Research Article |
Corresponding author: Andrey V. Frolov ( afrolov@zin.ru ) Academic editor: Christopher Majka
© 2023 Andrey V. Frolov, Lilia A. Akhmetova, Maria S. Vishnevskaya, Bogdan A. Kiriukhin, Olivier Montreuil, Fernando Lopes, Sergei I. Tarasov.
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:
Frolov AV, Akhmetova LA, Vishnevskaya MS, Kiriukhin BA, Montreuil O, Lopes F, Tarasov SI (2023) Amplicon metagenomics of dung beetles (Coleoptera, Scarabaeidae, Scarabaeinae) as a proxy for lemur (Primates, Lemuroidea) studies in Madagascar. ZooKeys 1181: 29-39. https://doi.org/10.3897/zookeys.1181.107496
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Dung beetles (Scarabaeidae, Scarabaeinae) are among the most cost-effective and informative biodiversity indicator groups, conveying rich information about the status of habitats and faunas of an area. Yet their use for monitoring the mammal species, that are the main providers of the food for the dung beetles, has only recently been recognized. In the present work, we studied the diet of four endemic Madagascan dung beetles (Helictopleurus fissicollis (Fairmaire), H. giganteus (Harold), Nanos agaboides (Boucomont), and Epilissus splendidus Fairmaire) using high-throughput sequencing and amplicon metagenomics. For all beetle species, the ⅔–¾ of reads belonged to humans, suggesting that human feces are the main source of food for the beetles in the examined areas. The second most abundant were the reads of the cattle (Bos taurus Linnaeus). We also found lower but significant number of reads of six lemur species belonging to three genera. Our sampling localities agree well with the known ranges of these lemur species. The amplicon metagenomics method proved a promising tool for the lemur inventories in Madagascar.
Beetles, coprophagy, gut content analysis, Madagascar, next-generation sequencing, scarabaeines
Madagascar is known for its unique biota characterized by an exceptionally high level of endemism at all taxonomic levels (
Dung beetles are a marquee focal group for global efforts to assess the status of biodiversity. They are among the most cost-effective and informative biodiversity indicator groups (
Recently, attempts have been made to use DNA metabarcoding with High-Throughput, or Next-Generation, sequencing (NGS) to identify taxa in material samples, such as plant species in herbivore dung, prey species in the gut contents of predators, or soil samples (
The main goal of the present study was to evaluate the amplicon metagenomics methods to find out what are the major food providers for a few model Madagascan dung-beetle species. We did not aim at studying the complete diet of these species, nor did we study differences in the diet among individuals or groups. Instead, the study was designed to include specimens of different taxa collected in a few different areas where both native and introduced animals are available as potential food producers for dung beetles. We were specifically interested in finding sequences of lemurs (Lemuroidea) because of their importance for nature conservation in Madagascar. For preparation of amplicon libraries, we used the primers for the 16S rRNA gene fragment, successfully utilized by
Examined material is housed in the collection of Muséum national d’Histoire naturelle, Paris (
Helictopleurus fissicollis (Fairmaire). Madagascar • 12 females and 5 males (
Helictopleurus giganteus (Harold). Madagascar • 5 females and 4 males (
Nanos agaboides Madagascar • 12 females and 6 males (
Epilissus splendidus Fairmaire. Madagascar • 2 females and 4 males (
The beetles we collected by standard pitfall traps baited with human feces (all except H. giganteus) and from cow dung pads (H. giganteus). The trap captured H. fissicollis contained a preservation solution with SDS and EDTA (
For the analysis, the preserved beetles were dissected under a stereomicroscope. Abdominal tergites were cut with micro-scissors and, if the gut had visible content, it was dissected and placed in Eppendorf micro-tube with 96% ethanol for DNA extraction. Gut content was extracted from eight specimens.
