Molecular Ecology Resources (2016) 16, 353–363
doi: 10.1111/1755-0998.12446
Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae PETRA KRANZFELDER,*,† TORBJØRN EKREM* and E L I S A B E T H S T U R * *Department of Natural History, NTNU University Museum, Norwegian University of Science and Technology, NO-7491, Trondheim, Norway, †Department of Entomology, University of Minnesota, 1980 Folwell Avenue, Saint Paul, MN 55108, USA
Abstract Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell-based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a speciesrich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpinâ Tissue XS Kit, DNeasyâ Blood and Tissue kit, and QuickExtractTM DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments. Keywords: aquatic macroinvertebrates, biodiversity assessment, COI, DNA barcoding, DNA isolation, species identification Received 8 May 2015; revision received 6 July 2015; accepted 13 July 2015
Introduction The effectiveness of DNA barcoding, metabarcoding and environmental barcoding as a routine practice in biodiversity monitoring and conservation is strongly dependent on the quality of the DNA extractions (Ivanova et al. 2006; Knebelsberger & St€ oger 2012). There are a variety of methods to isolate DNA from biological materials, but the ideal extraction technique should provide high DNA quality and quantity, as well as, be efficient in terms of cost and handling time, be suitable for high sample throughput and generate minimal hazardous waste (Chen et al. 2010). Traditional extraction methods, including phenol/chloroform (Nishiguchi et al. 2002) or DTAB-CTAB lysis (Phillips & Simon 1995), work efficiently for extracting DNA from animal tissues, Correspondence: Petra Kranzfelder, Fax: (612)-625-5299; E-mail: [email protected]
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providing the possibility to get DNA without damaging specimens, but use toxic chemicals and are time-consuming (Hajibabaei et al. 2005). Various commercially available DNA extraction kits, especially silica-membranebased methods, are becoming increasingly popular because of their ease-of-use for a wide range of biological samples, limited labour and ability to consistently produce high-quality DNA (Nishiguchi et al. 2002; Hajibabaei et al. 2005). However, some of the drawbacks of these kits include the expense and long incubation times (Ball & Armstrong 2008). Another rapid and low-cost DNA barcoding technique termed ‘direct PCR’ facilitates PCR amplification of target genes directly from samples without DNA extraction and purification; however, direct PCR success rates can be low for many taxa (Wong et al. 2014). Although these standardized DNA isolation methods can be used for a broad range of biological samples, some are still problematic for low yield or degraded samples (Zimmermann et al. 2008; Goldberg et al. 2015).
354 P . K R A N Z F E L D E R , T . E K R E M a n d E . S T U R Chironomidae (Insecta: Diptera) are a species-rich group of aquatic macroinvertebrates that are abundant and widely distributed in aquatic systems (Cranston 1995). Chironomid communities are considered valuable bioindicators of water quality due to their high species diversity, ubiquity and varying species sensitivity to environmental conditions (Ferrington et al. 2008; Marziali et al. 2010; Raunio et al. 2011). However, chironomid larvae are time-consuming and difficult to identify to genus or species, which has led to a debate about the importance of using chironomid data in biological monitoring programs (Rosenberg 1992; Cranston 2000; Rabeni & Wang 2001; Nijboer et al. 2005; Roque et al. 2010; Ruse 2010; Raunio et al. 2011). A efficient, effective, low-cost and easy-to-use form of sampling chironomids involves collections of pupal exuviae (Wilson & Ruse 2005; CalleMartınez & Casas 2006; Raunio et al. 2007; Bouchard & Ferrington 2011; Ca~ nedo-Arg€ uelles et al. 2012), which is the exoskeleton shed by the adult as it emerges on the surface of the water (Ferrington et al. 1991) (Fig. 1). Pupal exuviae accumulate behind obstructions, such as fallen trees or along banks through the action of wind or water current, and can be easily collected to give a comprehensive sample of chironomid communities (Ruse 2010). This collection method has several advantages over traditional benthic larvae sampling techniques: (i) applicability in nearly all types and conditions of aquatic environments, including deep, muddy rivers, canals and lakes, and riffles in shallow rivers; (ii) accumulated pupal exuviae represent taxa that have originated from a wide range of microhabitats; and (iii) sampling and sample sorting are easy and rapid (Wilson & Ruse 2005; Kranzfelder et al. 2015). Species identification is essential for aquatic bioassessment studies (Lenat & Resh 2001; Melo 2005; Marshall et al. 2006; Jones 2008), as some chironomid species inhabit waters of high quality, while others occupy poor
