Acta Sil va e et Ligni 132 (2023), 29–38 29 Original scientific article / Izvirni znanstveni članek THE INFLUENCE OF STORAGE CONDITIONS AND TOT AL DNA EXTRACTION PROTOCOL ON THE RESUL TS OF MOLECULAR ANALYSIS OF THE EUROPEAN SPRUCE BARK BEETLE (Ips typographus L.) VPLIV POSTOPKOV SHRANJEVANJA IN EKSTRAKCIJE CELOKUPNE DNA NA REZUL T ATE MOLEKULARNE ANALIZE OSMEROZOBEGA SMREKOVEGA LUBADARJA (Ips typographus L.) Zina DEVETAK 1 , Andreja KAVČIČ 2,3 , Maarten DE GROOT 4 , Barbara PIŠKUR 5 (1) Slovenian Forestry Institute, Department of Forest Protection, zina.devetak@gozdis.si (2) Slovenian Forestry Institute, Department of Forest Protection (3) Slovenia Forest Service, Regional Unit Brežice, andreja.kavcic@zgs.gov.si (4) Slovenian Forestry Institute, Department of Forest Protection, maarten.degroot@gozdis.si (5) Slovenian Forestry Institute, Department of Forest Protection, barbara.piskur@gozdis.si ABSTRACT One of the key steps of the molecular identification of bark beetles is obtaining a sufficient quantity of high-quality DNA extract. In this study, we investigated the influence of different storage procedures for Ips typographus (L.) specimens and various DNA extraction protocols on the quantity and quality of DNA intended for use in molecular diagnostics. Adult beetles were frozen at -20 °C, either dry or in ethanol. We tested four different protocols for DNA extraction. We compared the quantity of extrac- ted DNA and assessed its quality with PCR and Sanger sequencing. Different storage protocols had no significant effect on the quantity of DNA extracted. However, freezing specimens in ethanol provided higher-quality DNA for molecular applications. Only two of the extraction protocols produced sequenceable amplicons, and the difference in the amount of extracted DNA between them was not significant. We propose the optimal combination of storing specimens in ethanol at -20°C and using the Nucleospin Insect DNA extraction kit from Macherey Nagel, enabling a time-efficient identification process. Key words: early detection, specimen storage, total DNA extraction, PCR, polymerase chain reaction, Sanger sequencing, molecular diagnostics IZVLEČEK Za uspeh molekularne identifikacije podlubnikov je ključna zadostna količina kakovostnega ekstrakta DNA. Pri raziskavi smo se osredotočili na vpliv različnih postopkov shranjevanja osebkov Ips typographus (L.) in različnih protokolov ekstrakcije DNA na količino in kakovost DNA, namenjene za uporabo v molekularni diagnostiki. Odrasle hrošče smo zamrznili pri -20 °C na dva načina, v etanolu in na suho. Ekstrakcije smo opravili po štirih različnih postopkih. Primerjali smo količino pridobljene DNA. Kakovost ekstrahirane DNA smo ocenili s polimerazno verižno reakcijo in sekvenciranjem po Sangerju. Način shranjevanja ni statistično značilno vplival na količino pridobljene DNA. DNA višje kakovosti smo pridobili iz hroščev, ki so bili zamrzn- jeni v etanolu. Le dva postopka ekstrakcije DNA sta dala DNA, uporabno za sekvenciranje. V količini pridobljene DNA se pri sekvenciranju uspešna postopka nista pomembno razlikovala. Določili smo optimalno kombinacijo postopkov shranjevanja v etanolu pri -20 °C in ekstrakcije celokupne DNA z ekstrakcijskim kitom Nucleospin Insect DNA proizvajalca Macherey Nagel, ki omogoča časovno učinkovito identifikacijo. Ključne besede: zgodnje zaznavanje, shranjevanje osebkov, ekstrakcijski postopek celokupne DNA, PCR, polimerazna verižna reakcija, sekvenciranje po Sangerju, molekularna diagnostika GDK 145.76(Ips typographus L.):135--014 (045)=111 Received / Prispelo: 28. 08. 2023 DOI 10.20315/ASetL.132.3 Accepted / Sprejeto: 13. 12. 2023 1 INTRODUCTION 1 UVOD Bark beetles (Coleoptera: Curculionidae: Scolyti- nae) play a crucial role in forest ecosystems. The vast majority of species breed in dead and dying woody plant tissues, thus contributing to the cycling of organic matter (Machingambi et al., 2014). However, some bark beetle species (e.g., I. typographus L., Pityogenes chal- cographus B., I. duplicatus S.) can also cause significant ecological and economic damage in the form of tree mortality following exceptional climatic events (e.g., windthrow, snow damage, ice storms, and drought) 30 De v etak Z., Ka v č ič A., de Gr oo t M., Pišk ur B.: The infl uenc e o f st or a ge c ond i t ions and dna ex tr a ct ion pr o t oc ol on the ... (Avtzis