A non-destructive, fast, inexpensive, non-toxic chelating beads-based DNA extraction protocol for insect voucher specimens and associated microbiomes

A non-destructive, fast, inexpensive, non-toxic chelating beads-based DNA extraction protocol for insect voucher specimens and associated microbiomes | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 5 January 2025 V1 Latest version Share on A non-destructive, fast, inexpensive, non-toxic chelating beads-based DNA extraction protocol for insect voucher specimens and associated microbiomes Authors : Morgan Brown 0009-0006-0291-3008 , Sara Ottati 0000-0001-7907-8801 , and Valeria Trivellone [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173607115.51950228/v1 364 views 153 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Identifying a DNA extraction method that yields a high quantity and quality of DNA is a crucial component of molecular ecological studies; and the best suited method can vary greatly depending on research priorities. Here, we propose an extraction method which is suited for the non-destructive extraction of gut content DNA from single insect specimens with the goal of analyzing gut-associated microbiomes. We tested multiple factors, including the inclusion and exclusion of pre-lysis bleaching and post-lysis proteinase K inactivation. Two purification methods were used: a chelating beads-based method and a silica column-based method which served as a quality reference. Based on our findings, we recommend a method which includes pre-lysis bleaching, no proteinase K inactivation, and uses a chelating bead-based purification method. Our optimized protocol results in a high DNA yield suitable for downstream analyses including qPCR and next-generation sequencing. A non-destructive, fast, inexpensive, non-toxic chelating bead-based DNA extraction protocol for insect voucher specimens and associated microbiomes Running title: DNA extraction protocol for insect vouchers Morgan E. Brown 1, 2 , Sara Ottati 3, 4 , & Valeria Trivellone 1 1 Illinois Natural History Survey, Prairie Research Institute, University of Illinois Urbana-Champaign, Urbana-Champaign, Illinois | 2 Department of Entomology, University of Illinois Urbana-Champaign, Urbana-Champaign, Illinois | 3 Institute for Sustainable Plant Protection, National Research Council of Italy, Turin, Italy | 4 Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Centre for Plant Biology, Uppsala, Sweden Corresponding authors: V. Trivellone [email protected] , M. Brown [email protected] ABSTRACT Identifying a DNA extraction method that yields a high quantity and quality of DNA is a crucial component of molecular ecological studies; and the best suited method can vary greatly depending on research priorities. Here, we propose an extraction method which is suited for the non-destructive extraction of gut content DNA from single insect specimens with the goal of analyzing gut-associated microbiomes. We tested multiple factors, including the inclusion and exclusion of pre-lysis bleaching and post-lysis proteinase K inactivation. Two purification methods were used: a chelating bead-based method and a silica column-based method which served as a quality reference. Based on our findings, we recommend a method which includes pre-lysis bleaching, no proteinase K inactivation, and uses a chelating bead-based purification method. Our optimized protocol results in a high DNA yield suitable for downstream analyses including qPCR and next-generation sequencing. Keywords: vouchering, Auchenorrhyncha, phytoplasma, specimen curation, nucleic acids INTRODUCTION DNA extraction is a critical step in molecular ecological studies, and the selection of an effective extraction method is crucial to obtaining high quality DNA for downstream applications, such as quantitative polymerase chain reaction (qPCR) amplification and sequencing. Numerous methods for the extraction of nucleic acids have been proposed to accommodate a variety of sample types (Dairawan & Shetty, 2020), tailored to specific research priorities, including target organism, research goal, cost, and operator safety concerns (Shin, 2013). Detection of microbial associates in invertebrate hosts relies on efficient DNA extraction performed on the whole host body (Andriienko et al., 2024; Petersen & Osvatic, 2018). Recently, the use of insect vouchers preserved in museum collections has gathered attention due to their potential use in unveiling elusive microbe-host associations, particularly when studying insect-borne parasites (DiEuliis et al., 2016). Museum collections have become an increasingly important resource for understanding parasite diversity, ecology, and