense, and infrastructure demands of methods like HRM or multi-plex species-specific PCRs. High-quality DNA recovery from honeys and consistent amplification validate feasibility for routine testing. Species-level differentiation through distinct AseI profiles, supported by sequence identity and phylogenetics, directly answers the research question and enables reliable verification of labeling claims. The robustness to heating, long-term storage, and crystallization enhances applicability in real-world supply chains, including processed and commercial products. Broader impacts include supporting fair trade, consumer protection, geographic certification via species distributions, and conservation of wild bee species by curbing fraudulent substitution with lower-value honeys.

This work introduces a cost-effective, streamlined PCR-RFLP protocol for entomological authentication of honey, capable of distinguishing honeys from six Apis species using a single primer pair and the AseI endonuclease. The assay yields species-specific restriction profiles, validated by sequencing, phylogenetics, and extensive testing across processed and stored samples. It offers a practical reference framework for regulators and industry to implement honey origin verification and traceability. Future work could expand validation across wider geographic ranges and production contexts, increase sampling for underrepresented species, and extend the approach to subspecies or breeds to refine entomological certification standards.

- Sampling imbalance: only two A. andreniformis honey samples were available due to limited distribution and production, reducing statistical power for this species. - Very small restriction fragments (<50 bp) are difficult to resolve on agarose gels, and some expected minor fragments were reported as <50 bp, which may limit fine-grained pattern interpretation. - Validation focused on six Apis species from China; broader geographic and taxonomic validation (e.g., additional subspecies or closely related taxa) would strengthen generalizability.