Abstract: Cryopreservation is a practical strategy for conserving and managing invertebrate diversity. Degraded nucleic acids present the single greatest obstacle to successful molecular research programs. Since storage at cryogenic temperatures essentially stops biological degradation, it is likely that long-term cryopreservation will be a suitable method for storing tissues for current scientific applications and for future genomic studies. Although some damage may occur, for most genetic techniques cryopreservation is a valid option for long-term tissue storage. Other commonly used tissue preservation methods, such as storage in ethanol, are increasingly revealed to be inadequate even for present applications. A study undertaken by the Ambrose Monell Collection for Molecular and Microbial Research compared the quality of DNA isolates from Drosophila melanogaster frozen under various conditions. Samples were frozen with 6 cryoprotectants in combination with 4 freeze/thaw strategies. The Comet Assay was used to visualize the extent of DNA fragmentation. Spot applications of insect DNA are subjected to gel electrophoresis and a comet-like pattern of fragment migration is observed. The longer the tail, the greater the DNA damage. Results showed that DMSO, glycerol, and ethylene glycol were better than ethanol in protecting DNA from freeze/thaw damage. The slow freeze/slow thaw method caused the least amount of DNA damage with all cryoprotectants. Slow freezing with no cryoprotectant was found to be the best method for preserving DNA. The study was expanded to include a comparison of the effects of various combinations of temperatures and storage media on the preservation of DNA in beetles, which resist permeation by cryoprotectants. Samples were stored in buffer or ethanol, dry, or pretreated with a commercial preservative, RNAlater (Qiagen, Inc.), and frozen at -20oC and -160oC. DNA extractions were subjected to gel electrophoresis, and DNA was qualified and quantified. This study demonstrates the critical nature of specimen storage for biomolecule preservation.

FREEZING INSECTS:The effects of cryopreservation protocols on nucleic acid degradation.

Alva Wright, Joann Mercedes, Miriam Delarosa, J. Feinstein, A. Corthals Ambrose Monell Cryo-Collection, American Museum of Natural History, 79th Street at Central Park West, New

York, NY, 10024, USA (AMNH)

Lab Work and Facilities

Briefly, in the comet assay individual cell are lysed and their DNA unwounded. The DNA is then subjected to a gel electrophoresis procedure and this results in a “comet like” figure. This is due to the migration of (negatively charged) DNA fragments or DNA loops towards the positive pole. DNA “comets” can be visualized with a fluorescent microscope after staining with a fluorochrome (e.g. ethidium bromide). The tail length, tail DNA content or other parameters may be used to measure the extent of DNA damage.

Comet assay protocol

Comet assay visual results showing from the top left corner no DNA damage to high DNA damage

In the summer of 2002 an experimental study was undertaken at the Ambrose Monell Collection to compare the quality of DNA isolated from samples of a line of Drosophila melanogaster. Samples were frozen cryogenically using 6 cryoprotectants: 1.5 M DMSO, 1.5 M glycerol, 1.2 M ethylene glycol, 100% ethanol, 70% ethanol, and dry freezing (dry freezing was counted as a cryoprotectant). The cryoprotectants were tested using 4 freeze/thaw methods: fast freeze/fast thaw, slow freeze/slow thaw, fast freeze/slow thaw, and slow freeze/fast thaw. The Comet Assay was used to visualize the extent of DNA fragmentation. The longer the tail of the comet the greater the DNA damage. The results showed that DMSO, glycerol, and ethylene glycol were found to be better than ethanol. The slow freeze and slow thaw method worked the best for all of the cryoprotectants. However, slow freezing using no cryoprotectant was found to have the highest mean of DNA preservation.