evolution. They allow researchers to track past host-pathogen associations, document associations in collection sites that are no longer accessible, revise the taxonomic status of the associates, and resample the associations over time as new technologies become available (Colella et al., 2021; Dunnum et al., 2017; Nelder et al., 2024; Trivellone et al., 2021). Prior studies have compared the efficacy of different extraction methods, many of which guarantee homogeneous cellular digestion prior to DNA purification by crushing the whole or part of the insect body, thereby maximizing DNA yield (Asghar et al., 2015; Jangra & Ghosh, 2022; Junqueira et al., 2002). However, methods that rely on crushing are not suitable for long-lasting preservation of museum specimens. Several methods have been developed that successfully extract and purify DNA while leaving the insect exoskeleton intact (Andriienko et al., 2024; Carew et al., 2018; Castalanelli et al., 2010; Cilia et al., 2022; Favret, 2005; Gilbert et al., 2007; Hunter et al., 2008; Pons, 2006; Rowley et al., 2007). However, many of these methods utilize hazardous chemicals, such as phenols or chloroform (Guo et al., 2022; Lienhard & Schäffer, 2019). On the other hand, non-toxic protocols available as commercial kits may be costly, not widely available, require specialized equipment, or be unsuited for work on insects (Schiebelhut et al., 2017; Smith et al., 2003). Bead-based DNA isolation methods utilize negatively charged surfaces to purify nucleic acids by binding metal ions and positively charged proteins. These methods, such as those using chelating resin, are known to be non-toxic, cost-effective, and suitable for downstream application (Asghar et al., 2015; Hackett et al., 2024; Lienhard & Schäffer, 2019; Miura et al., 2017; Pacheco et al., 2023). Nevertheless, such protocols often employ a destructive approach, such as grinding or crushing the specimen’s body to improve penetration of cells containing nucleic acids. This approach is not suitable for museum specimens, which may be rare and irreplaceable, and for groups where taxonomic uncertainty requires vouchering to document the identity of the species under study. Instead, non-destructive methods can be implemented to keep the specimen intact. However, unlike destructive approaches, non-destructive methods may not guarantee homogeneous digestion of tissues, potentially affecting not only the final yield and/or quality of extracted DNA but also the efficient quantification of low-copy microorganisms. Moreover, non-destructive methods often rely on Proteinase K (PK), a broad-spectrum serine proteinase, to digest cellular proteins and thus assist in cell lysis during DNA extraction. PK is widely used for nucleic acid extraction and is commonly inactivated with heat after extraction to prevent further lysis activity and any negative impact on downstream analyses. Lienhard & Schäffer (2019), employing a destructive extraction method, found that DNA yield was greater in samples that did not undergo PK inactivation compared to samples that did undergo heat inactivation of PK. Furthermore, they did not report any disturbance in PCR analyses as a consequence of residual PK activity. The effects of PK inactivation on DNA yield under a non-destructive method and the efficacy of non-destructive DNA extraction methods for quantifying insect-associated microorganisms remain unknown. One important consideration when extracting DNA from insect samples for microbiome analyses is the potential presence of contaminants containing environmental DNA from various sources on external surfaces of the insect’s body. Greenstone et al. (2012) demonstrated that bleaching insect bodies prior to DNA extraction effectively eliminated potential sources of DNA contamination. Huszarik et al. (2023) further confirmed that bleaching reduces external contaminants and showed that DNA extracted from bleach-treated specimens could still be successfully amplified via PCR and sequenced via DNA metabarcoding. While specimens treated with bleach as described in these studies have been used successfully for analyzing insect gut content and characterizing associated microbiomes, the direct impact of bleaching on final DNA yield has not been extensively tested directly (Arora et al., 2018; Huszarik et al., 2023; Meyer & Hoy, 2008; Moreau, 2014). In this paper we aimed to optimize a protocol for the non-destructive extraction of nucleic acids from small insects preserved in museum collections, while providing a non-toxic, fast, inexpensive, and high-yield DNA extraction method suitable for microbiome analysis. To achieve this, we investigated