Samples

We used platypodine beetles of the genus Mitosoma Chapuis, 1865 [61] (Coleoptera: Curculionidae: Platypodinae) embedded in resin (Fig 1). Mitosoma is an endemic genus from Madagascar and is abundantly found embedded in resin drops from Hymenaea verrucosa (Angiospermae: Fabales: Fabaceae). We collected specimens in resins from a lowland forest close to the Pangalanes Canal, in Ambahy (Nosy Varika, Mananjary) (20º46’ S, 48º28’ W) and Andranotsara (Sambava) (14º37’ S, 050º11’ W) on the east coast of Madagascar. The Government of Madagascar authorised sampling (permit no. 160/13 /MEF/SG/DGF/DCB.SAP/SCB and no. 192/17/ MEEF/SG/DGF/DSAP/SCB.Re) and exportation (permit no. 186 N.EA10/MG13 and no. N. 249/17/MEEF/SG/DREEF-SAVA) of samples. The specimens embedded in resin were collected by X.D., E.P. and M.S.K. directly from H. verrucosa trees in Madagascar in October 2013 and September–October 2017, respectively. Resin samples were stored at room temperature until commencing the study in February 2019, which increased the risk of greater DNA degradation [27]. After the first experiments, all resin samples were stored at -20°C.

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Fig 1. Resin samples from Hymenaea trees in Madagascar with embedded platypodine beetle specimens of the genus Mitosoma, sampled during fieldtrips in 2013 and 2017.

https://doi.org/10.1371/journal.pone.0239521.g001

We used specimens of Mitosoma lobatum Schedl, 1961 [62], M. excisum Schaufuss, 1897 [63] and M. obconiceps Schedl, 1970 [64] as positive controls, collected in 2012 by B.J. when on a fieldtrip in Ranomafana National Park (Madagascar). Specimens were stored in >96% ethanol immediately after collection and preserved at -20°C prior to DNA extraction.

DNA extraction was performed at the Institute of Medical Microbiology, Immunology and Parasitology (University Clinic Bonn, Germany) from ten adult specimens in resin and four adult specimens in ethanol, all representing the same genus and body size, but different localities (see above) and ages (Table 1).

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Table 1. Specimens of Mitosoma sp. used for DNA extraction and DNA concentration of the eluted DNA [ng/μl].

https://doi.org/10.1371/journal.pone.0239521.t001

Only resin pieces with two or more complete specimens as syninclusions were selected in order to use some of them for analysis and store the other(s) at -20ºC at the Senckenberg Research Institute (Frankfurt, Germany) under the collection numbers SMF Be 13578–13584. We hoped thereby to halt degradation of the hypothetically preserved biomolecules [27] and to preserve the specimen(s) for future controls and experiments. These specimens are available to other researchers/laboratories upon request, for suitable research projects aimed at determining reproducibility. Different specimens from other resin samples collected at the same time (three from 2013 and four from 2017) were treated as independent replicates (Table 1, Fig 1).

Sample preparation and DNA extraction

All samples used in this study were entirely encased within H. verrucosa resin. The resins with insect inclusions were cut into small cubes with a sterile scalpel, leaving a few millimetres around the insect. The surfaces were sterilised with 0.1 M HCl and then washed in sterile water. All the following procedures were carried out under a laminar flow hood in a microbiology laboratory without any contact with entomological experiments. The resin cubes were ground with a micro-pestle in a 1.5 ml reaction tube to which 180 μl ATL-buffer with 20 μl proteinase K (Qiagen, Germany) was added. After incubation at 56°C for 72 h and occasional vortexing, the DNA was extracted using the DNeasy® Blood & Tissue kit (Qiagen, Germany), following the manufacturer’s instructions. DNA was eluted in DNase- and RNase free water and stored at -20°C until further experiments. DNA concentration was measured using the NanoDrop™ One/OneC microvolume-UV/VIS-spectrophotometer (Thermo Scientific, USA). DNA extraction from beetles in ethanol was performed using an identical procedure except for a reduced lysis step of 24 h. To test an alternative methodology some cubes were incubated in 5 ml 100% chloroform (AppliChem GmbH, Germany) for 3 days at 40°C to dissolve the resin. After the chloroform treatment the beetle was washed in ≥ 99% EtOH and further processed as described above. Precautions to eliminate contamination included regular disinfection of all surfaces and working materials with Freka®-NOL AF (Dr. Schumacher GmbH, Germany) and the use of dedicated protective clothing, equipment and reagents. In addition, prior to DNA extraction and amplification experiments, working materials and surfaces were cleaned with DNA-ExitusPlus™ IF (AppliChem GmbH, Germany) to avoid contamination with extrinsic DNA.