DNA was extracted by phenol-chloroform extraction method (
Raw reads were trimmed from adapter reads using bbduk. CutAdapt 4.3 was used to trim primer reads and to discard reads without primers (
To validate the identification of the hosts, we recovered a maximum-likelihood (ML) phylogeny with the most representative ASVs from each sample and reads obtained from GenBank for closely related species under the same genera. Reads were aligned, manually trimmed, and the ML phylogeny was recovered with MEGAX (
We have successfully amplified 16S rDNA marker from all samples with a total 363558 reads; 326522 reads passed through quality filtering and preprocessing procedures. Filtered reads were denoised and formed 150 ASVs. Taxonomy assignments produced by BLASTN were manually checked to address possible “overclassification” due to similarity of the marker (for example in Hominidae species). The results are summarized in Table
Results of the amplicon metagenomic analysis of the gut content of four Madagascan Scarabaeidae dung beetles species.
Mammal species | Nanos agaboides | Helictopleurus fissicollis | Epilissus splendidus | Helictopleurus giganteus |
---|---|---|---|---|
Homo sapiens | 55074 | 44855 | 80293 | 56315 |
Eulemur coronatus | 0 | 329 | 0 | 0 |
Eulemur fulvus | 0 | 0 | 28 | 63 |
Eulemur rubriventer | 9 | 13 | 1161 | 0 |
Eulemur sanfordi | 2853 | 14803 | 0 | 0 |
Hapalemur griseus | 0 | 0 | 212 | 0 |
Propithecus diadema | 3 | 6 | 425 | 0 |
Bos taurus | 13224 | 3895 | 12481 | 28376 |
Bovidae | 0 | 166 | 0 | 1 |
Canis lupus | 0 | 93 | 0 | 2 |
Sus scrofa | 256 | 700 | 323 | 427 |
Arvicolinae | 912 | 0 | 0 | 4 |
Ellobius talpinus | 427 | 223 | 1180 | 6 |
Lemmus lemmus | 8 | 7 | 0 | 3907 |
Rattus norvegicus | 412 | 189 | 1 | 2860 |
The total number of mammalian taxa identified includes 15 species with most of them belonging to primates (7 species including human). Small number of reads was classified as belonging to domestic animals (dog), and may also be considered contaminations, although dogs may occur in the studied areas. The Nanos agaboides sample yielded reads of Arvicolinae rodents, not occurring in Madagascar. The H. fissicollis sample yielded reads of Bovidae, having high but not 100% similarity with a number of ruminant taxa. The H. giganteus sample yielded reads of Norway lemming (Lemmus lemmus (Linnaeus)), which does not occur in Madagascar and is apparently a contamination (this sample was processed separately in the DNA extraction laboratory). All but H. giganteus samples yielded reads of northern mole vole (Ellobius talpinus (Pallas)). We consider these reads as contaminations, because these animals do not occur in Madagascar but they were studied in the past years in the molecular laboratory where the DNA was extracted.
The mammal species that we think were true sources of the food for the examined beetle species (Fig.
Results of the amplicon metagenomic analysis of the gut content of four Madagascan Scarabaeidae dung-beetle species (contamination reads are excluded). Left, number of effective tags per beetle sample. Right, proportion of effective tags per beetle sample, excluding human and cattle sequences.
The major challenge in studying dung-beetle gut content by molecular methods is that the DNA of the putative food producers is highly degraded and is generally in low quantity. At the same time, the total DNA is extracted from the samples of the gross guts dissected from the beetles, and it is hardly possible with the beetles of this size to separate gut content from the gut tissue. In addition, the gut content is normally full of symbiotic microorganisms. Therefore, using shotgun NGS sequencing for this DNA will be economically and computationally suboptimal since the great majority of the reads would belong to non-target organisms.
Our results demonstrated that instead of shotgun approach, the amplicon metagenomic method could be successfully applied to investigate the diet of Madagascan dung beetles and the composition of the local communities of animals that provide food resources to them.