the effects of pre-lysis bleaching and post-lysis proteinase K inactivation on DNA extractions using a bead-based method. Our study targeted the gut content in a group of phytophagous insects – leafhoppers (Hemiptera: Cicadellidae) – that are vectors of bacterial plant pathogens, i.e. phytoplasmas (Mollicutes) (Weintraub et al., 2019). MATERIALS AND METHODS Insect samples and associated bacterial pathogens. We used adults of the leafhopper Euscelidius variegatus (Kirschbaum) maintained in colonies at the Institute for Sustainable Plant Protection, National Research Council of Italy (IPSP-NRC) laboratory (Turin, Italy). This species is an efficient vector of the phytoplasma strains in the 16SrV phylogenetic group, subgroups C and D (hereafter FDp), under laboratory conditions. Healthy colonies of E. variegatus were reared on oat ( Avena sativa L.) and used to maintain FDp with sequential transmissions from broad beans to broad beans (Abbà et al., 2017; Rossi et al., 2020). The phytoplasma infection rate in E. variegatus was maximized by exposing the insects to phytoplasma infected broad beans for 7 days (acquisition access period), followed by 28 days on healthy broad beans (latency period). After completing the phytoplasma infection procedure, the putatively infected insects were preserved in 95% ethanol at -20° C. Experimental design The experiment uses a 2x2 factorial design to test the effects of pre-lysis bleaching and post-lysis PK inactivation on the final DNA yield and quantification of host-associated microorganisms (via qPCR) A total of 40 phytoplasma-infected E. variegatus individuals (20 females and 20 males) were randomly assigned to four treatments: (1) Bleaching + no PK inactivation; (2) No bleaching + no PK inactivation; (3) Bleaching + PK inactivation; and (4) No bleaching + PK inactivation. Each treatment included 10 biological replicates and 1 phytoplasma-free individual used as a negative control for phytoplasma quantification (see sub-heading 5). After the lysis step, each lysate sample was divided into two equal portions (paired samples) to complete the DNA extraction. One portion underwent a bead-based extraction method, while the other was processed using a commercial silica column-based method, which served as a reference for high-quality DNA recovery. A total of 80 paired samples were processed (see supplementary material SM-1). DNA Extraction 3.1 Preparation of samples and cell lysis A total of 40 phytoplasma-infected E. variegatus individuals were placed in single vials containing 95% ethanol (EtOH) prior to DNA extractions. For specimens treated with bleach, each individual was immersed in a 2.5% bleach solution for 5 minutes to eliminate potential contaminants from external body surfaces. Cell lysis was performed using TES buffer (20 mM Tris, 10 mM EDTA, 0.5% SDS, pH ~7.0) and Proteinase K. Each insect body was placed in a microcentrifuge tube containing 50 μL TES buffer and 2 μL PK, then incubated in a 55°C water bath for 24 hours to ensure thorough protein and enzyme digestion. Following incubation, the insect bodies were removed from the tubes and preserved in 95% EtOH for later examination and vouchering. Specimens were point-mounted, labeled, and deposited in the Illinois Natural History Survey Insect Collection. The extracted DNA samples were either subjected to PK inactivation by incubating at 95°C for 10 minutes or left without inactivation. Each sample was aliquoted (see Experimental Design) and assigned to one of the selected DNA purification strategies, chelating beads, or a commercial silica column-based kit. 3.2 Bead-based extraction Bio-Rad Chelex 100 Resin was used for bead-based DNA extractions. Chelex was added after incubation with the lysis buffer to precipitate undesired molecules, such as Mg²⁺ ions, leaving the DNA in the supernatant. Details of the Chelex suspension and precipitation protocol can be accessed at dx.doi.org/10.17504/protocols.io.bp2l6x54rlqe/v1 (steps 7-14). 