Primers

Primers and protocols were selected from previously published studies on the weevil subfamily Platypodinae [65–67], or newly designed based on Platypodinae DNA sequences. The primer pairs S3690F and A4285R, targeting the D2–D3 domains of the large nuclear ribosomal subunit (28S), and S2442F with A3014R, targeting the 3’ end of the mitochondrial cytochrome oxidase I (COI), efficiently detected DNA from extant Mitosoma samples and were therefore selected for further experiments (Table 2). Two additional COI primers (Table 2; COIRes F and COIRes R2; COInew) were designed based on sequences from the resin-embedded beetles collected in 2013. These enabled amplification of a much smaller fragments than the standardised primers used in previous studies (160 base pairs (bp) vs. more than 600 bp) and were more suitable for new experiments with older (and theoretically more fragmented) material.

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Table 2. Primer sequences for DNA extraction selected from the literature and two new sequences tested here.

https://doi.org/10.1371/journal.pone.0239521.t002

Polymerase Chain Reaction (PCR) amplification

The PCR reaction mixture used to amplify genes of interest was composed of 12.5 μl OneTaq® 2X Master Mix with Standard Buffer (New England Biolabs, Germany), 0.5 μl of each primer (10 μM) and 50 ng DNA, adding water to a final volume of 25 μl. Addition of 1 μl bovine serum albumin (BSA) (10 mg/ml) to the PCR reaction mixture improved efficiency and enabled a reduction in PCR cycles. In each PCR, a negative (sterile water) and a positive control (beetle DNA from specimens in ethanol) were included. PCR was performed in a Gradient LabCycler (SensoQuest, Germany) with the standard cycle program: initial denaturation step at 95°C for 5 min, followed by 30–50 cycles of denaturation at 95°C for 30 s, annealing at specific temperatures (Table 2) for 30 s and elongation at 68°C for 60 s, and a final elongation step at 68°C for 5 min. Further optimisation included different cycles and gradient PCRs to determine the annealing temperature. The PCR results were analysed by means of agarose gel electrophoresis (1.5%). The PCR products were purified using the GeneJet PCR Purification Kit or GeneJet Gel Extraction Kit (Thermo Scientific, USA), in case that fragments had to be extracted directly from the agarose gel, following the manufacturer’s instructions. Sanger sequencing was performed by Eurofins Genomics GmbH (Germany) with the amplification primers for 28S and COI (Table 2). Alignments between DNA sequences from beetles in EtOH and resin-embedded specimens to analyse the sequence data and investigate potential contamination with modern DNA were performed with Geneious R10 (https://www.geneious.com).

Authentication of DNA sequences

We applied the following criteria to authenticate the amplified DNA sequences recovered from the beetles [41, 68]:

1. Negative controls, extraction blanks and PCR negative controls should be devoid of specific PCR amplification products.
2. Amplified sequences should be consistently and reproducibly obtained from the same extract(s) from one specimen and from different samples of the same species to guarantee reproducibility.
3. All procedures should be repeated three times in different laboratory rooms (in our case, at the Institute of Medical Microbiology, Immunology and Parasitology).
4. All PCR products should be controlled and analysed via sequencing for quality, assembly and specificity.

Illustrations

General pictures of the resin samples were taken using a Nikon D3X with an AF-S Micro-NIKKOR 60 mm 1:2.8 G ED lens. Gel electrophoresis pictures were created using the FastGene FAS-Digi imaging system (NIPPON Genetics Europe). Detailed pictures of the beetle specimen were taken in the laboratory using a Keyence VHX1000 digital microscope under incident light (general body) and using a stereomicroscope in combination with a smartphone adapter. All figures were edited using CorelDraw-X8 software.

Original uncropped and unadjusted images underlying all gel results reported in this work may be found in the S1 Raw images.

Statistics

Significant differences between DNA concentrations from the analysed samples were calculated by an unpaired t-test using GraphPad Prism version 5.00 for Windows (GraphPad Software, La Jolla, California, USA; www.graphpad.com).