quality (Epler 2001). However, in many regions, it is difficult to identify chironomid pupal exuviae to species (morphologically) as most species descriptions and keys are based on adult males (Carew et al. 2007; Ekrem et al. 2007). Ekrem et al. (2007), among others, have found that partial mitochondrial gene cytochrome oxidase I (COI) gene sequences can be used to link different life stages of Chironomidae and DNA barcoding has been successful for identifying cryptic and undescribed chironomid species from different life stages: larvae, pupae and adults (Stur & Ekrem 2011; Anderson et al. 2013; Brodin et al. 2013; Meier et al. 2015). Yet, barcoding of chironomid pupal exuviae has been challenging due to the low quantities of genomic DNA available for extraction (Krosch & Cranston 2012). The exoskeleton of the pupal exuviae is made of extracellular chitin, the most widespread structural polysaccharide in nature (Merzendorfer 2006), and does not contain any nucleic acids. Therefore, genomic DNA yield from pupal exuviae relies on muscle tissue, hairs and epithelial cells lining the foregut, hindgut and tracheae that are left behind on the inner surface of the cuticle by the emerging adult. In addition, metabolic waste products from the pupal stage might be released by the adult upon emergence (Nation 2008). Trace DNA from chironomid pupal exuviae can be sufficient for PCR amplification and sequencing of target genes (Krosch & Cranston 2012). Miller et al. (1988) attempted to extract DNA from chironomid pupal exuviae using a modified salting out protocol, but had no success with this method. Recently, Krosch & Cranston (2012) were able to extract genomic DNA from 27 of 58 chironomid pupal exuviae using one extraction method (DNeasyâ Blood & Tissue Kit), but this study was limited by few specimens, one extraction method and lack of quantitative data. Consequently, there is no consensus regarding the most effective DNA isolation method for chironomid pupal exuviae or similar trace DNA samples. In this study, we compare the performance of five different DNA extraction methods and direct PCR in isolation of genomic DNA from chironomid pupal exuviae in terms of cost, handling time, DNA quantity, PCR success, sequence success and ability to sequence target taxa.
Materials and methods Sample collection and preparation
Fig. 1 Example of Chironomidae pupal exuviae.
Chironomidae pupal exuviae samples were collected using drift nets from Lake Lianvatnet (N 63.403°, E 10.318°) and Nidelva River (N 63.429°, E 10.379°) both in Trondheim, Sør-Trøndelag, Norway between August and October 2014. For Lake Lianvatnet samples, the net was pulled through the water surface on the lee side of
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D N A I S O L A T I O N F R O M I N S E C T S K I N S 355 the lake for approximately 10 min; for the Nidelva River samples, the net was left in the current at the edge of the river to collect passively between 10 and 240 min. A mixture of pupal exuviae ages were collected, but caution was used to collect from areas of fresh accumulation. Typically, any particular sample contains those pupal exuviae, which have emerged in the area in the last 24– 48 h (Coffman 1973). Pupal exuviae were picked from the nets and/or larval trays using un-sterilized forceps and put into absolute ethanol (>99.8%). Individual pupal exuviae were identified under a dissecting microscope, separated into sterile 1.5-mL microcentrifuge tubes filled with absolute ethanol and stored in dark conditions at 2 °C for up to 4 months. Before extraction, pupal exuviae were air-dried on a piece of paper and transferred into sterile 1.5-microcentrifuge tubes. This was performed in the pre-PCR room of a standard molecular laboratory at the NTNU University Museum.
DNA extraction For each of the five extraction protocols, total genomic DNA was isolated from 95 pupal exuviae belonging to 13 different taxonomic groups (Table 1). Each method had one negative control. Samples were extracted in batches of 24 samples at a time. The five DNA extraction methods included: a dodecyltrimethylammonium bromide/cetyltrimethylammonium bromide (DTAB/CTAB) protocol adapted from Nishiguchi et al. (2002), three commercially available kits: DNeasyâ Blood & Tissue Kit (Qiagen, Hilden, Germany), NucleoSpinâ Tissue XS Kit (Macherey-Nagel, D€ uren, Germany), E.Z.N.A.â Insect DNA Kit (Omega Bio-Tek Inc., Norcross, Georgia, USA), and an extraction solution: QuickExtractTM DNA Extraction Solution (Epicentre, Eindhoven, Netherlands).
Table 1 Number of pupal exuviae used from each chironomid taxonomic group Taxon
N
Tanypodinae Prodiamesa olivacea (Meigen 1818) Diamesinae Micropsectra spp. Chironomini Eukiefferiella sp. 1 Tvetenia calvescens (Edwards 1929) Eukiefferiella sp. 2 Orthocladius spp. Paracricotopus sp. Corynoneurini Tanytarsus spp. Cricotopus sp.
30 60 6 60 30 60 60 60 60 60 30 6 48
N, number of pupal exuviae.