et al., 2012; de Groot et al., 2018; Gandhi and Hofstetter, 2021). Worldwide, there are more than 6000 species of bark beetles (Linnakoski et al., 2012; Faccoli, 2015), of which around 90 are found in Slove- nia, with new reports continuously emerging (Titovšek, 1988; Jurc, 2003; Kavčič, 2018; Hauptman et al., 2019). The European spruce bark beetle (I. typographus) can cause considerable damage to Norway spruce (Gandhi and Hofstetter, 2021; Hlásny et al., 2021), which is one of the most economically important tree species in Europe (San-Miguel-Ayanz et al., 2022). Dur- ing the past century, Norway spruce (Picea abies, (L.) H. Karst.) has been extensively planted outside its nat- ural distribution range, where growing conditions for this species are suboptimal. With a warming climate and unfavourable weather events inducing higher stress and providing better living conditions for bark beetle development, there has been an increase in the frequency and extent of outbreaks (Netherer and Ham- merbacher, 2022). Identifying species morphologically or with molecular methods is often the first step in developing plans to manage such outbreaks and is an important part of the early detection of native as well as invasive alien species. The early detection of new and emerging pests is essential to prevent and limit damage, while correct identification is crucial for the implementation of ap- propriate control measures to further reduce the risk posed by pests (Morales-Rodríguez et al., 2019; Po- land and Rassati, 2019). Since measures, and therefore costs, are proportional to the threat posed by the iden- tified species to the local ecosystem, incorrect identi- fication can result in unnecessary measures with high economic and ecological costs (Lima et al., 2022; Chen et al., 2023). Historically, morphological identifica- tion was mainly used (Madden et al., 2019). With the rapid spread and introduction of species globally, tax- onomists are facing a growing challenge (Poland and Rassati, 2019). In recent decades, molecular methods have developed rapidly to aid in the identification of pests and other species (EPPO, 2021; Madden et al., 2019). Additionally, morphological methods are often limited to undamaged adult specimens, whereas mo- lecular methods enable identification even from sam- ples with missing morphological features and juvenile stages, sometimes even from materials containing only traces of the target DNA (e.g., frass, swabs from gal- leries) (Boykin et al., 2012; Taylor and Harris, 2012; Jörger and Schrödl, 2013; Albo et al., 2019; Morales- Rodríguez et al., 2019; Poland and Rassati, 2019), often referred to as environmental DNA, or eDNA (Ficetola et al., 2008; Taberlet et al., 2012; Gorički et al., 2017). Storage of samples prior to molecular analysis is an important part of the analytical process and can influ- ence the final result of the analysis (Ballare et al., 2019; Whitman et al., 2019; Martoni et al., 2021; Moškrič et al., 2023). The choice of DNA extraction protocol is also important, as it can influence the quantity and quality of the extracted DNA (Wang et al., 2019). Additionally, the presence of (ideally well-preserved) DNA in the sample is critical to the success of the extraction (Nagy, 2010). In addition to oxidation, the presence of water and high temperatures are the main causes of DNA degradation, as they promote enzymatic activity, with nuclease-type enzymes being the most problematic for DNA preserva- tion (Gemeinholzer et al., 2010; Nagy, 2010). Such con- ditions result in oxidative damage to the DNA molecule, molecular crosslinking, and fragmentation (Deagle et al., 2006), making the damaged DNA less useful for PCR and other molecular methods. For animal tissues, stor- age of DNA in a high concentration of ethanol is usually recommended (Gemeinholzer et al., 2010). In the present study, we compared two different ways of storing I. typographus specimens at -20°C: (i) dry storage and (ii) storage in 96% ethanol. Both stor- age protocols are relatively undemanding in terms of laboratory equipment, as they only require access to a standard freezer and require no elaborate prepara- tion apart from drying the specimen on filter paper in order to remove the ethanol prior to DNA extrac- tion. In addition, we tested four different total DNA extraction protocols based on