3.3 Silica column-based extraction The Qiagen DNeasy Blood & Tissue Kit was chosen as a quality reference due to its widespread use and established efficacy for extracting DNA from insects (Mullin et al., 2023). Since the manufacturer’s protocol does not account for non-destructive lysis, a slightly modified version was used to make the method more comparable to the bead-based protocol (see supplementary material SM-2). DNA yield and quality evaluation Since we targeted total DNA from insects and their associated microorganisms, specifically pathogenic bacteria that may be present in very low titers, we measured total DNA yield, integrity, purity, and microorganism quantification to ensure a comprehensive assessment. A total of 80 DNA templates were quantified using the Qubit 4 Fluorometer (Invitrogen) according to the manufacturer’s instructions. Total DNA yield (ng/sample) was calculated from the recorded concentrations. Using a NanoDrop Microvolume Spectrophotometer (Thermo Scientific), the purity of nucleic acids was evaluated by comparing the 260/280 absorbance ratio of a subset of 16 samples. If the 260/280 absorbance ratio is ~1.8 the DNA is considered optimal. Any deviation from this value indicates the presence of leftover contaminants (William et al. 1997). Gel electrophoresis was carried out on 10 samples that underwent the finalized protocol to estimate fragmentation. A mixture of 1% agarose in 1x TBE (tris, borate, EDTA) buffer was prepared. When possible, 100 ng of DNA was loaded into each well and electrophoresed before being photographed on a blue light box. A 1 kilobase pair ladder (New England Biolabs) was included as a reference for DNA weight. Quantification of phytoplasmas To verify the effect of each treatment (bleaching and PK inactivation) on absolute quantification of bacteria (i.e., phytoplasmas) associated with the insect body, we used a duplex qPCR assay. To quantify phytoplasma cells, universal primers 16S-fw (5’-CGTACGCAAGTATGAAACTTAAAGGA-3’), p16S-rv (5’-TCTTCGAATTAAACAACATGATCCA-3’ and TaqMan probe p16S-FAM (5’-FAM-TGACGGGAC-ZEN-TCCGCACAAGCG-IBFQ-3’) targeting the 16S rDNA gene of phytoplasmas (detailed in Christensen et al., 2004) were used. Absolute quantification was achieved by normalizing the phytoplasma genome unit on the ng of total DNA. To quantify insect DNA, the 18s rDNA was chosen as the endogenous insect target; primers Au18S_1719F qFw (5’-ACTGTGTGCATGGAATAATGGA-3’), Au18S_1852R (5’-TGCGACGATCCAAGAATTTCA-3’), and TaqMan probe Au_Probe_1796-Hex (5’-AGGGACAGGCGGGGGCATTCG-HEX-3’) were used. One μL of DNA (0.1 ng) was used in a reaction mix of 10 μL total volume, containing 1x TaqMan™ Universal PCR Master Mix (Invitrogen), 160 nM of each of the four primers and 160 nM of each of the two TaqMan probes. Each sample was run in duplicate in a CFX optimum Real-Time PCR Detection System (Bio-Rad). Cycling conditions were 95°C for 2 minutes and 50 consecutive cycles at 95°C for 15 seconds of denaturation followed by 1 min at 60°C of annealing and extension. In each qPCR plate, at least four serial 100-fold dilutions of pOP74 plasmid, harboring the target phytoplasma rDNA 16s genes, were included to calculate the phytoplasma load. Plasmid standard curve dilutions included in plates ranged from 10E+7 to 10 target copy numbers per μL and were prepared taking into account that 1 fg of plasmids harboring phytoplasma gene portion contains 194 molecules. For insect housekeeping gene detection, a standard curve was prepared using serial dilutions (11 ng/μL, 1.1 ng/μL, 0.11 ng/μL, and 0.011 ng/μL) of total DNA from phytoplasma-free specimens of E. variegatus reared in the IPSP-CNR lab colony. Dilution series plasmid and total DNA were used to calculate qPCR parameters (reaction efficiency and R2). Mean phytoplasma copy numbers in amplified samples were automatically calculated by CFX Maestro Software (Bio-Rad) and used to express phytoplasma amount as phytoplasma genome unit/ng of insect DNA. Data analysis To assess the effect of the three factors (bleaching, PK inactivation, and protocol type) on DNA yield and absolute quantification of phytoplasma, a linear mixed-effects model (LMM) was performed using the nlme R-package (Pinheiro & Bates, 2024). To account for inherent variability associated with individual identity, individual specimens were included as a random factor. Model assumptions were validated by examining residuals for normality and homoscedasticity (Winter 2013). Data were log-transformed for normalization. Data analysis and plotting were carried out in R version 4.4.0 (R Core Team, 2024). RESULTS Total DNA yield DNA extracted from bleached specimens (n = 40 samples) averaged 299 ng/sample (Standard Deviation - SD: 407) compared to non-bleached specimens (n = 40 samples) which averaged 417 ng/sample (SD: 578). DNA extracted from samples that underwent PK heat inactivation (n = 40 samples) averaged 264 ng/sample (SD: 364) while samples that did not undergo PK inactivation (n = 40 samples) averaged 452 (SD: 597). DNA processed using a bead-based protocol (n = 40 samples) yielded an average of 691 ng/sample (SD: 528) while DNA processed using a silica column-based protocol (n = 40 samples) averaged 25.6 ng/sample (SD: 33.7). For all DNA yield data, see supplementary material SM-3. A linear mixed effect model considering nested effects of each variable on