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For the DTAB/CTAB lysis protocol, DNA was extracted according to protocol outlined by Nishiguchi et al. (2002) with the following modifications: (i) tissue was digested at 68 °C in 600 lL of DTAB solution overnight. (ii) After digestion, pupal exuviae were removed carefully with a plastic pipette tip and transferred to absolute ethanol. (iii) Pellets were washed in 300 lL of 70% ethanol as suggested by Phillips & Simon (1995). (iv) Pellets were resuspended in 20 lL of TE buffer. For the three commercially available kits, DNA was extracted according to the manufacturer’s instructions with the following modifications: 1 DNeasyâ Blood & Tissue Kit: DNA was extracted similar to Krosch & Cranston (2012). (i) Tissue was digested with proteinase K overnight at 37 °C; (ii) after digestion, pupal exuviae were removed carefully with a plastic pipette tip and transferred to absolute ethanol; (iii) to maximize DNA yield, two successive elution steps (first elution: 70 lL, second elution: 30 lL), were performed for a final elution volume of 100 lL; (iv) incubation time of elution was increased to 5 min. 2 NucleoSpinâ Tissue XS Kit: (i) tissue was digested with Buffer T1 and proteinase K overnight at 37 °C; (ii) after digestion, pupal exuviae were removed carefully with a plastic pipette tip and transferred to absolute ethanol; (iii) incubation time of elution was increased to 5 min; (iv) elution fraction was incubated with open lid for 17 min at 75 °C. 3 E.Z.N.A.â Insect DNA Kit: (i) tissue was digested with CTL Buffer and proteinase K overnight at 37 °C; (ii) after digestion, pupal exuviae were removed carefully with a plastic pipette tip and transferred to absolute ethanol; (iii) to maximize DNA yield, two successive elution steps (first elution: 70 lL, second elution: 30 lL), were performed for a final elution volume of 100 lL; (iv) incubation time of elution was increased to 5 min. For the extraction solution, QuickExtractTM DNA Extraction Solution, DNA was extracted according to the manufacturer’s instructions with the following modification: tissue was digested with 100 lL of QuickExtractTM DNA Extraction Solution. During data analysis, DNA extracts were stored at 20 °C. After analysis, DNA extracts were moved to long-term storage at 80 °C. All reference material (DNA extracts & voucher specimens) for this study is deposited at the NTNU University Museum. DNA quantity was measured with a Qubitâ doublestranded DNA (dsDNA) High Sensitivity (HS) Assay Kit (Life Technologies, Eugene, OR, USA) and read a by Qubitâ 2.0 Fluorometer (Life Technologies, Eugene, OR, USA). For the DTAB/CTAB method, DNeasyâ kit, E.Z.N.A.â kit, and QuickExtractTM method 14 lL of
358 P . K R A N Z F E L D E R , T . E K R E M a n d E . S T U R Table 3 Evaluation of relative effectiveness of five DNA extraction methods and direct PCR Time (minutes)
DNA quantity (ng/lL)
PCR success (%)
Sequence success (%)
Target sequence success (%)
Method
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
DNeasyâ Blood & Tissue Kit NucleoSpinâ Tissue XS Kit E.Z.N.A.â Insect DNA Kit QuickExtractTM DNA Extraction Solution DTAB/CTAB lysis Direct PCR
44.65 44.80 48.04 4.45
0.36 0.24 1.13 0.26
0.010 0.019 0.003 0.020
0.006 0.056 0.006 0.055
97.89 98.94 83.16 83.15
14.43 10.26 37.62 37.62
28.42 38.95 17.89 36.84
45.34 49.02 38.54 48.49
17.89 14.74 4.21 12.63
38.53 35.64 20.19 33.40
47.10 1.90
0.25 0.00
0.000 N/A
0.003 N/A
0.00 2.11
0.00 14.43
0.00 2.11
0.00 14.43
0.00 1.05
0.00 10.26
Fig. 2 Comparison of DNA quantity (ng/ lL, log-transformed) of five DNA extraction methods. Black lines indicate median, 25th and 75th percentile, whiskers indicate 10th and 90th percentile, open dots indicate outliers, closed dots indicate mean. (a) Data shown with extreme upper outliers. (b) Data shown without extreme upper outliers.
(a)
(b)
PCR (P < 0.001). The DTAB/CTAB method resulted in no PCR success (Fig. 3). The NucleoSpinâ kit (x = 98.9%) and DNeasyâ kit (x = 97.9%) were statistically similar (P > 0.05) and had good PCR success. The E.Z.N.A.â kit (x = 83.2%) and QuickExtractTM method (x = 83.2%) were statistically similar (P > 0.05) and had moderate PCR success, but were statistically differed from the NucleoSpinâ kit and the DNeasyâ kit (P < 0.05). Direct PCR (x = 2.1%) had very low PCR success and was statistically different from all other methods (P < 0.001) (Table 4 and Fig. 3).