commercial extraction kits from two different manufacturers. The selected kits are all commercially available, time-efficient, and easy to use in a laboratory setting. We measured vari- ables such as the concentration and calculated the fi- nal amount of total DNA in the extracts. We used end- point PCR and subsequent Sanger sequencing of the amplicons to assess the quality of the extracted DNA and its usefulness in molecular identification. For the purposes of this study, DNA was deemed useful for mo- lecular identification if it could be used to produce a PCR amplicon that could in turn be sequenced, and the resulting chromatograms were of sufficient quality to enable reliable identification of the target via pairwise alignment to a reference sequence. Our results provide guidelines for the storage of specimens and the choice of total DNA extraction protocol for small arthropod samples intended for molecular analysis. 2 METHODS 2 METODE In May and June 2021, adult bark beetles were col- lected from Norway spruce logs kept in rearing tents. Acta Sil va e et Ligni 132 (2023), 29–38 31 Bark beetles were morphologically identified to the species level at the Laboratory of Forest Protection. Forty-eight specimens, identified as Ips typographus, were frozen, either dry or stored in 96% ethanol. After one month of storage, four different protocols were used to extract total DNA from individual bark beetle specimens according to the manufacturers’ in- structions (Table 1). For each extraction protocol, total DNA was extracted from three individual adult speci- mens per storage protocol, i.e., 12 adult specimens per storage protocol. The concentration of resulting total DNA extracts was quantified with a Biophotometer Plus (Eppendorf, Germany) and µCuvette (Eppendorf, Germany). DNA quantity was then calculated from the concentration of DNA and volume of the extracts. We tested two different sets of primers targeting the arthropod COI gene with an end-point PCR. The LCO1490/HCO2198 primer pair was developed by Fol- mer et al. (1994) and the Ips_F_cons/Ips_R_cons pair was developed by Becker et al. (2021) (Table 2). The PCR master mix was prepared using Amplitaq Gold 360 MM (ThermoFisher Scientific, Massachu- setts, USA): 25 µl of master mix, 1 µl of GC Enhancer, 1 µl of each primer, and 4 µl DNA (standardized to a concentration of 10 ng/µl) per 50 µl reaction. PCR products were separated on a 1% agarose gel, and in the case of the successful amplification of a product of expected size, purified using a Wizard SV Gel and PCR Clean-Up System (Promega, Wisconsin, USA) and then sequenced at a sequencing facility (Eurofins, Köln, Germany) in both forward and reverse directions us- ing the same primers as for the PCR. Sequences were visualized and manually edited using Geneious Prime ® v.2021.2.2. (Biomatters Ltd., Auckland, New Zealand). The obtained consensus sequences were compared to reference sequence MK314170 using MAFFT Align- ment implemented in Geneious Prime and deposited at GenBank. The amount of DNA extracted was normalized to the amount of input material. The normalized amount of DNA extracted from beetles stored in two different ways and using three different DNA extraction proto- cols was then compared using the statistical software R version 4.2.2 (R Core Team, 2022). In Protocol 4, the RNA carrier is used in the binding buffer during extrac- tion, resulting in residual RNA presence in the final DNA extract. UV-VIS photometry was used for the quantifi- cation of DNA extracts. As the method cannot discrimi- nate between RNA and DNA in the sample, the actual concentration of the extracts produced using Protocol 4 is lower than the one measured. For this reason, data produced with Protocol 4 were not included in the statistical analysis of the amount of DNA extracted us- ing the different protocols. Due to the non-normality of the acquired data, the Wilcoxon rank-sum test was used to check for differences between the two stor- age protocols. For differences between the extraction protocols, the non-parametric Kruskal-Wallis test was used, followed by the Dunn post-hoc test. The statisti- cal analysis and the creation of graphs were performed using the packages “FSA” (Ogle et al., 2022), “ggpubr” (Kassambara, 2022), and “ggplot2” (Wickham, 2016). 