Along the lower reaches of the Selangor River in West Malaysia, thousands of fireflies gather every night on mangrove trees, flashing in perfect synchrony as part of a pre-mating ritual that has made them one of the country’s best-known insects. Yet for all their fame, one basic question has gone unanswered: what do the adults actually eat? A new study has now taken a direct molecular approach to that mystery, extracting and sequencing DNA from the gut contents of adult Pteroptyx tener to identify the plants these beetles may have consumed before they were collected.
The researchers collected adult P. tener from the wild along three rivers on the west coast of Peninsular Malaysia: the Selangor River, the Sepetang River in Perak, and the Rembau River in Negeri Sembilan. The beetles were gathered from their display trees, the berembang mangrove Sonneratia caseolaris and the Malvacean Hibiscus tiliaceus. Before any DNA work began, the specimens were sterilised to strip away external contaminants such as pollen or plant residues, immersed in a solution of 0.5 percent sodium hypochlorite and Triton X-100, rinsed in double distilled water, and air-dried. Only then were the fireflies dissected and their entire alimentary canals removed for DNA extraction.
The team screened the gut DNA extracts with two kinds of genetic markers: plant chloroplast DNA markers, targeting the genes rbcL and matK and the trnH-psbA intergenic spacer, and invertebrate mitochondrial DNA markers, targeting the 16S rRNA and cytochrome oxidase subunit I genes. The mitochondrial screen served as a check for animal meals. It found nothing of the sort. Amplification of insect mitochondrial barcoding genes from the gut extracts showed the absence of non-P. tener DNA among the samples, and the mitochondrial sequences recovered from the guts were identical to those obtained from the legs, thorax and head of the fireflies themselves. Electropherograms showed no overlying or underlying peaks that would indicate mixed or multiple DNA sources. In other words, apart from the beetles’ own DNA, only plant DNA could be detected in the gut contents.
On the plant side, the results were more revealing. Approximately 26 plant genes, ranging from 124 to 744 base pairs in length, were amplified and sequenced from the gut DNA extracts of male and female P. tener. The fragments amplified with the three chloroplast markers corresponded to coding chloroplast and non-coding chloroplast-like genes. Gel images showed distinct PCR bands in many samples, including bands of 200 to 300 base pairs with the rbcL marker, 100 to 200 base pairs with trnH-psbA, and 300 to 500 base pairs with matK, although some amplifications produced smears rather than clean bands. Notably, the researchers were unable to amplify any plant DNA genes from the gut extracts of fireflies collected from the Rembau River.
One sequence stood out. A single query sequence, designated Query 1, was identical to the rbcL sequence of Heritiera littoralis, a Malvacean tree commonly found along riverine areas, which the researchers had barcoded from the firefly habitat itself. Before that match was made, the same sequence had shown 99 percent similarity to a GenBank sequence of Tilia paucustata. Several other plant meal sequences, including the accessioned sequences KX909584, KF950349 and KF950350 and Query 5, returned 97 to 99 percent similarity matches to GenBank sequences of Lawsonia inermis, a member of the Lythraceae. Intriguingly, the team could not locate L. inermis anywhere along the riverbanks of the Selangor River where the specimens were collected.
Perhaps the most striking result was what the guts did not contain. Despite the fact that P. tener spends a large portion of its adult life on S. caseolaris and H. tiliaceus, the researchers found no traces of either display tree in the firefly gut DNA extracts. The authors caution, however, that this does not rule these plants out as food sources, particularly if the beetles feed on nectaries, fruit or sap, which may not leave detectable DNA traces in the way that consumed plant tissue does. A majority of the query sequences, Queries 2 through 10, also failed to match any of the reference plant sequences from the riverine habitat, and the remaining sequences were non-coding, chloroplast-like DNA fragments.
Some of the unmatched sequences did find relatives in the public databases. Query sequences 6 through 10 showed similarity to sequences of Gonystylus bancanus, a Thymelaeacean that typically inhabits peat and freshwater swamps, and Aquilaria sp., a genus that is increasingly popular as a cultivated plant in Malaysia. Along with the L. inermis matches, these findings led the authors to speculate that P. tener may travel further inland to obtain these host plants, or that the plants may occur in difficult-to-access or isolated forest patches. The findings provide early indication that the fireflies are not restricted to the mangrove display trees where they congregate, but the authors stress that this remains an inference from DNA matches rather than a directly observed feeding behaviour.
The study’s methods reflect the practical realities of molecular gut-content analysis. The researchers compared their query sequences against reference DNA barcodes of riverine plants collected in botanical surveys along parts of the firefly-inhabited riverbank, targeting the families Malvaceae, Thymelaeaceae and Lythraceae, and against sequences in the NCBI GenBank database using BLAST. Voucher specimens of twigs, stems, flowers and fruits were collected from the habitat, preserved in silica gel, and identified to species level by comparison with material in the Kepong Herbarium at FRIM. Laboratory contamination was controlled by sterilising bench tops and pipettes with DNA remover and using filter tips, and only coding chloroplast DNA sequences longer than 200 base pairs were deposited into GenBank, generating accession numbers KX909577-99 and KX80349-50.
The authors are candid about the limitations of the approach. Most of the sequences had extremely low query coverage, between 6 and 24 percent of their gene length, even when the similarity index was high, which limits the confidence with which any single match can be assigned. Cost constraints meant that only a partial set of plants from the firefly habitat could be collected, barcoded and identified, and most tropical plants have still never been barcoded at all. Accurate species identification of land plants generally requires multiple DNA regions to be sequenced, and while rbcL and matK are the standard land plant barcoding genes, additional markers such as the trnH-psbA spacer and nuclear ribosomal internal transcribed spacers are sometimes needed to improve species resolution. Plant tissues also begin to break down after ingestion, and shorter DNA sequences are generally easier to detect than longer ones, so degradation in the gut can prevent accurate molecular analysis.
What the study does establish is a methodological foothold for a conservation-relevant question. The larvae of lampyrid fireflies require land snails for food, but the adult diet of P. tener has been a matter of speculation, with suggestions ranging from the nectar of S. caseolaris to the possibility that adults need no food at all. The new results point toward Heritiera littoralis as a plausible food plant, particularly since its bell-shaped flowers offer pollen as a reward and are thought to be beetle-pollinated, and since the tree grows in the vicinity of the firefly display trees. The authors suggest it is unlikely the beetles feed on the leaves and seeds of H. littoralis, which contain chemicals that interfere with insect growth and reproduction.
Subject of Research: Molecular analysis of gut contents to identify the food plants of the synchronous firefly Pteroptyx tener in Peninsular Malaysia.
Article Title: Elucidating food plants of the aggregative, synchronously flashing Southeast Asian firefly, Pteroptyx tener Olivier ( Coleoptera , Lampyridae )
Article References: Cheng, S., Chan, K.-M., Ishak, S.-F., Khoo, V., & Chew, M. Y. (2017). Elucidating food plants of the aggregative, synchronously flashing Southeast Asian firefly, Pteroptyx tener Olivier (Coleoptera, Lampyridae). BioRisk, 12, 25-39. https://doi.org/10.3897/biorisk.12.14061
Image Credits: AI Generated
Keywords: Pteroptyx tener, fireflies, gut content DNA analysis, chloroplast DNA barcoding, Heritiera littoralis, Sonneratia caseolaris, Selangor River, mangrove, molecular diet analysis, Malaysia, firefly conservation, rbcL
News Source: Gavin Prescott. (October 27, 2026). Gut DNA reveals the hidden plant meals of Malaysia’s synchronously flashing fireflies. Scienmag.
