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Limburger cheese, previously shown to attract femaleAnopheles gambiaeGiles, was solvent extracted and chemically fractionated into acid and non-acid fractions. The extracts and aliquots of headspace odour of the cheese were analysed by gas chromatography and electron impact mass spectrometry. Nineteen saturated and unsaturated aliphatic fatty acids, ranging in carbon chain length from C2to C18, were detected. The most abundant acids (>1 mg/g of cheese) identified in the acid extract were ethanoic, propanoic, butanoic, hexadecanoic and 9-octadecenoic acid. The same compounds were identified in analyses of headspace samples but only trace quantities of the less volatile acids (C10to C16) were present, whilst C18acids were absent. Behavioural responses of femaleA. gambiaetowards a range of dilutions of the acid extract (in diethyl ether) were recorded in a windtunnel bioassay. The undiluted extract was found to be repellent, but became highly attractive (P«0.001) at lower doses, and was still significantly attractive (P<0.001) when diluted 106times. A synthetic mixture of 12 of the more abundant aliphatic acids identified in the acid extract was found to be significantly attractive (P<0.001) when diluted 108times. Electroantennographic (EAG) studies showed significant and reproducible responses to (saturated) Limburger cheese headspace. At doses higher than 0.1%, the synthetic mixture of 12 acids elicited significantly higher EAG amplitudes than the solvent control (paraffin oil). EAG responses were recorded from mosquitoes stimulated with C5to C8acids, that were characterized by significant dose-dependencies. Weaker, though significant EAG responses were obtained with the less volatile acids (C9to C14). Only hexadecanoic acid did not elicit a detectable response. The electrophysiological and behavioural responses obtained with fatty acids isolated from Limburger cheese suggests that together they could act as a kairomone for femaleA. gambiae. The implications of this are discussed together with the occurrence and bacterial production of these compounds on human skin.

The distribution and ecology of mosquitoes of the Anopheles maculipennis complex were studied in the delta of the rivers Rhine and Meuse in the southwest of The Netherlands. The study area was previously malarious, with A. atroparvus being the only vector. 125 potential aquatic habitats of A. maculipennis were sampled, of which 47 (37.6%) contained larvae of this species complex. Larval densities varied from 7.4-325.93 larvae m-2. There was no correlation between chlorinity (@1000) of the water and presence and/or density of larvae. The presence of A. maculipennis was not associated with one particular aquatic floristic habitat, although larvae were often found together with floating algae (Enteromorpha spp.). Larvae were not found in areas experiencing tidal flooding. A newly developed PCR method was used for identification of the mosquito sibling species. Of 150 larvae examined, only 4 were identified as A. atroparvus. All other larvae examined were A. messeae. Adult mosquitoes were identified as A. messeae and 30 wild-caught mosquitoes had fed on domestic animals. Because most anophelines found in 1999 were A. messeae, it is concluded that the study area has undergone a dramatic ecological change since the previous anopheline investigations in 1935, causing the near extinction of A. atroparvus. This species was the only malaria vector in The Netherlands and therefore it is not expected that malaria can return to its former endemic status in the coastal areas of The Netherlands.

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