Sequence success High-quality sequences were obtained from 126 of the 349 sequenced pupal exuviae (36.1%). Sequence success was significantly different between the four DNA extraction methods and direct PCR (P < 0.001). The DTAB/ CTAB method resulted in no sequence success (Fig. 3). The NucleoSpinâ kit ( x = 38.9%), the QuickExtractTM method ( x = 36.8%) and the DNeasyâ kit ( x = 28.4%) were statistically similar (P > 0.05) and resulted in the highest sequence success. The E.Z.N.A.â kit ( x = 17.9%)
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D N A I S O L A T I O N F R O M I N S E C T S K I N S 361 Carrier DNA contamination occurs when a low number of contaminating molecules present in the extraction of amplification reagents absorb to plastic ware, but do not yield positive-PCR products. However, when low-quantity DNA extract, such as from our samples, is added, then the contaminating molecules on the plastic surface are displaced by the DNA extract and these contaminants can become available for PCR amplification (Handt et al. 1994). Low-quality or trace DNA samples compete with contaminant free DNA over the course of an experiment (Lusk 2014), but stricter laboratory precautions, such as routine UV irradiation of reagents and tools and positive pressure laboratory ventilation systems, could reduce chances of free DNA contamination from the laboratory (Willerslev & Cooper 2005; Champlot et al. 2010). While laboratory contamination is probable, the majority of the detected cross-contaminations most likely were introduced from the field. The source of the main nontarget DNA (Holopedium gibberum) is expected to be from the Nidelva River, as moderate densities of this cladoceran were observed to be floating on the surface of the water during fieldwork. Even the contamination with human DNA might have originated from the river, as there is periodical sewage run-off upstream of the sampling locality. During sample sorting, individual pupal exuviae were inspected under a dissecting microscope and discarded if they had evidence of another organism attached. Nonetheless, cells or free DNA from nontarget organisms could get stuck inside or attach to different parts of the pupal exuviae. It is therefore important to consider natural contamination when designing a sampling regime for molecular studies on chironomid pupal exuviae or other trace DNA samples, even if it requires an increase in sampling effort and laboratory processing. Additionally, forceps can be soaked in 50% bleach solution, rinsed in water and dried between samples to reduce cross-contamination (Goldberg et al. 2013). Chironomid pupal exuviae are among the most useful and cost-efficient life stages to include in aquatic biodiversity studies (Raunio et al. 2011; Ferrington & Coffman 2014; Kranzfelder & Ferrington 2015), but morphological species identification is challenging due to lack of species associations and comprehensive taxonomic keys. The association of chironomid pupal exuviae with adult counterparts using DNA barcodes can further increase the usefulness of this life stage in freshwater science. A detailed study of the identification success of chironomid pupal exuviae using DNA barcodes is under development and will be published elsewhere. The use of an effective protocol for DNA isolation of trace DNA in insect exuviae is crucial and we expect our results to be particularly useful for research focused on ecology, taxonomy and systematics of aquatic
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macroinvertebrates that shed their exoskeleton during metamorphosis. In addition, our results might be attractive for studies utilizing environmental DNA (eDNA) as high-throughput sequencing techniques enables nontarget contaminants to be easily filtered out from the resulting sequences.
Acknowledgements We would like to thank Erik Bostr€ om for assistance with practical study design and molecular laboratory work, James Speed for support with selecting statistical analyses and three anonymous reviewers for useful suggestions to the manuscript. We also give many thanks for the following funding sources that allowed this work to be completed: the Norwegian University of Science and Technology-University of Minnesota Fulbright Direct Exchange, the Marion Brooks-Wallace Graduate Fellowship of University of Minnesota’s Department of Entomology and the Torske Klubben Norwegian Luncheon Club Graduate Fellowship. This manuscript is published under the auspices of the NTNU University Museum, Trondheim, Norway.
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D N A I S O L A T I O N F R O M I N S E C T S K I N S 359 Table 4 Pairwise comparisons of four DNA extraction methods and direct PCR. (A) PCR success (n = 475), (B) sequence success (n = 475), and (C) target sequence success (n = 475) Pairwise comparisons (A) DNeasyâ – direct PCR E.Z.N.A.â – direct PCR NucleoSpinâ – direct PCR QuickExtractTM – direct PCR E.Z.N.A.â – DNeasyâ NucleoSpinâ – DNeasyâ QuickExtractTM – DNeasyâ NucleoSpinâ – E.Z.N.A.â QuickExtractTM – E.Z.N.A.â QuickExtractTM– NucleoSpinâ (B) DNeasyâ – direct PCR E.Z.N.A.â – direct PCR NucleoSpinâ – direct PCR QuickExtractTM – direct PCR E.Z.N.A.â – DNeasyâ NucleoSpinâ – DNeasyâ QuickExtractTM – DNeasyâ NucleoSpinâ – E.Z.N.A.â QuickExtractTM – E.Z.N.A.â QuickExtractTM – NucleoSpinâ (C) DNeasyâ – direct PCR E.Z.N.A.â – direct PCR NucleoSpinâ – direct PCR QuickExtractTM – direct PCR E.Z.N.A.â – DNeasyâ NucleoSpinâ – DNeasyâ QuickExtractTM – DNeasyâ NucleoSpinâ – E.Z.N.A.â QuickExtractTM – E.Z.N.A.â QuickExtractTM– NucleoSpinâ
