Maintaining DNA quality in stored-grain beetles caught in Lindgren funnel traps

Stevens, Mark M.; Warren, Glen N.; Mo, Jianchu; Schlipalius, David I.

doi:10.1016/j.jspr.2010.10.002

Cited by 13 publications

(8 citation statements)

References 23 publications

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“…Fragmentation of DNA after immersion in propylene glycol did not differ between samples that were dehydrated and those that were not dehydrated. Generally, dehydration by substitution is recommended, because dilution by water from insect tissues can increase the risk of fragmenting DNA (Quicke et al, 1999;Stevens et al, 2011;Ferro et al, 2013). In this study, no serious fragmentation of DNA was recorded because only small amounts of tissue were preserved in the tubes.…”

Section: Discussionmentioning

confidence: 71%

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Methods for retaining well-preserved DNA with dried specimens of insects

Nakahama¹,

Isagi²,

Itô³

2019

Eur. J. Entomol.

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To prevent degradation, DNA samples are generally preserved in 95-99.5% ethanol or acetone, or in a freezer, or a combination of these (preserved in 95-99.5% ethanol or acetone and stored in a freezer at −20°C to −80°C) because this prevents DNA degradation (Reiss et al., 1995;Quicke et al., 1999;Vink et al., 2005;Nasu et al., 2016). However, the maintenance and space costs associated with immersed or frozen specimens are greater than those associated with dried specimens. The preservation of frozen specimens requires large freezers, which are expensive and may have limited space for specimen preservation and immersed specimens must be regularly checked to ensure that the stock solution has not evaporated. In addition, morphological observations and dissections of 99% ethanol-immersed specimens are diffi cult due to dehydration (Naem et al., 2010). To overcome these problems, we tested whether DNA in the legs of dried specimens of insects can be preserved for a long time. To this end we suspended insects on a pin in 0.2-ml tubes with only the legs immersed in the preservation solution. We also considered the methods used for killing insects, because apart from dragonfl ies Abstract. Dried specimens of insects are increasingly seen as genetic resources. However, genetic analysis of dried specimens of insects is hampered by the deterioration of the DNA. In this study, we developed methods for preparing dried specimens of insects with well-preserved DNA, mainly for PCR-based genetic analysis. First, we compared the effects of either exposure to ethyl acetate vapour for from 10 min to 6 h or by freezing on the fragmentation of DNA in order to determine optimal length of time needed for killing insects using the above methods. Second, we compared the fragmentation of DNA after preservation by drying or immersion of legs in 99.5% ethanol or 99% propylene glycol in 0.2-ml tubes. We assessed degrees of fragmentation of DNA by determining polymerase chain reaction (PCR) success rates with primers for 313-, 710-and 1555-bp fragments using DNA that was collected immediately, and at one, six and 12 months after preparing the specimens. Differing times taken to kill insects did not affect the fragmentation of DNA. In dried specimens, DNA was seriously fragmented after one month, whereas that in legs prepared by immersion in 99.5% ethanol or 99% propylene glycol contained long fragments of DNA (1555 bp~) after 12 months. Propylene glycol was more suitable for preservation than ethanol, because the latter evaporates. Thus, to preserve insect DNA we suggest inserting the pin on which an insect is impaled into the hinged lid of a 0.2-ml tube containing 99% propylene glycol so that when the lid is closed the legs of the insect are preserved in the solution.

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Section: Discussionmentioning

confidence: 71%

“…Comparisons of those of methods (8) and (9) revealed the effects of dehydration on fragmentation of DNA in tissues. Dilution of preservative solutions by the water from insect tissues reportedly promotes DNA fragmentation (Quicke et al, 1999;Stevens et al, 2011;Ferro et al, 2013). Each method was carried out using 12 samples.…”

Section: Introductionmentioning

confidence: 99%

Methods for retaining well-preserved DNA with dried specimens of insects

Nakahama¹,

Isagi²,

Itô³

2019

Eur. J. Entomol.