the DNA yield data revealed that protocol was significantly correlated with the resulting DNA yield (p-value < 0.001). No effects of PK inactivation, bleaching, nor interaction effects within the variables were identified (Table 1). Total DNA quality The average value of 260/280 absorbance ratio measured for the samples extracted with the silica column-based protocol (Qiagen) was 1.99 (SD: 0.858, 8 individuals) whereas an average of 1.12 (SD: 0.0874) from the same paired samples was obtained using the bead-based extraction protocol (Chelex). These results indicate optimal values for silica column-based extracted samples and the presence of organic contaminants in bead-based extracted samples (see supplementary material SM-4). Gel electrophoresis of a subset of 10 samples showed that the inclusion of pre-lysis bleaching and exclusion of post-lysis PK inactivation had no observable effects on the integrity of DNA extracted with both non-destructive DNA extraction protocols (see supplementary material SM-5). qPCR of phytoplasmas associated with the tested insect samples Bleaching and PK inactivation had no significant effects on downstream qPCR analysis, while the extraction protocol slightly influenced the outcome of qPCR analysis (p-value < 0.05). When comparing all bleached (n = 40 samples) and unbleached treatments (n = 40 samples), the average value of phytoplasma quantification, expressed as genome units (GU)/ng of insect DNA, was 6.082E+5 (SD: 7.181E+5) and 5.031E+5 (SD: 7.181E+5), respectively. Treatments with PK inactivation (n = 40 samples) had a mean quantification of 5.193E+5 GU/ng insect DNA (SD: 6.782E+5), while treatments with no PK inactivation (n = 40 samples) resulted in a mean quantification of 5.921E+5 GU/ng insect DNA (SD: 7.578E+5). Samples extracted following the bead-based protocol resulted in a mean phytoplasma quantification of 6.360E+5 GU/ng insect DNA (SD: 8.104E+5), while column-based extraction protocol had a mean phytoplasma quantification of 4.752E+5 GU/ng insect DNA (SD: 6.057E+5). DISCUSSION Published nucleic acid extraction protocols are generally customized to address specific biological contexts and experimental goals, aiming to maximize both the quality and quantity of recovered nucleic acids. These criteria often include considerations for sample type, preservation methods, and the intended downstream applications such as qPCR, next-generation sequencing (NGS), or metabarcoding (Sheershika & Ram, 2024; Shin, 2013). In this study, our primary objective was to optimize a protocol for the efficient extraction of a group of bacteria (phytoplasmas), which are obligate intracellular plant pathogens, from insect vouchers while ensuring suitability for downstream analyses such as molecular quantification and sequencing. Our study demonstrates the applicability of a two-step approach: first, bleaching specimens to eliminate external contaminants before lysis and second, omitting the post-lysis step of PK inactivation through heating. Excluding PK inactivation reduces the total extraction time. While it is known that the heat step to inactivate PK does not adversely affect DNA yield if exposure to high temperatures is appropriate (10 minutes), our results showed that not inactivating PK resulted in a slightly higher total DNA yield compared to the inactivated treatment. However, this difference was not significant. Our results are consistent with the findings of Lienhard & Schäffer (2019), who retrieved an adequate amount of DNA using a Chelex extraction protocol, regardless of whether PK was inactivated or not. Efficient and complete lysis is crucial for maximizing DNA yield and ensuring accurate downstream detection of microorganisms such as phytoplasmas, which may otherwise be underestimated if host tissues are incompletely lysed. There was initial concern that leaving PK active in the extracted sample could interfere with downstream analyses. However, our study showed no significant difference in phytoplasma absolute quantification when comparing treatments with and without inactivation, suggesting that leaving the PK active does not compromise the detection and amount of gut-associated bacterial DNA. These results are in line with a previous study by Wang et al. (2019), which targeted a recombinant adeno-associated virus through qPCR and showed that cycle threshold values were identical regardless of whether PK was inactivated. While PK is commonly used solely to increase DNA yield, soaking specimens in PK has also been demonstrated to be an effective method for clearing soft tissue from arthropods. After this treatment, only the exoskeleton remains, allowing for clear visualization