OR
CI
P value
2162.46 229.52 4372.12 229.52 0.11 2.02 0.11 19.03 1.00 0.05
144.34–32.53e3 29.58–17.82e2 160.77–11.93e4 29.58–17.82e2 0.01–0.82 0.07–54.98 0.01–0.82 1.17–310.13 0.35–2.82 3.20e3–0.86
doi: 10.1111/1755-0998.12446
Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae PETRA KRANZFELDER,*,† TORBJØRN EKREM* and E L I S A B E T H S T U R * *Department of Natural History, NTNU University Museum, Norwegian University of Science and Technology, NO-7491, Trondheim, Norway, †Department of Entomology, University of Minnesota, 1980 Folwell Avenue, Saint Paul, MN 55108, USA
Abstract Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell-based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a speciesrich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpinâ Tissue XS Kit, DNeasyâ Blood and Tissue kit, and QuickExtractTM DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments. Keywords: aquatic macroinvertebrates, biodiversity assessment, COI, DNA barcoding, DNA isolation, species identification Received 8 May 2015; revision received 6 July 2015; accepted 13 July 2015
Introduction The effectiveness of DNA barcoding, metabarcoding and environmental barcoding as a routine practice in biodiversity monitoring and conservation is strongly dependent on the quality of the DNA extractions (Ivanova et al. 2006; Knebelsberger & St€ oger 2012). There are a variety of methods to isolate DNA from biological materials, but the ideal extraction technique should provide high DNA quality and quantity, as well as, be efficient in terms of cost and handling time, be suitable for high sample throughput and generate minimal hazardous waste (Chen et al. 2010). Traditional extraction methods, including phenol/chloroform (Nishiguchi et al. 2002) or DTAB-CTAB lysis (Phillips & Simon 1995), work efficiently for extracting DNA from animal tissues, Correspondence: Petra Kranzfelder, Fax: (612)-625-5299; E-mail: [email protected]
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providing the possibility to get DNA without damaging specimens, but use toxic chemicals and are time-consuming (Hajibabaei et al. 2005). Various commercially available DNA extraction kits, especially silica-membranebased methods, are becoming increasingly popular because of their ease-of-use for a wide range of biological samples, limited labour and ability to consistently produce high-quality DNA (Nishiguchi et al. 2002; Hajibabaei et al. 2005). However, some of the drawbacks of these kits include the expense and long incubation times (Ball & Armstrong 2008). Another rapid and low-cost DNA barcoding technique termed ‘direct PCR’ facilitates PCR amplification of target genes directly from samples without DNA extraction and purification; however, direct PCR success rates can be low for many taxa (Wong et al. 2014). Although these standardized DNA isolation methods can be used for a broad range of biological samples, some are still problematic for low yield or degraded samples (Zimmermann et al. 2008; Goldberg et al. 2015).
354 P . K R A N Z F E L D E R , T . E K R E M a n d E . S T U R Chironomidae (Insecta: Diptera) are a species-rich group of aquatic macroinvertebrates that are abundant and widely distributed in aquatic systems (Cranston 1995). Chironomid communities are considered valuable bioindicators of water quality due to their high species diversity, ubiquity and varying species sensitivity to environmental conditions (Ferrington et al. 2008; Marziali et al. 2010; Raunio et al. 2011). However, chironomid larvae are time-consuming and difficult to identify to genus or species, which has led to a debate about the importance of using chironomid data in biological monitoring programs (Rosenberg 1992; Cranston 2000; Rabeni & Wang 2001; Nijboer et al. 2005; Roque et al. 2010; Ruse 2010; Raunio et al. 2011). A efficient, effective, low-cost and easy-to-use form of sampling chironomids involves collections of pupal exuviae (Wilson & Ruse 2005; CalleMartınez & Casas 2006; Raunio et al. 2007; Bouchard & Ferrington 2011; Ca~ nedo-Arg€ uelles et al. 2012), which is the exoskeleton shed by the adult as it emerges on the surface of the water (Ferrington et al. 1991) (Fig. 1). Pupal exuviae accumulate behind obstructions, such as fallen trees or along banks through the action of wind or water current, and can be easily collected to give a comprehensive sample of chironomid communities (Ruse 2010). This collection method has several advantages over traditional benthic larvae sampling techniques: (i) applicability in nearly all types and conditions of aquatic environments, including deep, muddy rivers, canals and lakes, and riffles in shallow rivers; (ii) accumulated pupal exuviae represent taxa that have originated from a wide range of microhabitats; and (iii) sampling and sample sorting are easy and rapid (Wilson & Ruse 2005; Kranzfelder et al. 2015). Species identification is essential for aquatic bioassessment studies (Lenat & Resh 2001; Melo 2005; Marshall et al. 2006; Jones 2008), as some chironomid species inhabit waters of high quality, while others occupy poor