DNA extraction

DNA was extracted from beetle specimens of Mitosoma sp. and from specimens of the same genus preserved in resin of the same origin (H. verrucosa) but differing in age (2013 and 2017 fieldtrips) and collection site. DNA concentrations differed significantly between recent and resin-embedded beetles (Table 1). We observed a time-correlated decrease, albeit not significant, in the amount of DNA obtained from resin beetles (Table 1, Fig 2). The lowest DNA concentration was detected in resin beetles when the resin encasing the specimens had been dissolved in chloroform prior to DNA extraction (Table 1, Fig 2).

PCR amplification

Initial attempts to amplify DNA from two- and six-year-old resin samples, using the PCR protocol normally employed with recent DNA, were unsuccessful, whereas amplification of recent samples (beetles in ethanol) was positive with the original primer pairs COI and 28S. As DNA extraction from the resin samples was performed with completely ground insect together with remaining resin portions, it was suspected that the resin components might inhibit the PCR. This was tested by the addition of different proportions of resin to beetle samples at different time points during DNA extraction. Neither addition of resin to the sample before DNA extraction nor mixing of extracted DNA from resin and recent samples (Table 3) supported the hypothesis of PCR inhibition by resin components. In further experiments, BSA was added to the PCR mixture to avoid any possible inhibition of the DNA polymerase (Fig 3). The blood plasma protein efficiently binds to inhibitors and thereby prevents a negative impact of these molecules on the DNA polymerase [69–71]. After that, two different approaches to amplify the specific DNA fragments were tested: a) two consecutive PCRs of 30 cycles each with a PCR product purification step in between, and b) an increase in the number of PCR cycles up to 50. Both strategies resulted in positive amplifications (Fig 4). Whereas the addition of BSA increased the amplification efficiency of the primer combination targeting the large fragment of COI and enabled successful amplification with a lower number of cycles (≥ 35 cycles), no product could be detected for 28S with less than 45 PCR cycles (Fig 3).

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Fig 3. Gel electrophoresis of specific DNA fragments amplified with the original primer combinations COI and 28S with different numbers of cycles.

Comparison of PCR products with and without the addition of BSA to prevent DNA polymerase inhibition. Rb13 = Resin beetle (collected in 2013); Rb17 = Resin beetle (collected in 2017); (+) = positive control, DNA from beetles in ethanol; (-) = negative control, DNase- & RNase-free water; M = 100 bp DNA ladder (New England Biolabs).

https://doi.org/10.1371/journal.pone.0239521.g003

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Fig 4. Gel electrophoresis of specific DNA fragments amplified with original primer combinations COI and 28S.

A. Positive results with an increase in number of cycles to 50. Rb13 = Resin beetle (collected in 2013); Rb17 = Resin beetle (collected in 2017); (+) = positive control, DNA from beetle in ethanol; (-) = negative control, DNase- & RNase-free water; M = 100 bp DNA ladder (New England Biolabs). B. Two-step PCR with a PCR product purification step in between. Rb17² = Resin beetle (collected in 2017) after two consecutive PCRs of 30 cycles each; (+)¹ = positive control, DNA from beetle in ethanol after one PCR of 30 cycles; (+)² = positive control, DNA from beetle in ethanol after two consecutive PCRs of 30 cycles each; (-)¹ = negative control, DNase- & RNase-free water after one PCR of 30 cycles; M = 100 bp DNA ladder (New England Biolabs).

https://doi.org/10.1371/journal.pone.0239521.g004

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Table 3. Inhibition test performed with the primer combination COI for different proportions of DNA from beetles in ethanol and resin beetles.

https://doi.org/10.1371/journal.pone.0239521.t003

Negative amplification was obtained when the DNA from beetles in ethanol was absent, weak positive result when the proportion was low and positive in the remainder of tests, even when resin remains were included.