Beetle and fly juice

Results from 2002 (B. Webster) The results of the assay are presented in Figure 1 with the average tail moment for each CP. Samples subjected to the fast freeze/fast thaw protocol demonstrated an average tail moment that was significantly lower (P<0.002) than the average tail moment of samples subjected to the slow freeze/slow thaw protocol. Samples that were cryopreserved in permeating and "Neither" CPs (Table 1), using a fast freeze/fast thaw protocol had an average tail moment that was significantly (P<0.01,0.0001 respectively; Student's t test) lower than the tail moment for the same samples subjected to a slow freeze/slow thaw protocol (Fig 1). There was no significant difference in average tail moment in samples cryopreserved in nonpermeating CPs (Table 2) using the fast freeze/fast thaw protocol or average tail moments of the same samples cryopreserved using the slow freeze/slow thaw protocol. The data appear to mirror the general result of other studies of cryopreservation in that control tissue exhibited significantly lower DNA damage than the experimental samples (Fig 2). This is indicated by the fact that control tissue had a lower average tail moment than the average tail moments of the cryopreserved samples (P<0.001; Student's t Test). This is expected given the highly damaging effects of freezing on cells. Permeating CPs apparently performed best in the preservation of DNA during cryopreservation, as measured by the comet assay (Figure 1). The highest-performing treatments included cryopreservation of samples in 100 % acetone and 1.2M ethylene glycol, as well as the use of a "Neither" CP, the dessication pretreatment by acetone vapor drying. Significantly lower average tail moments were observed for these samples as compared to the mean value for all cryopreserved samples (P<0.01; Student's t Test). Cryopreservation in 1.5M DMSO also performed highly but its departure from the average treatment did not reach the P<0.003 threshold. As permeating intracellular CPs, these compounds also act to dehydrate cells and replace water in solvating membranes and proteins. The dehydration of cells may prevent damaging intracellular ice formation or contribute to vitrification of the cytosol if the concentration of the CP is high enough. The replacement of water may stabilize proteins and lipids during the harsh conditions of cryopreservation. It is unclear from the data which of these properties (dehydration or water replacement) is most important in exerting cryoprotective effects in preventing DNA strand lesion and breakage formation. The nonpermeating CPs showed a lesser degree of DNA protection. Although D-(+)-trehalose dihydrate is capable of dehydrating cells, it did not demonstrate a significant level of cryoprotection. Saline/chelating solutions similarly did not afford a significant degree of cryoprotection as measured by comet assay, including RNAlater, AMCC lysis buffer, and lithium formate monohydrate. Calf serum bovine, a complex mixture of mammalian biological components, also failed to adequately protect samples in the comet assay. Vitrification DMSO solution performed worse than other solutions with lower concentrations of permeating CPs.

Fruit flies (in this case, Drosophila heteroneura)

Modern host harvesting techniques.

Tenebrio Results

Results show that field samples left in ethanol at room temperature (3) in an environment similar to that encountered during field collection trips (i.e. left for 10 days at room temp.), yield very low quality (and molecular weight) DNA compared to samples frozen immediately in LN2 (1) and samples extracted right away after death. It is interesting to note as well that samples frozen immediately in LN2 (1) yield virtually the same quality DNA than that of the 'fresh kill' sample (2)

Electrophoresis gel showing the extracted Tenebrio DNA from 1: immediately frozen in Liquid Nitrogen

2: Fresh kill3: ETOH preserved at room temperature

- It was noted that the use of complete beetle did not yield good DNA results. We therefore advocate to use the legs, rather than the whole body. (Abdomen yields good DNA too, but could compromise results because of bacterial contamination

- Future studies will be made with the comet assay on different field collection buffer, such as RNA later, lysis buffer and other new preservant on the market.

- Insofar as insects are concerned, the results of both studies have shown that freezing is the best method to recover the highest molecular weight DNA

Discussion and future research

Contact: The Ambrose Monell Cryo Collection

212 769 5663212 496 3389

Common mealworm beetle:Tenebrio molitor

1 2 3

The Ambrose Monell Cryo-Collection Facilities

Figure 1 Figure 2

Table 11. Storage technique2. Cryoprotectant3.Storage TimeFreezing and thaw protocols