3 RESUL TS 3 REZUL T ATI The average mass of I. typographus specimens used for total DNA extraction was 10 mg per extraction. The calculated average amount with the standard deviation of total DNA extracted per gram of input Table 1: List of protocols and commercial kits used for total DNA extraction Preglednica 1: Seznam postopkov in komercialnih kitov, ki so bili uporabljeni za ekstrakcijo celokupne DNA Protocol name Kit and protocol used P1 Nucleospin DNA Insect Kit (Macherey Nagel, Düren, Germany) P2 DNeasy Blood and Tissue Kit (Qiagen, Venlo, Netherlands) P3 Qiagen QIAamp DNA Micro Kit (Qiagen, Venlo, Netherlands) without carrier RNA in the binding buffer P4 Qiagen QIAamp DNA Micro Kit (Qiagen, Venlo, Netherlands) with carrier RNA in the binding buffer Table 2: Primers and conditions used in the study (Folmer et al., 1994; EPPO, 2021; Becker et al., 2021) Preglednica 2: Začetni oligonukleotidi in pogoji, uporabljeni v raziskavi (Folmer et al., 1994; EPPO, 2021; Becker et al., 2021) Primers Primer sequence (5’-3’) Annealing temperature Expected amplicon length LCO1490 HCO2198 GGTCAACAAATCATAAAGATATTGG TAAACTTCAGGGTGACCAAAAAATCA 45°C (5 cycles) + 51°C (25 cycles) 709 bp Ips_F_cons Ips_R_cons CAAATATTGCCCATGAAGGAAC GCATCTGGATAATCTGAGTAACGTCG 55°C (35 cycles) 936 bp 32 De v etak Z., Ka v č ič A., de Gr oo t M., Pišk ur B.: The infl uenc e o f st or a ge c ond i t ions and dna ex tr a ct ion pr o t oc ol on the ... specimen was 191 ± 39 µg from frozen and 314 ± 80 µg from frozen in ethanol with the P1 extraction pro- tocol, 414 ± 112 µg from frozen and 618 ± 518 µg from frozen in ethanol with the P2 extraction protocol, 175 ± 50 µg from frozen and 148 ± 28 µg from frozen in ethanol with the P3 extraction protocol, and 405 ± 89 µg from frozen and 376 ± 4 µg from frozen in ethanol with the P4 extraction protocol (Table 3). It was demonstrated that there was a significant dif- ference between the amount of DNA extracted per gram of input specimen among the three assessed protocols, i.e., P1, P2, and P3 (Table 4). After conducting the Dunn post-hoc test, we found that only the use of P2 and P3 produced significantly different amounts of total DNA (Fig. 1, Table 4). The minimum/maximum amount of DNA produced from one gram of input specimen by P2 was 285 µg/480 µg for the frozen storage protocol and 268 µg/1213 µg for the samples frozen in ethanol. The P3 extraction protocol produced a minimum of 127 µg and a maximum of 226 µg of DNA per one gram of the frozen samples and a minimum of 115 µg and a maxi- mum of 167 µg of DNA per one gram of the samples frozen in ethanol. The P1 protocol was not indicated as producing significantly different amounts compared to both the P2 and P3 protocols (Fig. 1, Table 4). Using the Wilcoxon rank-sum test with continu- ity correction, no significant difference in the amount of DNA extracted between the storage protocols was found (df = 1, W = 35, p = 0.667). Using LCO1490/HCO2198 primers on the total DNA extracts from I. typographus specimens did not produce any amplicons with most of the samples used. Only two out of 24 DNA extracts tested provided use- ful sequences, with both obtained consensus sequenc- es belonging to parasitoid nematodes from the order Rhabditidae (results not shown). PCR using primers Ips_F_cons/Ips_R_cons were more successful, as most of the protocols we tested produced amplicons of expected size around 936 bp (Fig. 2). However, only amplicons produced using pro- tocols P1 and P2 were of sufficient quantity and quality to enable successful sequencing (Table 5). 4 DISCUSSION AND CONCLUSIONS 4 RAZPRAVA IN ZAKLJUČKI Our study confirmed that the choice of specimen storage method and total DNA extraction protocol can significantly affect the outcome of downstream mo- lecular processes in the molecular identification of Ips typographus. When analyzing the amount of DNA ex- tracted per gram of input material with each extraction protocol, the only significant difference was found be- tween the P2 and P3 protocols (Fig. 1, Table 4), while the amount of DNA extracted per gram of input mate- rial was not significantly affected by the different stor- age methods (Fig. 1). PCR using universal arthropod Table 3: Average amount and the standard deviation of to- tal DNA (µg) extracted from one gram of specimen with the different extraction protocols and storage conditions. P1: Nucleospin DNA Insect Kit (Macherey