Joumal of the American Mosquito Control Association, 14(1):58-60, 1998Copyright @ 1998 by the American Mosquito Control Association, Inc.

PRESERVATION OF ANOPHELINE MOSQUITOES FORDNA PROBE ANALYSIS

R. D. COOPER

Army Malaria Institute, Gallipoli Barracks, ENOGGERA, Queensland 4052, Australia

ABSTRACT, Anopheles (Cellia) farauti Laveran sensu stricto adults were subjected to a range of conditionssimilar to those experienced by trap-collected mosquitoes prior to processing. Squash blots of these specimenswere then analyzed using isotopic DNA probes. Freezing and thawing 6 times and dry storage on silica gel ina vacuum desiccator for up to 180 days did not affect the quality of the DNA. Exposure to conditions of highhumidity and high temperature for more than 2 h seriously reduced the quality of the DNA.

KEY WORDS Anopheline DNA, preservation, storage, DNA probe analysis

INTRODUCTION

The appearance of isomorphic species withinmany of the malaria vector taxa has necessitatedthe use of alternative techniques for species iden-tification, an essential prerequisite to any fieldstudy. Various techniques are available (Cooper etal. 1991); however, for the identification of largenumbers of field specimens, the most suitable meth-od we have found is that using squash blots andisotopic DNA probes. This method was applied tomembers of the Punctulatus Group collected fromPapua New Guinea (PNG) (Cooper et al. 1997) us-ing the encephalitis virus surveillance (EVS) lighttrap, a device similar to the CDC light trap but in-corporating CO, as bait (Rohe and Fall 1979). Al-though most specimens were positively identified,there was considerable variation in the strength ofthe signal, indicating that degradation of rhe DNAhad occured. With some specimens, the signal wasvery weak or nonexistent, and a positive identifi-cation could not be made.

The DNA in these specimens may have degradedafter collection. Because mosquito traps (EVS orCDC) are usually set before 1800 h and not re-trieved until 0600 h the following morning, thesemay contain specimens that died early in the eve-ning and have undergone decomposition for severalhours. Material collected during whole-night bitingcatches may suffer the same fate if the specimensare not preserved at intervals during the night.

DNA degradation may also occur during trans-port and storage of the specimens prior to process-ing. In the field, liquid nitrogen (LNr) or dry icemay not be available, and specimens may go fromwet ice to a domestic freezer (*2O"C) a number oftimes while in transit. Power supplies may also beunreliable and intermittent, allowing specimens tothaw out for various periods of time.

In this study, the degradation of mosquito DNA,following the death of the specimen, was examinedin mosquitoes exposed to high temperature and hu-midity, repeated freezing and thawing, and dry stor-age for long periods.

MATERIALS AND METHODS

Colony specimens of Anopheles farauti Laveransensu stricto (: An. farauti 1, one of 12 membersof the Punctulatus Group) were killed using chlo-roform and placed individually into Eppendorftubes to make 25 sets, with each set containing 5replicate tubes. Fifteen of these sets were main-tained at 26"C and 8O-85Vo RH. 2 sets were re-moved and frozen (-70"C) at l+-, th-, I-, 2-, 4-, 6-,8-, 10-, I2-, l4-,16-, 18-, 2O-,22-, and 24-h inter-vals. Three sets were stored in a silica gel chargeddesiccator under vacuum and removed and frozen(-70"C) after 30, 60, and 18O days. Five sers werefrozen at -70'C; each set was then brought to 26"C(taking approximately 5 min) then frozen again.This treatment was repeated, excluding a set eachtime, until the last set, at the time of processing,had been thawed and frozen 6 times. The remaining2 sets were frozen (-70oC) and used as controls.

Following the above treatments, all specimenswere squash blotted and probed using the An. far-auti s.s. genomic probe made by Cooper et al.(1991). The aMomens of the specimens wereplaced, 1 cm apart, onto a nylon membrane satu-rated with l07o SDS. Each abdomen was individ-ually squashed using a glass pestle and left for 1Omin. The DNA was then denatured in 0.5 M NaOH,1.5 M NaCl for 5 min; neutralized in 0.5 M Tlis,1.5 M NaCl buffer for 10 min; and then fixed tothe membrane by baking at 80"C for 2h. The mem-brane was then saturated with 0.1 M NaOH and anychitinous material was rubbed off. The membranewas rinsed in distilled water and prehybridized in40 ml of phosphate buffer (0.263 M Na"HPOo, 1.0mM EDTA tpH 8.01, 77o SDS, 17o bovine serumalbumin) for at least 2 h at 65"C. Ttre membranewas then hybridized using plasmid DNA containingthe An. farauti s.s. probe labeled by random prim-ing with 3'�P-dCTP The probe concentration was 10ng/ml of the phosphate buffer, and hybridizationwas run overnight at 65"C. The membrane was thenwashed in 0.1 X SSC, 0.17o SDS for 1 min at roomtemperature and then twice for 30 min at 65'C. Itwas then sealed in plastic wrap and exposed to au-

JoURNAL oF THE AMERTCAN Moseutro CoNrnol AssoclATIoN Vor-. 14, No. I

specimens were alive when the trap was retrievedin the morning.

Preservation methods have been studied for anumber of species of Diptera (Copeland et al. 1992,Post et al. 1,993, Stevens and Wall 1995). Theseworkers found that low-temperature storage (liquidnitrogen or -70'C) was the most suitable method,although good results were achieved with 1007o al-cohol and dry storage over silica gel. These studieswere conducted on extracted DNA and not squashblots. Cooper et al. (1991) showed that mosquitospecimens fixed in 707o isopropanol or ethanol orfrozen at -zO"C or -70"C were suitable for squashblot analysis. Therefore, if low-temperature storageis not available, alcohol or silica gel preservationwould be suitable for storage and shipping prior toprocessing.

If the problem of poor preservation involves onlya few specimens, then the more sensitive polymer-ase chain reaction-restriction fragment length poly-morphism (PCR-RFLP) analysis could be used.Beebe and Saul (1995) have developed a PCRmethod where the ribosomal DNA ITS2 (internaltranscribed spacer) region, once amplified and di-gested with Msp I, will produce products that canbe used to separate members of the PunctulatusGroup. However, since this method first requires aDNA extraction step and is more laborious andtime-consuming than using squash blots, it is un-suitable for processing large numbers of specimens.

The problem of preserving fleld-collected mate-rial may be overcome by modifying the trap designso that the specimens are preserved as they are col-lected. McDonald (1970) describes the use of analcohol light trap to collect Culicoides, and Mc-Donald (1980), using a New Jersey light trap (Ser-vice 1993) containing 707o methanol rather than akilling agent, successfully collected and preservedseveral species of mosquito. The EVS traps cur-rently used could be modified so that the specimensare retained in an alcohol-filled jar rather than acollection bag. However, consideration would haveto be given to the effect alcohol may have on thespecimens with regard to other studies, such as par-asite detection, host blood meal source, or morpho-logical studies.

ACKNOWLEDGMENTS

This paper is published with the approval of theDirector General of Army Health Services. I thankA. W. Sweeney and Tom Travis for preparing thefigures.

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