Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Microorganism-mediated behaviour of malaria mosquitoes
    Busula, Annette O. - \ 2017
    Wageningen University. Promotor(en): W. Takken, co-promotor(en): J. de Boer. - Wageningen : Wageningen University - ISBN 9789463431156 - 199
    culicidae - anopheles gambiae - anopheles arabiensis - mosquito-borne diseases - disease vectors - animal behaviour - host-seeking behaviour - plasmodium falciparum - hosts - man - cows - hens - odours - culicidae - anopheles gambiae - anopheles arabiensis - ziekten overgebracht door muskieten - vectoren, ziekten - diergedrag - gedrag bij zoeken van een gastheer - plasmodium falciparum - gastheren (dieren, mensen, planten) - mens - koeien - hennen - geurstoffen

    Host-seeking is an important component of mosquito vectorial capacity on which the success of the other behavioural determinants depends. Blood-seeking mosquitoes are mainly guided by chemical cues released by their blood hosts. This thesis describes results of a study that determined the effect of microorganisms – host skin bacteria as well as malaria parasites – on host-seeking behaviour of female Anopheles gambiae sensu stricto and An. arabiensis in Homabay county, western Kenya. Semi-field and field experiments were conducted to determine the response of mosquitoes with different host preference to synthetic and natural odour blends from three vertebrate hosts, a human, a cow and a chicken. Screen house experiments were conducted to test whether specific skin bacteria or a mix of skin bacterial volatiles from the three vertebrate hosts mediate mosquito response. A review chapter in this thesis discusses how malaria parasites can manipulate human hosts to enhance their own transmission, by making the hosts more attractive to mosquitoes. Another experiment, using a dual-choice olfactometer, determined whether infection with malaria parasites increases human attractiveness to malaria mosquitoes, and whether the attractiveness of infected humans is Plasmodium falciparum-stage specific. Here, the same children participated in the study during infection with malaria parasites and after treatment with antimalarial drugs, artemisinin lumefantrine. Cage assays were further used to test mechanisms of attractiveness of P. falciparum-infected individuals using body odours or skin bacterial volatiles collected from the children at the two time points. Overall results show that skin bacterial volatiles play an important role in guiding mosquitoes with different host preferences to their specific host. For An. gambiae s.s., high (microscopic) densities of P. falciparum gametocytes (and not parasite-free, submicroscopic gametocytes or asexual stages of Plasmodium parasites) results into higher attractiveness of hosts, and body odours play a role in attractiveness of P. falciparum-infected humans. The results may help to develop more effective health policies and enable targeted interventions towards the most attractive hosts, which could contribute to reductions in malaria transmission. Identification of general or common attractive volatiles produced by the natural hosts as well as those from the gametocyte carriers may contribute to the development of an improved synthetic odour blend that may be used for sampling of mosquitoes with different host preferences. The use of powerful attractive odorants may result in reductions of vector-borne diseases transmitted by mosquitoes.

    Host-plant resistance to western flower thrips in Arabidopsis
    Thoen, Manus P.M. - \ 2016
    Wageningen University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - Wageningen : Wageningen University - ISBN 9789462578807 - 191
    arabidopsis thaliana - host plants - insect pests - frankliniella occidentalis - defence mechanisms - pest resistance - genomics - genome analysis - host-seeking behaviour - optical tracking - data analysis - insect plant relations - arabidopsis thaliana - waardplanten - insectenplagen - frankliniella occidentalis - verdedigingsmechanismen - plaagresistentie - genomica - genoomanalyse - gedrag bij zoeken van een gastheer - optisch sporen - gegevensanalyse - insect-plant relaties

    Western flower thrips is a pest on a large variety of vegetable, fruit and ornamental crops. The damage these minute slender insects cause in agriculture through feeding and the transmission of tospoviruses requires a sustainable solution. Host-plant resistance is a cornerstone of Integrated Pest Management (IPM). Plants have many natural defense compounds and morphological features that aid in the protection against herbivorous insects. However, the molecular and physiological aspects that control host-plant resistance to thrips are largely unknown.