Nagel, Düren, Germa- ny); P2: DNeasy Blood and Tissue Kit (Qiagen, Venlo, Neth- erlands); P3: Qiagen QIAamp DNA Micro Kit (Qiagen, Venlo, Netherlands) without carrier RNA in the binding buffer; P4: Qiagen QIAamp DNA Micro Kit (Qiagen, Venlo, Netherlands) with carrier RNA in the binding buffer. Preglednica 3: Povprečna količina in standardni odklon ce- lokupne DNA (µg), pridobljene iz enega grama osebka glede na uporabljen postopek in način shranjevanja. P1: Nucleos- pin DNA Insect Kit (Macherey Nagel, Düren, Nemčija); P2: DNeasy Blood and Tissue Kit (Qiagen, Venlo, Nizozemska); P3: Qiagen QIAamp DNA Micro Kit (Qiagen, Venlo, Nizozem- ska) brez nosilne RNA v pufru za vezavo; P4: Qiagen QIAamp DNA Micro Kit (Qiagen, Venlo, Nizozemska) z nosilno RNA v pufru za vezavo. Storage protocol Total DNA extraction protocol P1 P2 P3 P4 Frozen 191 (± 39) 414 (± 112) 175 (± 50) 405 (± 89) Frozen in EtOH 314 (± 80) 618 (± 518) 148 (± 28) 376 (± 4) Table 4: Results of the Kruskal-Wallis and the Dunn post-hoc tests for comparing the amount of DNA extracted per gram of specimen with the extraction protocols P1, P2, and P3 Preglednica 4: Rezultati Kruskal Wallis-ovega in Dunn-ove- ga post-hoc testa za primerjavo količine ekstrahirane DNA iz enega grama osebka s postopki P1, P2 in P3 Kruskal Wallis test Dunn post-hoc test Chi square ( χ 2 ) Degrees of freedom P value Comparison P value 10.89 2 0.004 P1-P2 0.251 P1-P3 0.351 P2-P3 0.003 Acta Sil va e et Ligni 132 (2023), 29–38 33 primers LCO1490/2198 was not successful in amplify- ing I. typographus DNA. The use of Ips_F_cons/Ips_R_ cons primers enabled successful amplification across all storage methods and extraction protocols (Fig. 2), although gel bands produced by extracts from extrac- tion protocols P3 and P4 were only slightly visible, and only extraction protocols P1 and P2 enabled successful sequencing of the products (Table 5). Fig. 1: Amount of total DNA (µg) extracted per gram of speci- men by a) extraction protocol and b) storage method used Slika 1: Količina ekstrahirane celokupne DNA (µg) iz enega grama osebka glede na a) ekstrakcijski postopek in b) način shranjevanja Fig. 2: Amplicons from the PCR using Ips_F_cons/Ips_R_cons after separation (100 bp ladders used) Slika 2: Pomnožki, pridobljeni v polimerazni verižni reakciji z začetnima oligonukleotidoma Ips_F_cons/Ips_R_cons po ločevanju na gelu (uporabljena je bila lestvica 100 bp) 34 De v etak Z., Ka v č ič A., de Gr oo t M., Pišk ur B.: The infl uenc e o f st or a ge c ond i t ions and dna ex tr a ct ion pr o t oc ol on the ... When choosing a storage protocol for arthropod specimens, one of the factors that should be consid- ered is the usability of the samples for downstream processing. For specimens intended for morphologi- cal identification, dry freezing may not be suitable for long-term storage, as over time they may become too dehydrated to remain useful for morphological exami- nation (Whitman et al., 2019). However, for specimens intended for use in downstream molecular processes, one of the key considerations is the preservation of DNA quality. While storage in 96% ethanol may also be problematic for morphological applications (Quicke et al., 1999), it is generally considered suitable for use with specimens intended for downstream molecular applications, provided that the ethanol used is non- methylated (Whitman et al., 2019). While our data indicates no difference in the amount of DNA extracted per gram of input material between the two different storage protocols, there is evidence of a difference in the rate of DNA degradation between the two protocols. In Fig. 2, there is a clear vis- ual difference between the bands produced by extracts from specimens that were frozen dry and those frozen in ethanol. Since the extracts used were normalized to a concentration of 10 ng/µl, the difference in bands was not due to the starting amount of DNA but to its suitability for use in PCR. The difference in in DNA deg- radation between the two storage protocols may be due to the fact that ethanol denatures proteins, includ- ing those with DNase activity (Flournoy et al., 1996; Stein et al., 2013). While this could be disadvantageous as it also denatures histones and other structural pro- teins, which might consequently make the DNA struc- turally inaccessible for extraction, we resolved this by choosing