of the genitalia morphology, which is useful for species identification. The use of PK to prepare genitalia capsules for microscopic examination has been shown to be more effective than the commonly used potassium hydroxide (KOH) (Martinelli et al., 2017). PK has been observed to degrade the exoskeleton to a lesser extent than KOH, retaining more defined features such as texture and sclerotization patterns. This makes PK a viable and potentially preferable method for clearing soft tissue. Thus, the inclusion of PK in DNA extractions is especially suited for insect groups for which genitalic morphology is a key identifier, such as leafhoppers. Previous studies have shown the effectiveness of bleaching in reducing surface contamination without negatively impacting the integrity of target DNA. For instance, Greenstone et al. (2012) demonstrated that bleaching insect samples prior to DNA extraction enhanced the purity of microbial DNA, particularly for gut microbiome studies. Huszarik et al. (2023) also confirmed that bleaching is a reliable method for minimizing contaminants while preserving DNA yield and amplification efficiency. Despite the proven effectiveness of bleaching in eliminating external contaminant DNA, a critical concern has been whether the bleaching process may degrade the specimen’s own DNA (Kemp & Smith, 2005) and therefore reduce the final yield or interfere with downstream analyses. Our findings showed that bleaching neither decreased the total DNA yield, nor compromised downstream analysis such as qPCR quantification of associate low-titer microorganisms. Future studies should investigate whether active PK affects DNA quality during extended storage or repeated freeze-thaw cycles, particularly for applications requiring high-quality DNA or long-term sample preservation. Insect specimens preserved in museum collections potentially offer a wealth of genetic information on difficult to obtain species, which can enhance our understanding of many facets of biology, but such specimens often represent species that are rare and difficult to collect and/or localities that are difficult to access, so museum curators may be reluctant to allow destructive DNA sampling from specimens under their care. For these reasons, it is important to develop non-destructive DNA extraction methods which produce high yield and quality of DNA but leave the specimens themselves intact. In addition to our findings, others have also implemented a variety of strategies to extract DNA from museum specimens while keeping the insect bodies intact. Patzold et al. (2020) extracted DNA from museum specimens of lepidopteran type specimens that were 20 to 214 years old using a minimally-destructive method which utilized one whole leg from each specimen. Using this method, they retrieved an average DNA concentration of 12.1 ng/μL, a suitable amount to perform downstream analyses. Mullin et al. (2023) used a similar approach in which they used a single leg from bumblebee specimens that were up to 113 years old with the goal of obtaining genome-wide data to serve as a baseline when investigating population changes at a genetic level. DNA was successfully extracted using this method, although there were high levels of fragmentation with most DNA fragments containing less than 100 base pairs. Mullins et al. suggested that DNA degradation in specimens increases with time since death, which may cause issues when trying to amplify DNA from older specimens. Thomsen et al. (2009) extracted DNA from whole-body beetle museum specimens, including some from 1820 AD. They successfully extracted and amplified DNA for specimens of all ages. The main goal of this study was to provide an improved, non-destructive, fast, inexpensive, non-toxic chelating bead-based DNA extraction protocol optimized for insect museum vouchers and their associated microbiomes. Achieving a high DNA yield is critical for studies that require multiple downstream analyses, such as DNA sequencing or metagenomic studies, and for ensuring reproducibility in experiments. The silica column-based Qiagen kit that served here as an internal reference is known to be a reliable method for extracting high-quality DNA from insect specimens (Patzold et al., 2020). The inclusion of samples processed using silica columns allowed us to ensure that samples treated similarly, differing only in the use of chelating beads, could still yield comparable results. Consistent with prior studies (Casquet et al., 2012; Lienhard & Schäffer, 2019), samples purified with chelating beads consistently yielded higher quantities of DNA compared to those purified using silica columns. We also obtained