Control for contamination

Authentication procedures in aDNA studies are aimed at avoiding sample contamination with modern environmental DNA and detecting this should it occur. Therefore, each PCR product was purified and then sequenced by Sanger sequencing. To analyse the sequences several alignments were performed. Besides alignment of the sequences to Platypodinae sequences in the NCBI database BLAST to test for specificity, all products were checked for contamination with modern DNA. We especially examined the risk of inter-sample contamination from the positive control (beetles in ethanol) to the tested samples by multiple alignments of all sequences. Whereas the similarity between the sequences of insects extracted from resin in 2013 and 2017 was ≥ 97.4% with both primer combinations, a comparison between resin beetles and the sequence of beetles in ethanol yielded a similarity of only 73.7% for the original primer pair COI and 52.5% for primer combination 28S, respectively (Table 4), excluding any contamination with DNA from beetles in ethanol.

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Table 4. Multiple alignments of all analyzed sequences and the similarity value for the different combinations.

https://doi.org/10.1371/journal.pone.0239521.t004

Contamination occurring during analysis

A major handicap in aDNA research, especially when using resin samples, is contamination with modern environmental DNA [16–20]. The selection of gene sequences for DNA amplification is a crucial step before working with genetically unknown organisms, as is the case of extinct organisms. Here, the closest living relatives often form the basis for identification of homologous sequences. In this study, primers were selected and specifically designed for beetles of the subfamily Platypodinae [66, 67], to prevent amplification of contaminating modern DNA. Furthermore, our experiments were performed in a microbiology laboratory experienced in molecular methods, but which had never worked with entomological DNA before.

To avoid contamination with modern DNA in the experiments presented here, several control procedures at different stages of the isolation and amplification processes were included in the experimental setup. Besides precautions for working with DNA, all PCR products were purified, sequenced and controlled for sequence specificity and inter-sample contamination. Relatively long fragments of the 28S ribosomal subunit (~ 800 bp) and the mitochondrial gene oxidase (~ 600 bp) (Table 1, Fig 2) were successfully amplified.

To take into account aDNA fragmentation due to decay, the primer used for amplification should encase small sequences. A basic premise is that primers should amplify fragments up to about 160 bp, because longer aDNA sequences become damaged and degraded and have generally proved impossible to amplify [18, 41, 68]. We designed primers for the amplification of small fragments (~ 160 bp) (Table 2; COInew) for future experiments. The new primers were tested successfully in first experiments with resin-embedded platypodine specimens from 2017 (Fig 5) and therefore, build a fundament for future experiments with aDNA using the same insects after confirming the results obtained by longer fragments.

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Fig 5. Gelelectrophoresis of the specific DNA fragments amplified with primer combination COInew.

The primers were designed based on specific DNA sequences from resin-embedded beetles (collected in 2017) to amplify smaller DNA fragments. Rb17 = Resin beetle (collected in 2017); (-) = negative control, DNase- & RNase free water; M = 100 bp DNA ladder (New England Biolabs).

https://doi.org/10.1371/journal.pone.0239521.g005

Reproducibility

Entomological studies can easily implement most recommended aDNA authentication procedures to increase the degree of reliability [18–20]. However, other criteria, such as reproducibility or independent replication, are difficult to meet with insect samples due to the small size and frequent uniqueness of the samples. The methodology entailed with samples embedded in resin renders the research even more difficult. The establishment of authentication procedures for aDNA from small samples requires further consideration [27]. Since aDNA isolation in amber insects requires the complete destruction of fossil material, which is obviously undesirable when dealing with rare and very old species stored in museum collections, a more feasible approach would be to analyse several individuals of the species of interest or individuals of any related species of a group [68]. A taxonomic group with relatively abundant representatives would be the perfect target for these experiments; when a specimen is truly unique, samples cannot be used for this purpose because of obvious methodological and ethical problems [27]. The problem of reproducibility is solved here by the selection of insect samples in resin that contain co-specific syninclusions (minimum 2), with a comparable degree of preservation. After cutting the resin sample, the remaining syninclusions were stored at -20°C to maintain their stability as far as possible and thereby preserve the DNA for future tests. Although they do exist, it is difficult to find similar syninclusions in older resins. For rare or unique finds, the approved protocol is to archive independent similar results in two independent laboratories [17, 68]. However, it is extremely challenging to achieve this when using insects from resin or amber deposits because it is difficult to independently replicate DNA extraction from the same fossil insect. In addition, resins of different origin should be treated as independent experiments because of the different nature of their chemical compounds.