extraction protocols that include the use of a proteinase. Another possible reason for the differ- ence in degradation may be that ethanol also displaces water in tissues, thus preventing the water-mediated DNA degradation that would otherwise occur during dry storage, where water that is inherently present remains in the cells (Stein et al., 2013). Ice crystals, which can form when water is present in the cells dur- ing freezing, further exacerbate this degradation ef- fect. The storage protocol without ethanol produced only two readable sequences in our results, compared to five produced by specimens frozen in ethanol. In all seven cases, the consensus sequences were sufficiently identical to the reference sequence to enable identifi- cation of the specimens. The amplicon produced by the Ips_F_cons and Ips_R_cons primers is relatively long, Table 5: Results of Sanger sequencing and subsequent chromatogram analysis of Ips typographus specimens using Ips_F_cons/Ips_R_cons primers (F – frozen, F+EtOH – frozen in ethanol), including the GenBank Accession Numbers of the obtained sequences Preglednica 5: Rezultati sekvenciranja osebkov Ips ty- pographus po Sangerju z uporabo začetnih oligonukleotidov Ips_F_cons/Ips_R_cons in sledeče analize kromatogramov (F – zamrznjen, F+EtOH – zamrznjen v etanolu), vključno z Gen- Bank akcesijskimi številkami pridobljenih sekvenc Extraction protocol Storage protocol PCR product con- centration [µg/ml] Consensus sequence Length (trimmed) %HQ % Identity to ref. sequence (MK315170) GenBank Accessi- on Number P1 F 66.8 no - - - - P1 F 68.6 yes 889 94.0 99.8 OR458594 P1 F 60.9 yes 889 97.6 99.9 OR458590 P1 F + EtOH 80.2 yes 885 98.4 99.8 OR458592 P1 F + EtOH 81.3 yes 878 96.2 99.9 OR458591 P1 F + EtOH 83.2 yes 851 91.2 99.9 OR458595 P2 F 59.7 no - - - - P2 F 73.1 no - - - - P2 F 64.1 no - - - - P2 F + EtOH 64.2 no 769 81.0 99.7 OR458593 P2 F + EtOH 83.7 yes 889 90.2 99.7 OR458589 P2 F + EtOH 61.6 no - - - - P3 F 64.3 no - - - - P3 F + EtOH 60.7 no - - - - P3 F + EtOH 59.8 no - - - - P4 F + EtOH 74.3 no - - - - P4 F + EtOH 60.4 no - - - - P4 F + EtOH 65.6 no - - - - Acta Sil va e et Ligni 132 (2023), 29–38 35 at 936 bp. If the specimens were intended for down- stream applications using primers that produced am- plicons of shorter length, the effect of storage should be even less pronounced. During our study, DNA belonging to endoparasites was also amplified in the tested samples. It is known that species of the Ips genus often host endopara- sites (Hoffard and Coster, 1976), which are also in- cluded in the broad target spectrum of the general LCO1490/HCO2198 primers. However, host-specific primers are more useful for molecular identification in the presence of endoparasites. As previously pro- posed (Becker et al., 2021), the results demonstrate the limitations of using universal primer pairs such as LCO1490/HCO2198 with some arthropod samples. This approach might work for other Scolytinae taxa without the problems of non-target amplification, as demonstrated by Marinč et al. (2019). In their study, five kits for total DNA extraction on beetles of the ge- nus Ambrosiodmus were compared, including DNeasy Blood and Tissue and Nucleospin DNA Insect, which were also used in our study. The authors found these two kits to be the most useful of the five kits tested in terms of DNA yield for the DNeasy Blood and Tissue Kit, and time- and cost- efficiency for the Nucleospin DNA Insect Kit. Both kits also performed well in our comparisons, although the amount of DNA extracted was not significantly different between the two kits. The DNA extracted with the Nucleospin DNA Insect Kit proved to be more useful for sequencing applica- tions (Table 5). In conclusion, both kits performed well in the PCR on different Scolytinae taxa in both studies, although our results indicate the importance of verify- ing the kits for sequencing applications, as sequencing was not equally successful using extracts from differ- ent kits. Protocols P3 and P4 both include overnight lysis at 56°C, which may also contribute to DNA degradation, explaining why the PCR using extracts from these two protocols was less successful, yielding only slightly vis- ible bands (Fig. 2) and no useful sequencing results (Table 5). However, Rohland (2007) proposed, based on ancient DNA, that the increased DNA degradation