Corresponding author: Shawn Cheng (
Academic editor: Josef Settele
The aggregative, synchronously flashing firefly,
Cheng S, Chan K-M, Ishak S-F, Khoo V, Chew MY (2017) Elucidating food plants of the aggregative, synchronously flashing Southeast Asian firefly,
The synchronous firefly,
In temperate regions, fireflies have been reported to feed on a diverse range of host plants such as ginger lilies, honey-dew, pomegranate and floral segments of the milkweed,
Unfortunately, fireflies are not afforded the same attention, funding or structured research programmes given to insects of economic importance such as agricultural, livestock or stored product pests. The situation is also dire for
Aggregative, synchronously flashing firefly,
Adult
Botanical surveys to search for members of the
PCR was performed on a GeneAmp 9700 Thermal Cycler (Applied Biosystem, Foster City, CA) in a reaction containing 1µl of template DNA (10ng/µl), 0.6µl of each primer, 5.0µl of 2× Transtaq Hifi Supermix (TransGene Biotech, Beijing) and 3.4µl of Ultrapure ddH2O (Invitrogen, USA). The following parameters were used to perform the PCR: initial denaturation at 95°C for 3 minutes, denaturation at 95°C for 1 minute, annealing for 1 minute at 47°C for matK, trnH and rbcL, extension at 72°C for 1 minute and 30 seconds, final extension at 72°C for 10 minutes. Amplification of insect barcoding genes used the following conditions: initial denaturation at 95°C for 3 minutes, denaturation at 95°C for one minute, annealing for 1 minute at 48°C for the 16S rRNA and cytochrome oxidase subunit I genes, extension at 72°C for 1 minute and 30 seconds, final extension at 72°C for 10 minutes. The primers used to amplify the three different genic regions (rbcL, trnH-psbA and matK) from the gut contents extracted from
DNA and PCR products were electrophoresed on 0.85% agarose and 2.0% agarose gels, respectively, and visualised under ultraviolet light after staining with GelRed (Biotium, USA). Unincorporated primers and dNTPs were removed from the PCR products with shrimp alkaline phosphatase (USB ExoSAP-IT PCR Product Cleanup, Affymetrix, USA) following the manufacturer’s protocol. The PCR products were then used in Big-Dye® Terminator ver. 3.1 cycle sequencing reactions after which they were injected into a 3130xl Genetic Analyzer (Applied Biosystems). Sequencing was performed in both the forward and reverse direction after which the sequences were assembled in Sequencher ver. 4.9 (Gene Codes Corp., Ann Arbor, MI). The following GenBank accession numbers KX909577-99 and KX80349-50 were generated from this study. The identity of the DNA sequences were determined by comparing the query sequences against reference DNA sequences on the National Centre for Biotechnology Information (NCBI) database (Madden 2003) using the Basic Local Alignment Search Tool (BLAST) and the reference DNA barcodes of riverine plants in the vicinity of the firefly habitat.
Amplification of insect mtDNA barcoding genes from gut DNA extracts with 16S rRNA and cox 1 markers showed the absence of non–
Several samples showed distinct PCR bands, for example, in lanes 5 through 8, lanes 16 through 23 (rbcL genes), lanes 25 through 28 (trnH-psbA genes) and lanes 33 through 38 (matK genes) (Figure
PCR amplification of rbcL, trnH-psbA and matK genes from
Amplification of trnH-psbA gene from
Amplification of rbcL and matK genes from
Approximately 26 plant genes between 124 and 744 bp in size were amplified and sequenced from the gut DNA extracts of male and female
We found no traces of
Table of relationships between plant DNA sequences detected in the gut contents of
Only plant DNA could be detected in gut DNA extracts from
The larvae and adults of some beetles have mouthparts that have been adapted for feeding on fluids. The fluids are absorbed via canals or internal ducts located at the tip of their mandibles (
Some firefly species remain in the larval stage for a longer period but are short-lived as adults. Others however spend a considerable amount of time in the adult stage after having only spent a brief period in the larval stage.
Plants play an important role in the life-cycle of insects. Plants are utilised as a stage for their mating, as food or for egg–laying (
It is probably unlikely that
Three approaches are generally available to determine the hosts or food plants of fireflies. These include conducting a DNA analysis of their gut contents; observational studies in the field and/or laboratory; or the use of biochemical methods to detect sugars and/or cellulose in their gut. Observational studies may help in the identification of the host plants of the firefly, however, some clues as to where to look would be helpful. Clear choices of host plants would be plants or flowers that are constantly frequented by the insect. Sugar and cellulose assays such as the anthrone test or calcofluor fluorescent staining (
More recent studies however have employed the use of DNA barcodes amplified from the gut content of insects to determine their plant hosts (e.g.,
There were limits as to what we could barcode from the firefly habitat due to the significant amount of cost required to collect, barcode and identify plants from the area, which was beyond our project funding ability. In addition, most tropical plants have still not been barcoded. Accurate species identification of land plants also requires multiple DNA regions or loci to be sequenced. Plastid genes such as the rbcL and matK are important DNA barcoding genes for land plants (CBOL Plant Working Group, 2009). However, genes such as the plastid intergenic spacer trnH-psbA and nuclear ribosomal internal transcribed spacers (ITS) are sometimes required to increase species resolution (
We thank the Selangor State Government, in particular, the Honourable Elizabeth Wong (Executive Councillor in Charge of Tourism, Consumer affairs, and Environment), Mrs. Farah Liyana Binti Mohamed Nor and Mrs. Mas Sakdah Binti Kamaruzzaman (Economic Planning Unit, Selangor State Government) for providing FRIM with the research grant entitled “Genetic Research on the Sungai Selangor Fireflies” (Grant No: 53 -31-08-02-006) for the study. We would also like to extend our deepest thanks to the villagers of Kampung Kuantan (Selangor), Kampung Dew (Perak) and Sungai Timun (Rembau, Negeri Sembilan) for assistance rendered to the Project Team during the sampling phase of the project. We also thank Lee Chai-Ting, Norita Mihat, Maggie Sudo Pui-San and members of the Molecular Genetics Laboratory (FRIM) for technical and administrative assistance on the Project.
Supporting Information S1. 16S ribosomal RNA sequence alignment
Link:
molecular data
Supporting Information S2. Cytochrome oxidase subunit 1 sequence alignment
Link:
molecular data