quality (Epler 2001). However, in many regions, it is difficult to identify chironomid pupal exuviae to species (morphologically) as most species descriptions and keys are based on adult males (Carew et al. 2007; Ekrem et al. 2007). Ekrem et al. (2007), among others, have found that partial mitochondrial gene cytochrome oxidase I (COI) gene sequences can be used to link different life stages of Chironomidae and DNA barcoding has been successful for identifying cryptic and undescribed chironomid species from different life stages: larvae, pupae and adults (Stur & Ekrem 2011; Anderson et al. 2013; Brodin et al. 2013; Meier et al. 2015). Yet, barcoding of chironomid pupal exuviae has been challenging due to the low quantities of genomic DNA available for extraction (Krosch & Cranston 2012). The exoskeleton of the pupal exuviae is made of extracellular chitin, the most widespread structural polysaccharide in nature (Merzendorfer 2006), and does not contain any nucleic acids. Therefore, genomic DNA yield from pupal exuviae relies on muscle tissue, hairs and epithelial cells lining the foregut, hindgut and tracheae that are left behind on the inner surface of the cuticle by the emerging adult. In addition, metabolic waste products from the pupal stage might be released by the adult upon emergence (Nation 2008). Trace DNA from chironomid pupal exuviae can be sufficient for PCR amplification and sequencing of target genes (Krosch & Cranston 2012). Miller et al. (1988) attempted to extract DNA from chironomid pupal exuviae using a modified salting out protocol, but had no success with this method. Recently, Krosch & Cranston (2012) were able to extract genomic DNA from 27 of 58 chironomid pupal exuviae using one extraction method (DNeasyâ Blood & Tissue Kit), but this study was limited by few specimens, one extraction method and lack of quantitative data. Consequently, there is no consensus regarding the most effective DNA isolation method for chironomid pupal exuviae or similar trace DNA samples. In this study, we compare the performance of five different DNA extraction methods and direct PCR in isolation of genomic DNA from chironomid pupal exuviae in terms of cost, handling time, DNA quantity, PCR success, sequence success and ability to sequence target taxa.
Materials and methods Sample collection and preparation
Fig. 1 Example of Chironomidae pupal exuviae.
Chironomidae pupal exuviae samples were collected using drift nets from Lake Lianvatnet (N 63.403°, E 10.318°) and Nidelva River (N 63.429°, E 10.379°) both in Trondheim, Sør-Trøndelag, Norway between August and October 2014. For Lake Lianvatnet samples, the net was pulled through the water surface on the lee side of
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D N A I S O L A T I O N F R O M I N S E C T S K I N S 355 the lake for approximately 10 min; for the Nidelva River samples, the net was left in the current at the edge of the river to collect passively between 10 and 240 min. A mixture of pupal exuviae ages were collected, but caution was used to collect from areas of fresh accumulation. Typically, any particular sample contains those pupal exuviae, which have emerged in the area in the last 24– 48 h (Coffman 1973). Pupal exuviae were picked from the nets and/or larval trays using un-sterilized forceps and put into absolute ethanol (>99.8%). Individual pupal exuviae were identified under a dissecting microscope, separated into sterile 1.5-mL microcentrifuge tubes filled with absolute ethanol and stored in dark conditions at 2 °C for up to 4 months. Before extraction, pupal exuviae were air-dried on a piece of paper and transferred into sterile 1.5-microcentrifuge tubes. This was performed in the pre-PCR room of a standard molecular laboratory at the NTNU University Museum.
DNA extraction For each of the five extraction protocols, total genomic DNA was isolated from 95 pupal exuviae belonging to 13 different taxonomic groups (Table 1). Each method had one negative control. Samples were extracted in batches of 24 samples at a time. The five DNA extraction methods included: a dodecyltrimethylammonium bromide/cetyltrimethylammonium bromide (DTAB/CTAB) protocol adapted from Nishiguchi et al. (2002), three commercially available kits: DNeasyâ Blood & Tissue Kit (Qiagen, Hilden, Germany), NucleoSpinâ Tissue XS Kit (Macherey-Nagel, D€ uren, Germany), E.Z.N.A.â Insect DNA Kit (Omega Bio-Tek Inc., Norcross, Georgia, USA), and an extraction solution: QuickExtractTM DNA Extraction Solution (Epicentre, Eindhoven, Netherlands).
Table 1 Number of pupal exuviae used from each chironomid taxonomic group Taxon
N
Tanypodinae Prodiamesa olivacea (Meigen 1818) Diamesinae Micropsectra spp. Chironomini Eukiefferiella sp. 1 Tvetenia calvescens (Edwards 1929) Eukiefferiella sp. 2 Orthocladius spp. Paracricotopus sp. Corynoneurini Tanytarsus spp. Cricotopus sp.
30 60 6 60 30 60 60 60 60 60 30 6 48
N, number of pupal exuviae.