and reduced proteinase K activity over time in longer lysis steps are compensated for by the greater DNA re- lease over time. In our study, the volumes of elution buffers were standardized across the protocols (100 µl). As the amount of starting material was quite low (average weight of 10 mg per I. typographus specimen), reduc- ing the volume of elution buffer and multiple elution steps with the same buffer could be explored in further studies, as this may improve the amount of DNA eluted. As the chosen kits were all based on silica col- umn extraction with a similar chemical background, further research should be performed to include dif- ferent types of extraction methods, such as solution- based CTAB (Saghai-Maroof et al., 1984; Doyle and Doyle, 1987) and phenol-chloroform (Chomczynski and Sacchi, 1987) extractions or resin-based Chelex extractions (Walsh et al., 1991). Additionally, manual or automatic extraction methods based on magnetic or paramagnetic beads (Alderton et al., 1992) should be tested. Additionally, including more samples for each treatment would make the results even more statisti- cally robust. The choice of protocols for specimen storage prior to extraction is important and should be tailored to the intended downstream applications. Based on our re- sults, freezing specimens in ethanol produced similar amounts of DNA as dry freezing, although the former proved more suitable for sequencing applications. Similarly, the choice of commercially available extrac- tion kits and protocols should be based on evidence demonstrating their suitability for the specific type of samples and downstream applications, as different protocols do not produce similar results on the same type of samples. Of the four DNA extraction protocols tested, Protocol 1 using the Nucleospin DNA Insect Kit (Macherey Nagel, Düren, Germany) proved to be the most useful for relatively small arthropod sam- ples intended for PCR and sequencing applications. As observed, different extraction protocols produce DNA extracts of varying quality in terms of their usefulness for sequencing applications. Molecular methods are gaining popularity for the identification of existing and emerging forest pest species, as the time component is crucial, especially in the early detection of invasive species. Because such methods rely on obtaining a sufficient quantity and quality of the target DNA, this work represents a crucial step in the process of en- suring reliable results and thus improving the tools needed to protect our forests from current and future biological threats. 5 SUMMARY 5 POVZETEK Podlubniki (Coleoptera: Curculionidae: Scolytinae) kot promotorji kroženja organske snovi igrajo ključno vlogo v gozdnih ekosistemih. Nekatere vrste lahko po izjemnih podnebnih dogodkih povzročijo tudi veliko okoljsko in gmotno škodo. Navadna smreka (Picea abies, (L.) H. Karst.) je ena izmed ekonomsko najbolj pomembnih drevesnih vrst v Evropi. Zaradi pretek- 36 De v etak Z., Ka v č ič A., de Gr oo t M., Pišk ur B.: The infl uenc e o f st or a ge c ond i t ions and dna ex tr a ct ion pr o t oc ol on the ... lega sajenja na območja, manj primerna za njeno rast, je lahko dodatno dovzetna za izbruhe osmerozobega smrekovega lubadarja (Ips typographus, L.). Ob pojavu izbruhov te in drugih vrst škodljivih organizmov je identifikacija povzročitelja pomembna za načrtovanje omejitve izbruha in sanacije. Prav tako je ključnega pomena za zgodnjo detekcijo invazivnih tujerodnih vrst, kar omogoči zmanjšanje ali celo prepreči škodo, ki bi lahko nastala zaradi neomejenega širjenja na nova območja. Molekularne metode so tu odlična podpora tradicionalnim morfološkim identifikacijskim meto- dam, saj lahko omogočijo identifikacijo neodvisno od razvojnega stadija in celo na poškodovanih osebkih ali v sledovih. Shranjevanje vzorcev pred molekularno analizo in izbira ekstrakcijskega postopka lahko vplivata na količino in kakovost pridobljene DNA. V raziskavi smo primerjali dva načina shranjevanja primerkov I. ty- pographus, suho zamrzovanje in zamrzovanje v 96 % etanolu. V primerjavo ekstrakcijskih postopkov pa smo vključili štiri različne postopke na osnovi komercialno dostopnih ekstrakcijskih kitov dveh različnih proizva- jalcev. P1 - Nucleospin DNA Insect Kit (Macherey Nagel, Düren, Nemčija); P2 - DNeasy Blood and Tissue Kit (Qiagen, Venlo, Nizozemska); P3 - Qiagen QIAamp DNA Micro Kit (Qiagen, Venlo, Nizozemska) brez nosilne RNA v pufru za vezavo; P4 - Qiagen QIAamp DNA Mi- cro Kit (Qiagen, Venlo, Nizozemska) z nosilno RNA v pufru za vezavo. Ekstrakcije celokupne DNA smo z navedenimi kiti opravili po navodilih proizvajalcev. Z UV spektrofotometrom smo izmerili koncentracijo in izračunali končno količino pridobljene DNA, ki smo jo nato normalizirali glede na vhodno maso osebka, iz katere je bila pridobljena. Kvaliteto DNA in njeno upo- rabnost za molekularno identifikacijo pa smo ocenili s pomočjo polimerazne verižne reakcije in sekvenciran- ja po Sangerju z dvema različnima paroma začetnih oligonukleotidov. DNA smo ocenili kot uporabno za molekularno identifikacijo, če je v polimerazni verižni reakciji dala pomnožke, s pomočjo katerih smo s sek- venciranjem prišli do kromatogramov, ki so bili dovolj kakovostni za zanesljivo identifikacijo tarčnega oseb- ka s pomočjo primerjave z referenčno sekvenco. DNA smo pridobili v 24 postopkih iz posameznih osebkov I. typographus, po 12 na postopek shranjevanja in po 3 na postopek ekstrakcije celokupne DNA. Iz statistične analize količine ekstrahirane DNA smo zaradi nosilne RNA v ekstraktu in narave kvantifikacije izločili pro- tokol P4. Kljub temu, da se količina pridobljene DNA na gram vhodnega materiala med obema postopkoma shranjevanja ni statistično razlikovala, pa je bila raz- lika v uporabnosti DNA za PCR očitna iz vizualne ocene pomnožkov na gelu. Enaka vhodna količina DNA je namreč v isti polimerazni verižni reakciji dala na vi- dez očitno bolj svetle pomnožke pri postopku shran- jevanja v etanolu kot pri postopku shranjevanja brez etanola. Ekstrakti, pridobljeni iz osebkov, shranjenih v etanolu, so na gelu dali pomnožke z večjo intenziteto kot tisti, ki so bili rezultat suhega zamrzovanja. Raz- lika v kvaliteti DNA je bila lahko rezultat ohranitvene vloge etanola, ki v tkivih zamenja vodo in tako prepreči škodo, ki bi drugače nastala ob prisotnosti vode. Poleg tega pa etanol denaturira proteine, vključno s tistimi, ki imajo sposobnost razreza ali drugačnega vpliva na DNA. Statistična primerjava postopkov ekstrakcije ce- lokupne DNA pa je pokazala, da se pridobljena količina DNA na gram vhodnega materiala statistično razlikuje le med protokoloma P2 in P3. Prvi preizkušeni par začetnih oligonukleotidov, LCO1490/HCO2198, se ni izkazal za uporabnega, saj smo uporabne sekvence pridobili le pri dveh od 24 ekstraktov, le-te pa so pri- padale parazitskim ogorčicam iz reda Rhabditidae. Ta oligonukleotidni par je bil predhodno že uspešno uporabljen pri drugih vrstah podlubnikov, kar dodatno nakazuje na to, da je izbiro postopkov treba prilagoditi tarčni taksonomski enoti. Bolj uspešna je bila polime- razna verižna reakcija z začetnima oligonukleotidoma Ips_F_cons/Ips_R_cons, saj smo pri večini preizkušenih postopkov pridobili pomnožke pričakovane dolžine okoli 936 baznih parov. Kljub temu so bili za nadaljnje sekvenciranje uporabni le pomnožki, pridobljeni s pro- tokoloma P1 in P2. Med raziskavo smo potrdili, da lahko izbira postop- kov shranjevanja in ekstrakcije celokupne DNA občutno vpliva na izid molekularnih postopkov za identifikaci- jo I. typographus. Izbor postopkov mora posledično sloneti na eksperimentalnih podatkih za načrtovani tip vzorcev in vrste molekularne analize. V naši raziskavi se je za optimalno izkazala uporaba kombinacije shranjevanja v etanolu in ekstrakcijskega postopka P1, pri katerem smo uporabili ekstrakcijski kit Nucleospin DNA Insect Kit nemškega proizvajalca Macherey Nagel. Ker molekularne metode pridobivajo pri pomembnos- ti v hitri identifikaciji že obstoječih in novo vnesenih vrst gozdnih škodljivcev, je naša raziskava eden izmed ključnih korakov pri zagotavljanju zanesljivosti rezul- tatov. To pa nam bo v prihodnosti omogočilo širjenje nabora orodij, s pomočjo katerih ščitimo naše gozdove pred obstoječimi in še prihajajočimi škodljivci. Acta Sil va e et Ligni 132 (2023), 29–38 37 ACKNOWLEDGEMENTS ZAHVALA The authors acknowledge that the study was sup- ported by Research Programme P4-0107, which is financed through the Slovenian Research and Innova- tion Agency and by the Administration of the Repub- lic of Slovenia for Food Safety, Veterinary Sector and Plant Protection (contract No. C2337-21-000016 and C2337-22-000020). 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