It should be noted that in all samples 2/3–4/5 of the reads belong to humans. Part of the reads might result from the contamination by the bait, although the traps were designed in such a way as to minimize possible contact of the beetles with bait. Even if the beetles manage to consume bait, it may contribute to the total reads but not substitute the reads from the dung consumed before. In addition, since the beetles are fixed immediately or shortly after they arrived to traps, the foodstuff from the bait may not go through digestive system to get to the hindgut. The contamination is also possible during the laboratory work. However, we believe the human contaminations can be small or negligible percent of all reads we encountered.
The second most abundant reads belong to cattle (Bos taurus Linnaeus). Not surprisingly, they are the most abundant in the H. giganteus sample, where the beetle was collected from a cattle dung pad. But a reasonable amount of reads were also found in the H. fissicollis sample from Amber Mountain National Park, collected by pitfall traps a few kilometers inside the park.
A high number of reads of humans (more than 2/3 in our analyses) and cattle, which was similar for different samples, regardless of the taxonomy of the beetles and collecting localities, shows that humans and cattle can be considered as the main food producers at least for some native Madagascar dung beetle taxa.
In all our samples (except for Epilissus splendidus) we revealed the sequences of Norway rat (Rattus norvegicus (Berkenhout)). However, with the data available we cannot be sure that in all these cases rats were the real food producers for the beetles. It is possible that in case of H. giganteus at least a part of sequences might be a result of contamination in the laboratory, which previously processed rodent samples. Helictopleurus giganteus sample also yielded reasonable number of lemming reads, which are definitely a result of contamination.
However, the revealed sequences of our main focal taxon, lemurs, cannot be contaminations because no experiments were carried out with Madagascan primates in the involved laboratories. Also, the known and predicted distribution of the six lemur species (
Our results demonstrated that Madagascan dung beetles can be considered a promising indirect tool for monitoring lemurs. They can easily be collected by standardized pitfall traps baited with ready available human feces. They can also be used in other regions, but more research is needed to assess the species-specificity of trophic relationships between beetles and mammals, as well as to evaluate the primers that are most suitable for the target mammal taxa.
With the development of amplicon metagenomics methods, they can be used for more complex analyses in the future. For example, as opposed to lemurs and other mammals, dung beetles are represented in natural history collections in large numbers from numerous localities, and they have been accumulated during decades. Thus, the rich dung beetle collections putatively contain information about occurrence of dung producer species in a particular area, sometimes not existing as a natural habitat any longer.
We are thankful to the Malagasy Institute of Conservation of Tropical Ecosystems (MICET) for the support in the acquisition of permits and logistic support during the expedition to Madagascar. Michele Rossini and Thomas Merrien (Finnish Museum of Natural History, Helsinki, Finland) helped with fieldwork. Aleš Bezděk (Institute of Entomology ASCR, České Budějovice, Czech Republic) commented the draft manuscript. This research was funded by Russian Science Foundation, grant number 22-24-00715 to A.F. The scarab beetle collection of the Zoological Institute is maintained under the Russian State research program 122031100272-3.
The authors have declared that no competing interests exist.
No ethical statement was reported.
Russian Science Foundation, grant number 22-24-00715.
Conceptualization: A.F., S.T., O.M., L.A.; methodology: A.F., S.T., F.L.; bioinformatics analysis: B.K.; field work: A.F. and S.T.; specimen identification: O.M.; specimen curation: L.A.; sample preparation, M.V.; draft preparation: A.F.; draft review and editing: all authors; project administration: A.F.; funding acquisition: A.F.
Andrey V. Frolov https://orcid.org/0000-0002-6724-6828
Lilia A. Akhmetova https://orcid.org/0000-0002-2151-1278
Maria S. Vishnevskaya https://orcid.org/0000-0002-2634-2610
Bogdan A. Kiriukhin https://orcid.org/0000-0001-8840-2976
Olivier Montreuil https://orcid.org/0000-0003-2682-7594
Fernando Lopes https://orcid.org/0000-0002-1246-2777
Sergei I. Tarasov https://orcid.org/0000-0001-5237-2330
The data presented in the study are deposited in the NCBI Sequence Read Archive (SRA) database, accession number PRJNA958125.