Taking into account all the authentication procedures to increase the degree of reliability and avoid contamination in aDNA studies [17–20, 27, 68], we summarise here the optimal method that we employed, which we consider should become standard practice in taphonomic research on DNA preservation in modern and ancient resins:

1. Analyse the sample in a DNA laboratory that has not previously worked with organisms similar to the target specimens, preferably with previous experience in aDNA research.
2. Select a taxonomic group with abundant representatives as syninclusions in modern resin, sub-fossil resin or amber.
3. Include negative controls, extraction blanks and PCR negative controls.
4. Use specific primers for the target specimens instead of more general primers which amplify a broader range of genomes.
5. Include a sequencing analysis for quality, assembly and specificity control.
6. Repeat the experiment, ideally with similar specimens from the same sample. Close taxonomic groups from the same sample and corroboration with a sequencing process may be sufficient to demonstrate reliability and discard contamination.
7. Include a detailed list of the protocols used in the experiments with a clear discussion of the authentication procedures employed.

The use of chloroform in DNA extraction from specimens embedded in resin

The dissolution of resin, sub-fossil resin and amber in chloroform is a typical procedure to release the embedded insects for further analysis [21, 72]. Penney et al. [21] were unable to detect any insect DNA via next generation sequencing after the use of chloroform to dissolve the resin, and thus concluded that DNA was absent. Chloroform:phenol mixtures are frequently used for DNA extraction [73–75] and one might assume that the difference in age between the youngest sample analysed in Penney et al. [21] and the oldest one in this study (6 years vs. 60 years) could be a key factor in our successful amplification. Surprisingly, we found that the use of chloroform to dissolve the resin surrounding the specimens compromised DNA concentration (Table 1, Fig 2). During work with DNA samples, the use of 70% ethanol also poses a problem as DNA precipitates at this ethanol concentration [47]. Therefore, only ≥ 80% ethanol should be used to sterilise resin surfaces. Büsse et al. [51] reported the amplification of DNA sequences from specimens preserved inside sub-fossil resin samples, but the lack of information about the methodology used with the specimens embedded in resin precludes comparisons. Further experiments are required to obtain more strongly supported conclusions about the effects of chloroform and ethanol on DNA extraction from resin-embedded material.

Dehydration of specimens

The life-like preservation of many amber inclusions is accompanied by preservation of ultrastructural details such as muscle fibres [76], mitochondria [77], brain tissue [3], and internal genitalia [78]. After the death of an organism, enzymes such as DNases rapidly degrade the DNA, but some repair mechanisms, if active, can revert this process. Under rare circumstances, tissue rapidly desiccates after death [8, 79] and DNA is absorbed by a mineral matrix [80]; in addition, rapid burial may change surrounding conditions, significantly reducing or completely inhibiting enzymatic and microbial degradation [81, 82]. On such occasions, slow but still manifest chemical processes start to affect the DNA [18, 83, 84]. Cano [85] stated that sugars such as arabinose, galactose and sucrose, which are components of natural resins, increase osmotic pressure on cells and thereby draw out the water, which results in tissue dehydration and inhibition of biochemical reactions. Additionally, alcohols and terpenes may act as fixatives to preserve tissues.

Dehydration and preservation in resin or permafrost are the three modes of preservation that retain more life-like organisms than any other kind of fossil, maintaining tissues over time [3]. While promising results have been obtained from permafrost-preserved invertebrates [43], none of the claims of DNA isolated from million-year-old amber samples could be independently replicated [21, 47]. In a recent study, amplified fossil genomes were extracted from birch pitch [60], another organic plant substance like resin, but obtained by heating birch bark. Supposedly, volatile compounds cause thorough and rapid dehydration and fixation of tissue once the trapped organism comes into contact with the resin [3]. This contrasts with our observations of resin-embedded Mitosoma sp. specimens from Madagascar (Fig 6). Two-year-old ground specimens still contained apparently flesh-like tissue without any indication of dehydration or shrinkage (Fig 6C). Therefore, hypothetical dehydration in the resin is not as rapid as was thought and must be regarded as the result of a process lasting for several years. Although the mono- and sesquiterpenoid volatile compounds in resins are known to have antimicrobial and enzyme inhibiting characteristics [23, 86, 87], it is possible that the di- and triterpenoid non-volatile compounds, some of which are also antimicrobial, are crucial to stabilise tissue over longer timescales. The lack of preservation of other molecules, such as chitin and lignin, which are more time-resistant than DNA, in Miocene amber-embedded insects and plants [88] has also been used to argue against the preservation of DNA in these particular fossils [18]. However, ß-1,3 and ß-1,4-linked polysaccharides, and specifically N-acetylglucosamine residues from chitin, have been detected in fungal mycelia in resinicolous fungi from Spanish amber [89]. One explanation for the better preservation of aDNA in bones than in resins may be hydroxyapatite. This mineral, which predominates in bone, is known for its very strong binding affinity to DNA [15, 80, 90]. Our observations indicate that there is still no satisfactory explanation for preservation in amber, but imply that water is available in the system for longer than previously thought, which has a negative effect on DNA stability in specimens embedded in resin.

