Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from Kenyan villages using permethrin-impregnated nets.

The permethrin tolerance (PT) of a population of the mosquito Anopheles gambiae (Diptera: Culicidae) increased following the introduction of permethrin-impregnated nets for malaria control in certain villages near Kisumu, western Kenya. Using a biochemical test that indirectly measures oxidases associated with permethrin resistance, we found that this population had higher oxidase levels than a comparison population from villages without impregnated nets. Mosquitoes from a colony of An. gambiae selected for PT, the RSP (reduced susceptibility to permethrin) strain, were exposed to permethrin with or without the oxidase inhibitor piperonyl butoxide (PB). Significantly higher mortality rates occurred when permethrin was synergized by PB, presumably by suppression of oxidases responsible for PT. An unselected (UNS) colony of An. gambiae that was more susceptible than RSP in a permethrin-susceptibility bioassay (i.e. LT50 22 min for UNS, vs. 42min for RSP) was compared with the RSP colony for levels of oxidases and esterases. The levels of both enzymes were very significantly higher in the RSP strain (P<0.0001). We speculate that use of impregnated nets selected for higher oxidase and esterase levels in An. gambiae to metabolize permethrin acquired from the nets. Both oxidase and esterase mechanisms could confer cross-resistance to other pyrethroids.

The effects of a tsetse DNA virus infection on the functions of the male accessory reproductive gland in the host fly Glossina morsitans centralis (Diptera; Glossinidae).

Freshly deposited third instar Glossina morsitans centralis larvae were infected with the tsetse DNA virus by microinjection, and at emergence adult males were separated from the females and fed on rabbit blood every second day for 8 days. A control group treated with sterile saline were handled similarly. They were dissected, and comparative observations made on the appearance and size of the accessory reproductive glands (ARG) in infected and control males. Regularly fed 8-day-old males from infected and control groups were mated to 2-day-old normal females obtained from the insectay. After separation from copula, the females were dissected and the uteri examined for the presence and quality of the spermatophore. The spermathecae were also examined for insemination. ARG tissues from the control and virus infected regularly fed 8-day-old male flies were fixed and processed for electron microscopic studies. The ARGs from control flies were found to be milky in appearance, whereas those from virus-infected flies were transparent in most parts. The ARGs from virus-infected males were significantly smaller in diameters (F = 42.26, p < 0.0001) and shorter (F = 200.4, p < 0. 0001) than those of the controls. Most of the virus-infected males failed to form a complete spermatophore, whereas almost all the controls formed complete spermatophore as observed in the uteri of the female mates (Chi2 = 111.661, p < 0.0001). The infected males that formed partial spermatophores and those that did not form any at all failed to inseminate their female mates. Histological studies of the ARGs revealed some lesions in the epithelial cells characterized by degeneration of cytoplasmic organelles and detachment of the muscle layer from the basal plasma membrane. However, no virus particles were observed in the affected cells.

The effects of a DNA virus infection on the reproductive potential of female tsetse flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae).

Reproductive anomalies associated with the tsetse DNA virus infection in the female tsetse hosts, Glossina morsitans centralis Machado and Glossina morsitans morsitans Westwood, inoculated with the virus during the 3rd instar larval stage were studied and the data compared to those obtained from the control females injected with sterile physiological saline. Virus infected flies had significantly longer first and second pregnancy cycles (P < 0.0001) and produced pupae that were of significantly less weight in milligrams (P < 0.0001) compared to controls. Transmission of the virus to progeny was not absolute and only 21% of G. m. centralis and 48% of G. m. morsitans first progeny flies from infected females developed salivary gland hypertrophy as a result of transmission from mother to progeny. The virus infected females produced significantly fewere pupae compared to the controls during the experimental period (P < 0.00001).

A simple method for maintaining, detecting and recovering virulent Leishmania donovani in hamsters.

