Research Article |
Corresponding author: Shawn Cheng ( shawn@frim.gov.my ) Corresponding author: Kar-Men Chan ( chankarmen@frim.gov.my ) Academic editor: Josef Settele
© 2017 Shawn Cheng, Kar-Men Chan, Shah-Fadir Ishak, V. Khoo, M.Y. Chew.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Cheng S, Chan K-M, Ishak S-F, Khoo V, Chew MY (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
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The aggregative, synchronously flashing firefly, Pteroptyx tener congregates on a nightly basis on Berembang trees (Sonneratia caseolaris) growing along the lower reaches of the Selangor River (West Malaysia). Every night, the males and females of this species engage one another in a pre-mating ritual of flash communication. Little is known of the dietary requirements of the adults of P. tener apart from suggestions that these beetles feed on the nectar and sap of mangrove trees. The drastic reduction in their numbers in recent years has sparked an urgency to understand their dietary needs. Here, we report on a series of probing experiments where we sequenced and analysed DNA fragments obtained from the gut contents of adult P. tener specimens. We detected coding and non-coding chloroplast DNA (cpDNA) gene sequences in the gut DNA extracts of P. tener. One DNA sequence was in reasonably good condition to allow us to match it to the cpDNA sequence of a Malvacean, that is, Heritiera littoralis, a common inhabitant of estuarine habitats. We also detected the DNA sequences of plants (cultivated and natural) that may have come from hamlets or isolated freshwater swamps located further inland. The findings reported here provide early indication that P. tener may be able to travel further inland to search for their hosts. Future research should focus on visually confirming if P. tener feeds on H. littoralis and obtaining a more complete reference DNA database of plants in the firefly habitat.
Pteroptyx tener , host, firefly, plants, chloroplast DNA
The synchronous firefly, Pteroptyx tener Olivier (Coleoptera: Lampyridae), congregates in the thousands in estuaries in several locales throughout Peninsular Malaysia. Here, they perform their synchronous flashing behaviour throughout the year (Figure
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, Asclepias syriaca (L.) (
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 P. tener in Malaysia, as the country’s best-known insect species is threatened by waste pollution and destruction of its breeding habitat for the purpose of commercial planting of oil palms (
Adult P. tener fireflies were collected from the wild from Selangor, Sepetang (Perak) and Rembau Rivers (Negeri Sembilan) along the west coast of Peninsular Malaysia from their display trees, namely, S. caseolaris and Hibiscus tiliaceus (Malvaceae). Prior to DNA extraction, specimens were sterilised to remove contaminants such as pollen or plant residues from their bodies. Fireflies were sterilised by immersing them in a solution containing 0.5% sodium hypochlorite and 0.01µl/ml Triton X-100 (Fisher BioReagents™, USA) and agitating them for 1 minute (in
Botanical surveys to search for members of the Malvaceae, Thymelaeaceae and Lythraceae, were carried out along parts of the riverbank inhabited by P. tener. Target plant families were ascertained earlier from preliminary laboratory screening of the gut contents of P. tener (unpublished). Voucher collection of twigs and stems with flowers and fruits, were made of the flora from the firefly habitat. DNA samples of the voucher materials, in the form of leaves of each targeted plant species were preserved in zip-lock bags containing silica gel. Plant voucher specimens were identified to the species-level by comparing them with identified materials in the Kepong Herbarium in FRIM.
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 P. tener were rbcL-aF (5’-ATGTCACCACAAACAGAGACTAAAGC-3’) (Levin et al., 2003) and rbcLa-724r (5’-TCGCATGTACCTGCAGTAGC-3’) (Fay et al., 1997); trnH-f (5’-CGCGCATGGTGGATT CACAATCC-3’) (Tate and Simpson, 2003) and psbA-r (5’-GTTATGCATGAACGTAAT GCTC-3) (Sang et al., 1997); and the maturase K gene using the primer pair matK-f (5’-GTACAGTAGTTTTGTGTTTACGAG-3’) and matK-r (5’-ACCCAGTCCATCTGGAAAT CTTGGTTC). The cytochrome oxidase subunit I or cox1 gene was amplified with the primer pair HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) and LCO1490 (5’-GGTC AACAAATCATAAAGATATTGG-3’) while the 16S ribosomal RNA gene was amplified with the primer pair 16S-ar-JJ (5’-CGCCTGTTTATTAAAAACAT-3’) and 16S-1472-JJ (5’-GGTCCTTTCGTACTAA-3’) (Folmer et al. 1994).
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–P. tener DNA among our samples (Suppl. materials 1and 2). MtDNA gene sequences obtained from the gut DNA extracts of P. tener were found to be identical to the DNA sequences obtained from the legs, thorax, and head of the firefly. We detected no overlying or underlying peaks in all electropherograms which would indicate the presence of mixed or multiple DNA samples in the material we analysed. Separately, the gel image in Figure
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 P. tener. Note: “+” for positive control; “r.b.” for reagent blank; “L.” for 100 bp DNA ladder.
Amplification of trnH-psbA gene from P. tener from Selangor and Perak*. Note: “+” for positive control; “r.b.” for reagent blank; “L.” for 100 bp DNA ladder.
Approximately 26 plant genes between 124 and 744 bp in size were amplified and sequenced from the gut DNA extracts of male and female P. tener (Figure
We found no traces of S. caseolaris or Hibiscus tiliaceus in firefly gut DNA extracts (the display trees utilised by P. tener adults). A majority of our query DNA sequences (Queries 2-10) obtained from the gut DNA extracts of P. tener also did not match reference plant sequences from the riverine habitat of P. tener. The comparison of our other query sequences, that is, Query Sequences 6 to 10, showed similarity to the sequences of Gonystylus bancanus and Aquilaria sp. from GenBank. Gonystylus bancanus typically inhabits peat and freshwater swamps while Aquilaria is gaining popularity as a cultivated plant species in Malaysia. The remaining DNA or query sequences were non-coding, chloroplast-like DNA sequences. The results of the BLAST analysis to reference sequences on the GenBank database are shown in Figure
Table of relationships between plant DNA sequences detected in the gut contents of P. tener, reference DNA plants from the firefly habitat, and DNA sequences from GenBank. BLAST analysis results include the identity of the matching plant species, gene, query cover and similarity index (after forward slash sign).
Only plant DNA could be detected in gut DNA extracts from P. tener, apart from its own DNA. Although P. tener spends a large portion of its adult life on S. caseolaris and H. tiliaceus, the plants were not found in their gut DNA extracts. However, this does not preclude the plants from being utilised for food by fireflies especially if the beetles fed on nectaries, fruit or sap of these plants. In our initial investigation (unpublished), plant sequences detected in the gut of the fireflies returned a match to the DNA sequences of Thymelaeaceae, Malvaceae and Lythraceae. The results of this experiment however showed that a single plant DNA sequence obtained from P. tener was identical to the rbcL sequence of H. littoralis (Malvaceae). The discovery of several DNA sequences that were highly similar to the DNA sequence of L. inermis (Lythraceae), a non-native, but relatively common garden plant in Malaysia, G. bancanus (Thymelaeaceae), an inhabitant of peat and freshwater swamps, and Aquilaria sp., a popular, cultivated plant species in Malaysia, has led us to speculate that P. tener travels further inland to obtain these host plants. Alternately, the plants may be in difficult to access or isolated forest patches.
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 P. tener feeds on the leaves and seeds of H. littoralis which contain chemicals that interfere with the growth and reproduction of insects (
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
Data type: molecular data
Supporting Information S2. Cytochrome oxidase subunit 1 sequence alignment
Data type: molecular data