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Brugia pahangi: Exsheathment and Midgut Penetration in Aedes aegypti

1984; Wiley; Volume: 103; Issue: 4 Linguagem: Inglês

10.2307/3226478

2325-5145

Bruce M. Christensen, Daniel R. Sutherland,

Insect symbiosis and bacterial influences

Exsheathment and midgut penetration by microfilariae (mff) of Brugia pahangi were studied in intact Aedes aegypti (black-eyed Liverpool strain) and in isolated midguts maintained in vitro in Aedes saline. Penetration of the midgut occurred within 150 min postingestion (PI) of an infective blood meal, with the majority (60%) migrating out from 61-105 minutes. Penetration occurred anywhere on the midgut, and mff were capable of penetrating the foregut, hindgut, and Malpighian tubules. Approximately 60% of the mff ingested migrated out of the midgut in the mosquitoes examined. Exsheathment of mff rarely occurred within the midgut. Nearly 97% of 894 mff examined from the midgut after 5-150 minutes PI were sheathed, and approximately 75% of 288 mff retained their sheaths after midgut penetration. Data suggest that the sheath is likely weakened or broken at the anterior end of mff during midgut penetration, and that they completely free themselves of the sheath in the hemocoel. Results from inoculation experiments indicate that mff are capable of exsheathing in the hemocoel without midgut exposure, and that occasionally they can develop within the microfilarial sheath. The development of Brugia pahangi (Buckley & Edeson, 1956) in natural and experimental laboratory vector mosquitoes has been studied by a number of workers (Edeson et al., 1960; Ewert, 1965; Schacher, 1962; Schacher & Khalil, 1965). Esslinger (1962) indicated that microfilariae of B. pahangi exsheath in the blood meal of Anopheles quadrimaculatus (Say, 1824) almost immediately after engorgement, and therefore agreed with earlier statements regarding exsheathment of Brugia malayi (Brug, 1927) in mosquitoes (Feng, 1936). Following Esslinger's study, Ewert (1965) stated that exsheathment precedes migration in mosquitoes that permit complete development of B. pahangi. Preliminary observations of B. pahangi in Aedes aegypti (Linnaeus, 1762) (black-eyed Liverpool strain) in our laboratory, however, suggested that exsheathment rarely occurred within the mosquito midgut in this vector-parasite system. Our studies were conducted to clarify these preliminary observations. MATERIALS AND METHODS Aedes aegypti (black-eyed Liverpool strain) used in these studies were from a laboratory colony originally obtained from the University of London in 1977. Larvae were maintained in lots of 300 in distilled water in white enamel pans (25 x 42 x 7 cm) and fed a slurry of finely ground fish food (Tetramin?). Pupae were harvested by hand, and after emergence, adult females were separated and maintained in lots of 50 in 0.473-liter ice cream cartons with fine-mesh marquisette coverings. Cotton pads, moistened in 0.3 M sucrose The authors sincerely appreciate the technical assistance of Ms. Becky Lasee and Mr. Keith Forton. This study was supported by National Institutes of Health Grant AI 19769. TRANS. AM. MICROSC. Soc., 103(4): 423-433. 1984. ? Copyright, 1984, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.165 on Wed, 20 Apr 2016 05:56:17 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. solution, were placed on the marquisette. All mosquitoes were maintained in an environmental chamber at 26.5 ? 1?C and 75 ? 10% RH under a 16-h light: 8-h dark photoperiod. Fourto six-day-old A. aegypti were exposed to jirds, Meriones unguiculatus (Milne-Edwards, 1867), with microfilaremias of B. pahangi ranging from 192-1,008 microfilariae/20 ,l1 blood. Infected jirds were obtained from John W. McCall (School of Veterinary Medicine, University of Georgia) through a program supported by the U.S.-Japan Cooperative Medical Sciences ProgramNIAID. Jirds were anesthetized with a mixture of ketamine-HCl and Rompun administered intramuscularly (IM) before exposure to mosquitoes. Immediately following blood feeding, mosquitoes were dissected and the intact digestive tract (foregut, midgut, and hindgut with Malpighian tubules attached) was placed in a depression slide containing Aedes saline (Hayes, 1953) in a volume sufficient to completely cover the midgut. Dissected midguts were discarded if blood leakage was noted from any part of the digestive tract removed. Microscopical studies. Dissected midguts were observed at 25-50 x with the aid of a stereomicroscope, and when microfilariae (mff) began emerging into the saline, the entire midgut was fixed, for light microscopy, in Bouin's solution for 48-72 h. Fixed midguts were dehydrated in ethanol, embedded in plastic (JB-4), sectioned serially at 3 tm, and stained with Harris' hematoxylin and eosin. Midguts with emerging mff were processed for scanning electron microscopy by fixation in ice-cold 3% Karnovsky's solution in sodium cacodylate buffer overnight, and post-fixed in 2% OsO4 for 2 h. Specimens were dehydrated in ethanol, critical-point dried, coated with gold palladium, and examined with a JEOL JSM-U3 scanning electron microscope at 20 kV. Exsheathment studies. To determine if exsheathment of mff occurred within the midgut, dissected midguts were held for 5, 30, 60, 120, or 150 min postingestion (PI) of the blood meal, and then were dissected in 2% formalin. All mff remaining within the midgut were examined for the presence of a sheath using phase-contrast optics. The presence or absence of sheaths on mff following midgut penetration was determined by continuously observing individual midguts in Aedes saline with the aid of a stereomicroscope. As each parasite penetrated into the saline, the time and site of penetration were noted, and each mff was immediately removed with a micropipette and transferred to a drop of 2% formalin on a microscope slide. The resultant slides were examined by phase-contrast microscopy and the presence or absence of a sheath was noted. To ascertain if the results we obtained from midguts in vitro were representative of what occurs in vivo, we exposed mosquitoes to a jird with a microfilaremia of 946 mff/20 ,1, held them for 30 or 60 min, and then removed the intact midgut in Aedes saline. The midgut was transferred to a microscope slide, immediately dissected in 2% formalin, and mff recovered were examined for the presence of a sheath. The remaining mosquito body and fluids were thoroughly teased apart in Aedes saline, covered with a coverglass, and all mff 424 This content downloaded from 157.55.39.165 on Wed, 20 Apr 2016 05:56:17 UTC All use subject to http://about.jstor.org/terms VOL. 103, NO. 4, OCTOBER 1984 that had already penetrated the midgut were evaluated for the presence of a sheath. To determine if sheathed mff could develop and (or) exsheath within the hemocoel of mosquitoes, sheathed mff were isolated and concentrated from jird blood by diluting blood with distilled water (1:9) and centrifuging. Supernatant was discarded, and washing and centrifuging were repeated three times with the last wash containing Aedes saline instead of water. Five to 10 mff were intrathoracically inoculated into individual mosquitoes through the neck membrane as previously described (Christensen, 1981; Green et al., 1980). Inoculated mosquitoes were dissected daily through 5 days postinoculation.