The ability of hamsters (Mesocricetus auratus) to retain amastigotes of Leishmania donovani at cutaneous sites was examined. Following intradermal inoculation of L. donovani stationary phase culture promastigotes in fore and hind footpads, nasal area and belly skin, cultures of aspirates taken fortnightly from these sites showed that amastigotes can survive in the skin for up to 10 months without visceralizing. Hairless cutaneous sites were better at retaining L. donovani amastigotes than the hairy belly skin. L. donovani promastigotes cultivated from aspirates of sites of inoculation were highly virulent. The skin is suggested as one of the sites where viscerotropic L. donovani can remain cryptic for a long time before the infection either visceralizes or is aborted. Skins of hamsters when inoculated intradermally can serve as an easy site for maintaining, detecting and recovering virulent L. donovani without killing the hamster.

Long-term use of permethrin-impregnated nets does not increase Anopheles gambiae permethrin tolerance.

Previous use of permethrin-impregnated bednets (mosquito nets) and curtains in four Kenyan villages for one year, 1990-91, raised the permethrin LT50 of Anopheles gambiae to 2.4-fold above its baseline value, designated permethrin tolerance (PT), as measured by exposure to 0.25% permethrin-impregnated papers in W.H.O. test-kits. During 1992-93, with ongoing use of permethrin-impregnated nets and curtains, PT regressed slightly compared with the contemporary susceptibility level of An.gambiae from non-intervention villages, to 1.8-fold in 1992 and only 1.6-fold in 1993. Thus the selection pressure of impregnated nets for PT in An.gambiae appears to be minimal in our study villages, although the impact of permethrin was demonstrated by a significantly lower parous-rate of An.gambiae females in the intervention (63-66%) than in non-intervention (79%) villages, and by reduced malaria transmission (reported elsewhere). In a selected stock of An.gambiae from the study area, PT did not affect the susceptibility to deltamethrin, fenitrothion, propoxur or DDT. Bioassays described herein provide easy procedures for field-monitoring of mosquito susceptibility/tolerance/resistance to insecticides used for net impregnation in operational programmes.

The chemotactic effect of Phlebotomus duboscqi (Diptera: Psychodidae) salivary gland lysates to murine monocytes.

The possibility that salivary gland lysates of Phlebotomus duboscqi are able to attract vertebrate monocytes was investigated. In vitro studies showed that salivary gland lysates of P. duboscqi, the vector of Leishmania major in Kenya, are chemotactic to mouse peritoneal monocytes. This attraction of monocytes by vector salivary gland lysates may form part of the mechanisms through which sandfly saliva ensures successful parasitization of macrophages in a susceptible host by Leishmania parasites.

Reduced susceptibility of Anopheles gambiae to permethrin associated with the use of permethrin-impregnated bednets and curtains in Kenya.

Susceptibility of the malaria vector Anopheles gambiae to permethrin decreased following the installation of mosquito nets impregnated with 0.5 g permethrin per square metre in four villages near Kisumu, Kenya. During the first year that permethrin-impregnated bednets and curtains were in place, the exposure time to 50% mortality (LT50) increased 2.5-fold from 13 to 33 min, while the LT50 for An.gambiae was unchanged in two other villages where no intervention measures were used. Two years after permethrin-impregnated mosquito nets were distributed the LT50s for An.gambiae were 28, 28 and 16 min, respectively, in the villages with bednets, curtains and with no such intervention. Using a colony of An.gambiae derived from females collected in the villages using permethrin-impregnated mosquito nets, we lengthened the LT50 from 28 to 41 min in two generations by exposing all females to permethrin-treated papers for 60 min and rearing offspring of the survivors. Permethrin-impregnated bednets and curtains are intended to reduce vectorial capacity. Reduced susceptibility to permethrin could counter this beneficial effect.

Effect of Plasmodium falciparum on blood feeding behavior of naturally infected Anopheles mosquitoes in western Kenya.