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Fig 6. Different specimens of Mitosoma sp. preserved in resin from Hymenaea verrucosa collected in the Sambava region (Madagascar) in 2017.

A. Lateral view of a complete specimen. B. Dorsal view of a complete specimen. C. Photomicrograph of the sectioned head of one Mitosoma beetle in resin, showing the still fleshy internal tissue after two years inside the resin.

https://doi.org/10.1371/journal.pone.0239521.g006

Future approach to determine the time limits of DNA preservation in resin specimens

There are broad areas where further progress in studying aDNA from resin-embedded insects can be expected. Decay experiments provide a useful means to investigate variations in the preservation quality of different resin types [26] and offer a promising subject for future analysis. The preservation of inner structures of insects was highly compromised after only a few months embedded in different kinds of resin [26], but to the best of our knowledge, DNA preservation has never been tested. A comparison of DNA preservation in dead specimens of varying ages (such as those from museums and collections) [91], or specimens fixed in paraffin or embedded epoxy resin from less than one hundred years ago, would be highly interesting. Based on the positive results from this study, future experiments could investigate the limits of the DNA preservation in resin embedded specimens in short-time intervals. For future approaches with samples highly sensitive to contaminations, it will be required further adjustments of the methodology. For instance, the DNA concentration was measured via a microvolume-UV/VIS-spectrophotometer in this study, which lacks specificity, being unable to distinguish between DNA (single- and double-stranded), RNA and nucleotides, and is prone to contamination. Therefore, we recommend to use a fluorometer, which is highly accurate detecting fluorescent nucleic acids. Additionally, DNA of older resin embedded specimens should be analysed with more sensitive next generation sequencing technologies such as shotgun sequencing. This method is more specific, enabling distinction between modern contaminations and ancient DNA and provides further possibilities, e.g. analysis of fragmentation or degradation patterns. Proteins are thought to be more stable than DNA [92, 93] and seem to provide similar phylogenetic information [2]. Some experiments detected biomolecules such as chitin-protein complexes in insect cuticle from sub-fossil resin but failed to do so when Dominican amber samples were tested [88], while other more recent studies found amino acids from fossil feathers in amber from almost 100 million years BP [58]. Therefore, proteins may be a promising target for the study of deep time specimens [94]. However, if the objective is DNA, it may be more effective to amplify mitochondrial DNA since this is present in higher copy numbers than nuclear DNA, rendering it a more promising candidate for genetic analyses of aDNA [43–45].

The experiments presented here were performed using the commercial DNeasy® Blood & Tissue Kit (Qiagen, Germany), which was recommended for the extraction of beetle DNA [65, 66]. Other studies have also tested DNeasy® extraction systems, which are based on the binding of DNA to silica membranes, for other types of sample, such as bones or other insects [69, 95]. As a beetle embedded in resin is a mixture of two sample types, tissue (insect) and environmental (resin), selection of the most suitable extraction kit must be carefully assessed. Environmental kits offer the advantage of additional steps to avoid PCR inhibitors [96–98], whereas tissue kits are specifically designed for DNA extraction from tissue samples. Other aspects such as choice of DNA polymerase can significantly affect PCR efficiency [69], and should be further investigated to identify the ideal DNA extraction and PCR amplification protocol.

The interaction between a fossil sample and its environment following death of the specimen determines the preservation state of the biomolecules; age per se is not an absolute indicator of quantity or quality, at least over a longer timescale [18]. Storage conditions also play a crucial role in preservation, and dry, cold conditions are best for DNA preservation [27]. Since each case is unique, attending to our results, no biological sample should be disregarded as a potential source for recovery [15].