SHORT COMMUNICATION A New Fixing Technique Using Rice Vinegar Prior to Mosquito Dissection (Diptera: Culicidae)

Insects preserved in fluid have traditionally been placed in 70–80% ethyl alcohol (Borror and DeLong, 1971). When internal structures such as ovaries require examination, FAA or Kahle’s solution, a combination of alcohol, formaldehyde, and acetic acid, is often used (Pennak, 1953). Recent studies involving viral testing of insects and DNA analysis recommend preservation in 95–100% ethanol (Reeves and Korecki, 2004). Unfortunately, none of these fixative techniques are effective for good dissection of reproductive structures in the soft, flexible, and often fluid filled abdomens of mosquitoes. Monitoring mosquitoes can be an important aspect of disease surveillance, and has become more prevalent since the rapid spread of West Nile virus throughout the USA. We conducted a study of mosquitoes in Maryland that were captured using CO2 baited light traps, unbaited light traps, and aspirated from 1.8 m 3 brown saran cages during the unusually wet 2003 season (McCardle et al., 2004). In addition to surveying the 23 mosquito species present, their numbers, sex ratios and whether they were gravid or blood fed, we also determined parity (evidence of previous egg laying). Dissection was needed to identify parous females by examining the state of tracheal coils in the ovaries (Detinova, 1962). We found that abdominal organs could be easily removed intact from freshly killed mosquitoes. However, prolonged freezing (over a week), drying, or preservation in an alcoholic liquid reduced the probability of a successful dissection because of internal cellular breakdown, contraction, and distortion. Stronger preservation measures, such as FAA or Kahle’s solution, could not be used due to chemical sensitivities in one of the authors. There are additional problems associated with these stronger preservatives. Formaldehyde has recently been linked to increased risks of nasopharyngeal cancer (Hauptmann et al., 2004a) and leukemia (Hauptmann et al., 2004b). Transporting 70%þ alcohol on airplane flights is prohibited by the United States Transportation Security Administration in carry-on or checked luggage. There is no guarantee of obtaining high quality ethanol once outside of the United States. Some countries even require special permits to purchase this highly flammable liquid to prevent its use in terrorist activities. In our efforts to avoid fixatives having these problems, we considered rice vinegar as a reasonable alternative to stronger preservatives for short periods of insect preservation after collection. Vinegar, in general, is available worldwide at a reasonable price. The ‘‘bite’’ in vinegar is provided by acetic acid, which is also a component of the FAA preservative. Finally, rice vinegar has also been shown to have bacteriostatic properties at acidities greater than 0.1% (Entani et al., 1997). We found rice vinegar to be an excellent fixing solution, which allowed us to preserve large collections of newly caught mosquitoes until they could be accurately dissected. We collected the insects from our traps between 9:00 and 11:00 A.M. and processed them that afternoon. Trap catches were frozen for up to one-half hour to kill the contents before we separated out the mosquitoes. Next, we separated the mosquitoes and identified them to species at 103magnification with a stereomicroscope. A subset of these mosquitoes were placed in 5 ml of rice vinegar (4.3% acidity) (Marukan Rice Vinegar, Marukan Vinegar (U.S.A.) Inc.) and refrigerated for later dissection. Mosquitoes engorged with blood did not preserve well in vinegar, so they were not fixed with this method during our study. With refrigeration, specimens were in good condition for valid dissection for six weeks or longer. Unrefrigerated samples showed no deterioration for about a week.