a significantly higher amount of phytoplasma DNA when using chelating beads. Our results indicate that the chelating bead-based protocol is suitable for retrieving an acceptable amount of total DNA, even though the quality is slightly inferior. Overall, when comparing DNA quality between treatments and for each protocol separately, comparable results were retrieved. While we did not attempt to sequence the extracted DNA, others have shown that samples collected using non-destructive methods can be successfully metabarcoded (Carew et al., 2018; Martoni et al., 2022). This emphasizes the viability of such non-destructive methods for studies of the microbiomes of museum specimens. Based on our findings and those of previous studies, for gut-associate microbiome studies we recommend a protocol that includes a pre-lysis bleaching step, uses PK without a post-lysis heat inactivation step, and utilizes a bead-based purification method. The final optimized protocol can be viewed at dx.doi.org/10.17504/protocols.io.bp2l6x54rlqe/v1. This protocol is suited for the non-destructive extraction of DNA from museum arthropod specimens, particularly for extractions targeting gut content and microbial genome and for specimens representing species for which identification requires the preservation of genitalic morphology ACKNOWLEDGEMENTS We would like to thank Chris Dietrich for his useful feedback on the manuscript and for his support as a graduate advisor to the first author. 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Available from: http://arxiv.org/pdf/1308.5499.pdf DATA ACCESSIBILITY AND BENEFIT-SHARING Data accessibility DNA yield, absolute phytoplasma quantification, and NanoDrop data are available in the supplementary materials. Insect specimen voucher data are available on the Illinois Natural History Survey Insect Collection TaxonWorks database (catalog numbers 1071801 to 1071844). Benefit-sharing Benefits generated: Insect specimens used were acquired through a research collaboration with the National Research Council of Italy (see MATERIALS AND METHODS), with the main collaborator listed as a co-author and other collaborators acknowledged. AUTHOR CONTRIBUTIONS M.E.B. contributed to designing and performing research, analyzing data, and writing the manuscript. S.O. contributed to designing and performing research, providing insect specimens, analyzing data, and writing the manuscript. V.T. contributed to designing and performing research, analyzing data, and writing the manuscript. All authors approved the final manuscript. TABLES AND FIGURES DNA yield Value SE t-value p-value (Intercept) 6.67 0.28 23.68 0.000 Bleach (yes vs no) -0.20 0.40 -0.49 0.626 PK in † (yes vs no) -0.33 0.40 -0.82 0.413 Prot ‡ (Qiagen vs Chelex) -3.76 0.36 -10.57 0.000 Interaction term (Bleach & PK in) -0.98 0.56 -1.74 0.091 Interaction term (Bleach & Prot) 0.89 0.50 1.77 0.085 Interaction term (PK in & Prot) -0.35 0.50 -0.70 0.491 Interaction term (Bleach & PK in & Prot) 0.57 0.71 0.80 0.426 Absolute phytoplasma quantification (Intercept) 12.324 0.964 12.781 0.000 Bleach (yes vs no) 0.519 1.364 0.380 0.706 PK in (yes vs no) 0.424 1.364 0.311 0.757 Prot (Qiagen vs Chelex) -0.632 0.250 -2.526 0.0161 Interaction term (Bleach & PK in) -3.008 1.928 -1.560 0.128 Interaction term (Bleach & Prot) 0.444 0.354 1.255 0.218 Interaction term (PK in & Prot) -0.182 0.354 -0.515 0.610 Interaction term (Bleach & PK in & Prot) 0.436 0.500 0.872 0.390 Table 1. Results of linear mixed effect model for DNA yield and absolute phytoplasma quantification. Significant factors with a p-value < 0.05 are in bold. † Proteinase K inactivation, ‡ Protocol. Information & Authors Information Version history V1 Version 1 05 January 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords auchenorrhyncha nucleic acids phytoplasma specimen curation vouchering Authors Affiliations Morgan Brown 0009-0006-0291-3008 University of Illinois Urbana-Champaign Illinois Natural History Survey View all articles by this author Sara Ottati 0000-0001-7907-8801 Institute for Sustainable Plant Protection National Research Council View all articles by this author Valeria Trivellone [email protected] University of Illinois Urbana-Champaign Illinois Natural History Survey View all articles by this author Metrics & Citations Metrics Article Usage 364 views 153 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Morgan Brown, Sara Ottati, Valeria Trivellone. A non-destructive, fast, inexpensive, non-toxic chelating beads-based DNA extraction protocol for insect voucher specimens and associated microbiomes. Authorea . 05 January 2025. 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