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For the DTAB/CTAB lysis protocol, DNA was extracted according to protocol outlined by Nishiguchi et al. (2002) with the following modifications: (i) tissue was digested at 68 °C in 600 lL of DTAB solution overnight. (ii) After digestion, pupal exuviae were removed carefully with a plastic pipette tip and transferred to absolute ethanol. (iii) Pellets were washed in 300 lL of 70% ethanol as suggested by Phillips & Simon (1995). (iv) Pellets were resuspended in 20 lL of TE buffer. For the three commercially available kits, DNA was extracted according to the manufacturer’s instructions with the following modifications: 1 DNeasyâ Blood & Tissue Kit: DNA was extracted similar to Krosch & Cranston (2012). (i) Tissue was digested with proteinase K overnight at 37 °C; (ii) after digestion, pupal exuviae were removed carefully with a plastic pipette tip and transferred to absolute ethanol; (iii) to maximize DNA yield, two successive elution steps (first elution: 70 lL, second elution: 30 lL), were performed for a final elution volume of 100 lL; (iv) incubation time of elution was increased to 5 min. 2 NucleoSpinâ Tissue XS Kit: (i) tissue was digested with Buffer T1 and proteinase K overnight at 37 °C; (ii) after digestion, pupal exuviae were removed carefully with a plastic pipette tip and transferred to absolute ethanol; (iii) incubation time of elution was increased to 5 min; (iv) elution fraction was incubated with open lid for 17 min at 75 °C. 3 E.Z.N.A.â Insect DNA Kit: (i) tissue was digested with CTL Buffer and proteinase K overnight at 37 °C; (ii) after digestion, pupal exuviae were removed carefully with a plastic pipette tip and transferred to absolute ethanol; (iii) to maximize DNA yield, two successive elution steps (first elution: 70 lL, second elution: 30 lL), were performed for a final elution volume of 100 lL; (iv) incubation time of elution was increased to 5 min. For the extraction solution, QuickExtractTM DNA Extraction Solution, DNA was extracted according to the manufacturer’s instructions with the following modification: tissue was digested with 100 lL of QuickExtractTM DNA Extraction Solution. During data analysis, DNA extracts were stored at 20 °C. After analysis, DNA extracts were moved to long-term storage at 80 °C. All reference material (DNA extracts & voucher specimens) for this study is deposited at the NTNU University Museum. DNA quantity was measured with a Qubitâ doublestranded DNA (dsDNA) High Sensitivity (HS) Assay Kit (Life Technologies, Eugene, OR, USA) and read a by Qubitâ 2.0 Fluorometer (Life Technologies, Eugene, OR, USA). For the DTAB/CTAB method, DNeasyâ kit, E.Z.N.A.â kit, and QuickExtractTM method 14 lL of
358 P . K R A N Z F E L D E R , T . E K R E M a n d E . S T U R Table 3 Evaluation of relative effectiveness of five DNA extraction methods and direct PCR Time (minutes)
DNA quantity (ng/lL)
PCR success (%)
Sequence success (%)
Target sequence success (%)
Method
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
DNeasyâ Blood & Tissue Kit NucleoSpinâ Tissue XS Kit E.Z.N.A.â Insect DNA Kit QuickExtractTM DNA Extraction Solution DTAB/CTAB lysis Direct PCR
44.65 44.80 48.04 4.45
0.36 0.24 1.13 0.26
0.010 0.019 0.003 0.020
0.006 0.056 0.006 0.055
97.89 98.94 83.16 83.15
14.43 10.26 37.62 37.62
28.42 38.95 17.89 36.84
45.34 49.02 38.54 48.49
17.89 14.74 4.21 12.63
38.53 35.64 20.19 33.40
47.10 1.90
0.25 0.00
0.000 N/A
0.003 N/A
0.00 2.11
0.00 14.43
0.00 2.11
0.00 14.43
0.00 1.05
0.00 10.26
Fig. 2 Comparison of DNA quantity (ng/ lL, log-transformed) of five DNA extraction methods. Black lines indicate median, 25th and 75th percentile, whiskers indicate 10th and 90th percentile, open dots indicate outliers, closed dots indicate mean. (a) Data shown with extreme upper outliers. (b) Data shown without extreme upper outliers.
(a)
(b)
PCR (P < 0.001). The DTAB/CTAB method resulted in no PCR success (Fig. 3). The NucleoSpinâ kit (x = 98.9%) and DNeasyâ kit (x = 97.9%) were statistically similar (P > 0.05) and had good PCR success. The E.Z.N.A.â kit (x = 83.2%) and QuickExtractTM method (x = 83.2%) were statistically similar (P > 0.05) and had moderate PCR success, but were statistically differed from the NucleoSpinâ kit and the DNeasyâ kit (P < 0.05). Direct PCR (x = 2.1%) had very low PCR success and was statistically different from all other methods (P < 0.001) (Table 4 and Fig. 3).
