Anopheles crucians Wiedemann. 204. Anopheles punctipennis (Say). 31. Anopheles quadrimaculatus Say. 34. Coquillettidia perturbans (Walker). 81.
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SHORT COMMUNICATION A New Fixing Technique Using Rice Vinegar Prior to Mosquito Dissection (Diptera: Culicidae)1 BETH B. NORDEN, RALPH E. WEBB,2 P. WESLEY MCCARDLE, AND GEOFFREY B. WHITE Chemicals Affecting Insect Behavior Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, Beltsville, Maryland 20705 KEY WORDS:
Fixative, mosquito dissection, rice vinegar
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 m3 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 103 magnification with a stereomicroscope. A subset of these mosquitoes were placed in 5 ml of rice vinegar (4.3% acidity) (MarukanÒ 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.
1 2
Mention of a product in this paper does not constitute an endorsement by the USDA or the authors. To whom correspondence should be addressed.
Accepted 28 October 2004; revised 17 February 2005 Ó 2005 Kansas Entomological Society
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Fig. 1. Examples of parous and nulliparous ovaries from Maryland mosquitoes caught during May–Sept. 2003. A. An Ochlerotatus triseriatus ovary with arrows pointing to the coiled tracheae characteristic of nulliparous females. B. A Culex pipiens ovary with arrow pointing to the uncoiled tracheal characteristic of parous females. All mosquitoes were dissected on a glass microscope slide. The abdomen of a mosquito being dissected was removed from the body as close to the mesonotum as possible (approximately the 1st or 2nd abdominal segment) and placed in a drop of distilled water. The abdomen’s proximal end was grasped with forceps while another pair of forceps was used to grasp the distal end (at the 6th or 7th segment) just above the cerci. The internal organs, usually the reproductive organs, hindgut and midgut, were removed by gently pulling the forceps apart. The ovaries were separated from the gut contents and slightly stretched for better viewing of the trachial structures after the ovaries had dried. A female was considered parous if, under 1003 magnification from a light microscope, the tracheal scenes were clearly spread (Detinova, 1962) (Fig. 1). Table 1 lists the number of females of each mosquito species we preserved in rice vinegar and dissected with good results. Table 1. Species of Maryland mosquitoes (n ¼ 1266) fixed in rice vinegar and successfully dissected during May–Sept. 2003. Species name
Number dissected
Aedes albopictus Skuse Aedes vexans (Meigen) Anopheles crucians Wiedemann Anopheles punctipennis (Say) Anopheles quadrimaculatus Say Coquillettidia perturbans (Walker) Culex erraticus (Dyar & Knab) Culex pipiens Linnaeus Culex territans Coquillett Ochlerotatus canadensis canadensis (Theobald) Ochlerotatus japonicus (Theobald) Ochlerotatus triseriatus (Say) Psorophora ferox (von Humbolt) Uranotaenia sapphirina (Osten Sacken)
101 76 204 31 34 81 24 290 63 167 9 78 12 96
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Five species during the 2003 season usually had distended abdomens containing more fluid than seen in the other mosquitoes. These included: Aedes vexans (Meigen), Anopheles crucians Wiedemann, Anopheles quadrimaculatus Say, Coquillettidia perturbans (Walker), and Ochlerotatus canadensis canadensis (Theobald). Even these moister species preserved well in the rice vinegar and were readily dissected. Further, these same mosquitoes were sometimes found with several species of mites (Arrenuridae and Pionidae) attached to their cuticles. The mites also were preserved well in rice vinegar, and were easily mounted with natural presentation on microscope slides. We have found rice vinegar to be an inexpensive, easily available, and effective fixative for mosquito preservation prior to dissection or parasite collection and we recommend this new technique for future studies of soft bodied, water-laden insects. Acknowledgments We wish to acknowledge student interns Jamie Hurley and Kelechi Ego-Osuala for their help in the insect preparation and mosquito identification. We also thank Suzanne Batra, Arnold Norden, and an anonymous reviewer for their editorial assistance with the manuscript. Literature Cited Borror, D., and D. DeLong. 1971. An Introduction to the Study of Insects. Holt, Rinehart, and Winston, New York. 812 pp. Entani, E., M. Asai, S. Tsujihata, Y. Tsukamota, and M. Ohta. 1997. Antibacterial action of vinegar against foodborne pathogenic bacteria including Escherichia coli o157:H7 (Part 1). Examination of bacteriostatic and bactericidal activities. Kansenshogaku Zasshi. 71:443–450. Detinova, T. 1962. Age-grouping Methods in Diptera of Medical Importance. World Health Organization. Monograph 47. Geneva, Switzerland. 216 pp. Hauptmann, M., J. H. Lubin, P. A. Stewart, R. B. Hayes, and A. Blair. 2003. Mortality from lymphohematopoietic malignancies among workers in formaldehyde industries. Journal of the National Cancer Institute 95:1615–1623. Hauptmann, M., J. H. Lubin, P. A. Stewart, R. B. Hayes, and A. Blair. 2004. Mortality from solid cancers among workers in formaldehyde industries. American Journal of Epidemiology 159:1117–1130. McCardle, P. W., R. E. Webb, B. B. Norden, and J. R. Aldrich. 2004. Evaluation of five trapping systems for the surveillance of gravid mosquitoes in Prince George’s County, Maryland. Journal of the American Mosquito Control Association 20:254–260. Pennak, R. 1953. Fresh-water Invertebrates of the United States. The Ronald Press Co., New York. 769 pp. Reeves, W., and J. Korecki. 2004. Ochlerotatus japonicus japonicus (Theobald) (Diptera: Culicidae), a new invasive mosquito for Georgia and South Carolina. Proceedings of the Entomological Society of Washington 106(1):233–234.