Feeding behavior was compared between infected and uninfected field-collected groups of Anopheles gambiae sensu lato and An. funestus from western Kenya. A significantly greater percentage (81%) of Plasmodium falciparum-infected An. gambiae s.l. females probed on experimental hosts (hamsters) than did uninfected females (38%). Among those females that initiated probing, there was no effect of infection status on the ability to take a bloodmeal. Plasmodium falciparum-infected An. gambiae s.l. probed more often (mean = 4.0) and for a longer time (mean = 277 sec) than did their uninfected counterparts (mean = 2.4 probes and mean probing time = 214 sec). Results for the small number of An. funestus that fed followed the same trend. Among infected An. gambiae s.l. females, there was no effect of sporozoite density on either the number of probes made or the total probing time. Among uninfected females, there was no difference in feeding behavior between nulliparous and parous females. In laboratory experiments, female age had no effect on blood-feeding behavior. Our findings provide evidence that natural malaria infection modifies the feeding behavior of Anopheles females.

Malaria transmission potential of Anopheles mosquitoes in the Mwea-Tebere irrigation scheme, Kenya.

1. Anopheles arabiensis Patton and An. funestus Giles were identified as vectors of Plasmodium falciparum malaria in the Mwea-Tebere irrigation scheme, Kenya. An. arabiensis was the only member of the An. gambiae complex identified from chromosome characteristics. Other Anopheles species found included An. pharoensis Theobald, An. rufipes Gough and An. coustani Laveran. Survival rates per gonotrophic cycle for An. arabiensis averaged 0.37 during the short rains (October-November), 0.49 during the dry season (February) and 0.78 during the long rains (May-June). Vectorial capacities were correspondingly low due to low survival rates and a high degree of zoophily. The average duration of infective life for P. falciparum was 0.2 days for both An. arabiensis and An. funestus. In contrast, entomological inoculation rates were comparatively high: 6-8 infective bites/man/month. An. pharoensis averaged 110 bites/man/night during the short rains; 1/999 (0.1%) was positive by ELISA for P. falciparum circumsporozoite antigen, but the ELISA evidence is not conclusive for vector incrimination. In correspondence with clinical observations, the transmission of P. malariae and P. ovale is unlikely due to the low vector survival rates. The observed anomaly between low vectorial capacities and high entomological inoculation rates demonstrates the importance of accurately estimating vector sporozoite rates to monitor unstable malaria transmission in irrigated areas.

Seasonal population changes and malaria transmission potential of Anopheles pharoensis and the minor anophelines in Mwea Irrigation Scheme, Kenya.

A study in 1984 and 1985 showed that Anopheles gambiae s.l. and An. pharoensis were the major anophelines in Mwea Irrigation Scheme, Kenya, constituting 83.86% and 15.69% of the catch respectively. Four minor species made up the remaining 0.45%. The irrigation phase of the rice cultivation cycle in August, which linked the flooding effects of the two rainy seasons, resulted in major population increases of An. pharoensis and enabled continuous breeding for up to 9 months per year. The average of mean monthly proportions of unfed, bloodfed, and gravid females was 26.6, 58.8, and 14.6% respectively. The Plasmodium falciparum sporozoite rates for An. pharoensis were 1.3% by ELISA and 0.68% by dissection, while those for An. funestus were 1.7% by ELISA and 1.25% by dissection. An. pharoensis can contribute to the epidemiology of Malaria in the Mwea area.

Evaluation of Melia volkensii extract fractions as mosquito larvicides.

A standardized fraction of Melia volkensii fruit kernel extract was tested against Anopheles arabiensis mosquito larvae. The LC50 in 48 hr was 5.4 micrograms/ml. At low concentrations this fraction had growth inhibiting activity producing prolonged larval instars, and lethal effects during ecdysis. Further fractionation of the standardised fraction yielded seven bands on preparative Thin Layer Chromatography. The two most lipophilic bands had acute toxic effects on the larvae, the next two bands had growth inhibiting effects, while the three trailing bands had no effect at all. The acute toxicity and growth inhibiting effects were destroyed by heat during the drying of the fruits.