Sequence success High-quality sequences were obtained from 126 of the 349 sequenced pupal exuviae (36.1%). Sequence success was significantly different between the four DNA extraction methods and direct PCR (P < 0.001). The DTAB/ CTAB method resulted in no sequence success (Fig. 3). The NucleoSpinâ kit ( x = 38.9%), the QuickExtractTM method ( x = 36.8%) and the DNeasyâ kit ( x = 28.4%) were statistically similar (P > 0.05) and resulted in the highest sequence success. The E.Z.N.A.â kit ( x = 17.9%)
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D N A I S O L A T I O N F R O M I N S E C T S K I N S 361 Carrier DNA contamination occurs when a low number of contaminating molecules present in the extraction of amplification reagents absorb to plastic ware, but do not yield positive-PCR products. However, when low-quantity DNA extract, such as from our samples, is added, then the contaminating molecules on the plastic surface are displaced by the DNA extract and these contaminants can become available for PCR amplification (Handt et al. 1994). Low-quality or trace DNA samples compete with contaminant free DNA over the course of an experiment (Lusk 2014), but stricter laboratory precautions, such as routine UV irradiation of reagents and tools and positive pressure laboratory ventilation systems, could reduce chances of free DNA contamination from the laboratory (Willerslev & Cooper 2005; Champlot et al. 2010). While laboratory contamination is probable, the majority of the detected cross-contaminations most likely were introduced from the field. The source of the main nontarget DNA (Holopedium gibberum) is expected to be from the Nidelva River, as moderate densities of this cladoceran were observed to be floating on the surface of the water during fieldwork. Even the contamination with human DNA might have originated from the river, as there is periodical sewage run-off upstream of the sampling locality. During sample sorting, individual pupal exuviae were inspected under a dissecting microscope and discarded if they had evidence of another organism attached. Nonetheless, cells or free DNA from nontarget organisms could get stuck inside or attach to different parts of the pupal exuviae. It is therefore important to consider natural contamination when designing a sampling regime for molecular studies on chironomid pupal exuviae or other trace DNA samples, even if it requires an increase in sampling effort and laboratory processing. Additionally, forceps can be soaked in 50% bleach solution, rinsed in water and dried between samples to reduce cross-contamination (Goldberg et al. 2013). Chironomid pupal exuviae are among the most useful and cost-efficient life stages to include in aquatic biodiversity studies (Raunio et al. 2011; Ferrington & Coffman 2014; Kranzfelder & Ferrington 2015), but morphological species identification is challenging due to lack of species associations and comprehensive taxonomic keys. The association of chironomid pupal exuviae with adult counterparts using DNA barcodes can further increase the usefulness of this life stage in freshwater science. A detailed study of the identification success of chironomid pupal exuviae using DNA barcodes is under development and will be published elsewhere. The use of an effective protocol for DNA isolation of trace DNA in insect exuviae is crucial and we expect our results to be particularly useful for research focused on ecology, taxonomy and systematics of aquatic
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macroinvertebrates that shed their exoskeleton during metamorphosis. In addition, our results might be attractive for studies utilizing environmental DNA (eDNA) as high-throughput sequencing techniques enables nontarget contaminants to be easily filtered out from the resulting sequences.
Acknowledgements We would like to thank Erik Bostr€ om for assistance with practical study design and molecular laboratory work, James Speed for support with selecting statistical analyses and three anonymous reviewers for useful suggestions to the manuscript. We also give many thanks for the following funding sources that allowed this work to be completed: the Norwegian University of Science and Technology-University of Minnesota Fulbright Direct Exchange, the Marion Brooks-Wallace Graduate Fellowship of University of Minnesota’s Department of Entomology and the Torske Klubben Norwegian Luncheon Club Graduate Fellowship. This manuscript is published under the auspices of the NTNU University Museum, Trondheim, Norway.
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D N A I S O L A T I O N F R O M I N S E C T S K I N S 359 Table 4 Pairwise comparisons of four DNA extraction methods and direct PCR. (A) PCR success (n = 475), (B) sequence success (n = 475), and (C) target sequence success (n = 475) Pairwise comparisons (A) DNeasyâ – direct PCR E.Z.N.A.â – direct PCR NucleoSpinâ – direct PCR QuickExtractTM – direct PCR E.Z.N.A.â – DNeasyâ NucleoSpinâ – DNeasyâ QuickExtractTM – DNeasyâ NucleoSpinâ – E.Z.N.A.â QuickExtractTM – E.Z.N.A.â QuickExtractTM– NucleoSpinâ (B) DNeasyâ – direct PCR E.Z.N.A.â – direct PCR NucleoSpinâ – direct PCR QuickExtractTM – direct PCR E.Z.N.A.â – DNeasyâ NucleoSpinâ – DNeasyâ QuickExtractTM – DNeasyâ NucleoSpinâ – E.Z.N.A.â QuickExtractTM – E.Z.N.A.â QuickExtractTM – NucleoSpinâ (C) DNeasyâ – direct PCR E.Z.N.A.â – direct PCR NucleoSpinâ – direct PCR QuickExtractTM – direct PCR E.Z.N.A.â – DNeasyâ NucleoSpinâ – DNeasyâ QuickExtractTM – DNeasyâ NucleoSpinâ – E.Z.N.A.â QuickExtractTM – E.Z.N.A.â QuickExtractTM– NucleoSpinâ
OR
CI
P value
2162.46 229.52 4372.12 229.52 0.11 2.02 0.11 19.03 1.00 0.05
144.34–32.53e3 29.58–17.82e2 160.77–11.93e4 29.58–17.82e2 0.01–0.82 0.07–54.98 0.01–0.82 1.17–310.13 0.35–2.82 3.20e3–0.86