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“…3 days (RT) evaporation step prior to DNA extraction was ommitted as PG does not inhibit PCR; communities are highly similar and even showed higher proportion of arthropod reads and higher richness than samples conserved with >99% lab grade ethanol not to be underestimated amount of water and other compounds (Nagy 2010;Goetze and Jungbluth 2013;Borges et al 2016), but chemical interactions and their effects on medium-to long-term DNA integrity are largely unknown. In addition to high temperatures, water dilution of PG in principle leads to a generally lower DNA integrity (Stevens et al 2011;Höfer et al 2015;Patrick et al 2016;Nakamura et al 2020), since water can lead to the hydrolysis of nucleic acids. Since most reviewed studies did not indicate the nature of the propylene glycol used, or field and storage temperatures, it was difficult to compare study outcomes.…”

Section: Studymentioning

confidence: 99%

Application of propylene glycol in DNA-based studies of invertebrates

Weigand¹,

Desquiotz²,

Weigand³

et al. 2021

MBMG

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High-throughput sequencing (HTS) studies on invertebrates commonly use ethanol as the main sample fixative (upon collection) and preservative (for storage and curation). However, alternative agents exists, which should not be automatically neglected when studies are newly designed. This review provides an overview of the application of propylene glycol (PG) in DNA-based studies of invertebrates, thus to stimulate an evidence-based discussion. The use of PG in DNA-based studies of invertebrates is still limited (n = 79), but a steady increase has been visible since 2011. Most studies used PG as a fixative for passive trapping (73%) and performed Sanger sequencing (66%; e.g. DNA barcoding). More recently, HTS setups joined the field (11%). Terrestrial Coleoptera (30%) and Diptera (20%) were the most studied groups. Very often, information on the grade of PG used (75%) or storage conditions (duration, temperature) were lacking. This rendered direct comparisons of study results difficult, and highlight the need for further systematic studies on these subjects. When compared to absolute ethanol, PG can be more widely and cheaply acquired (e.g. as an antifreeze, 13% of studies). It also enables longer trapping intervals, being especially relevant at remote or hard-to-reach places. Shipping of PG-conserved samples is regarded as risk-free and is authorised, pinpointing its potential for larger trapping programs or citizen science projects. Its property to retain flexibility of morphological characters as well as to lead to a reduced shrinkage effect was especially appraised by integrative study designs. Finally, the so far limited application of PG in the context of HTS showed promising results for short read amplicon sequencing and reduced representation methods. Knowledge of the influence of PG fixation and storage for long(er) read HTS setups is currently unavailable. Given our review results and taking difficulties of direct methodological comparisons into account, future DNA-based studies of invertebrates should on a case-by-case basis critically scrutinise if the application of PG in their anticipated study design can be of benefit.

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“…Some studies have reported that propylene glycol could be an effective alternative to ethanol in terms of DNA preservability (Nagy, ; Ferro & Park, ; Moreau et al, ). However, there has been insufficient evaluation of DNA preservability in propylene glycol, with a lack of cross‐taxonomic comparisons (Nagy, ; Stevens et al, ; Moreau et al, ).…”

Section: Introductionmentioning

confidence: 99%

Propylene glycol: a promising preservative for insects, comparable to ethanol, from trapping to DNA analysis

Nakamura

Tamura

Taki

et al. 2020

Entomologia Exp Applicata

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Recent advances in DNA analysis allow us to identify an unprecedented number of insect samples collected by mass sampling techniques such as insect traps. In these circumstances, a preservative that can be applied from trap to storage is necessary to prevent degradation of DNA before analysis and save on the cost of labor for collecting insects from traps. Propylene glycol has a prominent feature as a trap solution. We aimed to examine the DNA preservability of 98% propylene glycol at 2 weeks and more than 6 months after initial collection in comparison with 99.5% ethanol, which is commonly used for storage of specimens for genetic analysis. We compared amplification performance of PCR targeting a specific region of the mitochondrial cytochrome c oxidase subunit I (COI) gene in the orders Hymenoptera, Diptera, and Coleoptera using two extraction methods varying in extraction efficiency. Even after 6 months, more than 75% of samples were recognized to have succeeded in PCR amplification irrespective of preservatives by the extraction method with higher extraction efficiency. It suggested that mitochondrial DNA was preserved in both solutions. However, dim bands in the electrophoreses of PCR products increased with time in extracts by another method with lower extraction efficiency. In Diptera and Coleoptera, the rate of dim bands increased more rapidly for ethanol‐preserved than for propylene glycol‐preserved specimens, indicating higher DNA preservability of propylene glycol over time for these taxa. On the other hand, in Hymenoptera, the preservatives did not affect PCR amplification performance. Considering its safer characteristics and high DNA preservability in a wide range of taxa, propylene glycol can be a promising solution from trapping of insects to storage for genetic analysis.

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Maintaining DNA quality in stored-grain beetles caught in Lindgren funnel traps

Cited by 13 publications

References 23 publications

Methods for retaining well-preserved DNA with dried specimens of insects

Methods for retaining well-preserved DNA with dried specimens of insects

Application of propylene glycol in DNA-based studies of invertebrates

Propylene glycol: a promising preservative for insects, comparable to ethanol, from trapping to DNA analysis

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