    A novel and powerful tool to study host-plant resistance to insects in natural populations is genome-wide association (GWA) mapping. GWA mapping provides a comprehensive untargeted approach to explore the whole array of plant defense mechanisms. The development of high-throughput phenotyping (HTP) systems is a necessity when large plant panels need to be screened for host-plant resistance to insects. An automated video-tracking platform that could screen large plant panels for host-plant resistance to thrips, and dissect host-plant resistance to thrips in component traits related to thrips behavior, was developed. This phenotyping platform allows the screening for host-plant resistance against thrips in a parallel two-choice setup using EthoVision tracking software. The platform was used to establish host-plant preference of thrips with a large plant population of 345 wild Arabidopsis accessions (the Arabidopsis HapMap population) and the method was optimized with two extreme accessions from this population that differed in resistance to thrips. This method can be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations. EthoAnalysis, a novel software package was developed to improve the analyses of insect behavior. There were several benefits from using EthoAnalysis to analyze EthoVision data. The detailed event statistics that could be extracted from EthoAnalysis allows researchers to distinguish detailed differences in moving and feeding behavior of thrips. The potential of this additional information is discussed in the light of quantitative genetic studies.

    Stress resistance was studied in the HapMap population on a total of 15 different biotic and abiotic stresses ranging from biotic stresses like insects and nematodes, to abiotic stresses like drought and salt. A multi-trait GWA study to unravel the genetic architecture underlying plant responses to the different stresses was performed. A genetic network in this study revealed little correlation between the plant responses to the different insect herbivores studied (aphids, whiteflies, thrips and caterpillars). For thrips resistance a weak positive correlation with resistance to drought stress and Botrytis, and a negative correlation with resistance to parasitic plants were observed. One of the surprising outcomes of this study was the absence of shared major QTLs for host-plant resistance and abiotic stress tolerance mechanisms. RESISTANCE METHYLATED GENE 1 (RMG1) was one of the candidate genes in this multi-trait GWA study that could be controlling shared resistance mechanisms against many different stresses in Arabidopsis. RMG1 is a nucleotide-binding site Leucine-rich repeat (NB-LRR) disease resistance protein and its potential relation to several resistance/tolerance traits was successfully demonstrated with T-DNA insertion lines.

    The 15 stresses were used in a comparison with a metabolomics dataset on this Arabidopsis HapMap population. It was discovered that levels of certain aliphatic glucosinolates correlated positively with the levels of resistance to thrips. This correlation was further investigated with the screening of a RIL (Recombinant Inbred Line) population for resistance to thrips, several knockout mutants and the analysis of co-localization of GWA mapping results between glucosinolates genes and thrips resistance. In a GWA analysis, the C4 alkenyl glucosinolates that correlated the strongest with thrips resistance mapped to the genomic regions containing genes known to regulate the biosynthesis of these compounds. However, thrips resistance did not co-localize with any of the GSL genes, unless a correction for population stratification was omitted. Additional screening of a Cvi x Ler RIL population showed a QTL for thrips resistance on chromosome 2, but no co-localization with any known glucosinolate genes, nor with thrips resistance loci identified by GWA mapping. Knock-out mutants and overexpressors of glucosinolate synthesis genes could also not confirm a causal link between glucosinolates and resistance to thrips. It is possible that the crucial factors that control resistance to thrips may not have been present in sufficient quantities or in the right combinations in the mutants, RILs and NIL screened in this study. Alternatively, the correlation between thrips feeding damage and glucosinolate profiles could be based on independent geographical clines. More research should be conducted to assess which of these explanations is correct.

    In the general discussion, the results from this thesis are discussed in a broader perspective. Some prototypes of new phenotyping platforms that could further aid screening for resistance to thrips in the future are presented. Natural variation in host-plant resistance to thrips is compared to the variation in host-plant resistance to aphids and caterpillars. The geographic distribution of host-plant resistance to thrips is not evident in the other insects, in line with the distribution of glucosinolate profiles and other climate factors. The chapter concludes with some suggestions for future research in the field of host-plant resistance to thrips.

    Foraging behaviour of parasitoids in multi-herbivore communities
    Rijk, M. de - \ 2016
    Wageningen University. Promotor(en): Marcel Dicke, co-promotor(en): Erik Poelman. - Wageningen : Wageningen University - ISBN 9789462576377 - 217
    016-3931 - parasitoids - parasitoid wasps - feeding behaviour - plant-herbivore interactions - hosts - host parasite relationships - host preferences - host-seeking behaviour - cotesia glomerata - pieris brassicae - brassica oleracea - parasitoïden - sluipwespen - voedingsgedrag - plant-herbivoor relaties - gastheren (dieren, mensen, planten) - gastheer parasiet relaties - gastheerpreferenties - gedrag bij zoeken van een gastheer - cotesia glomerata - pieris brassicae - brassica oleracea

    Foraging behaviour of parasitoids in multi-herbivore communities

    Parasitic wasps, or parasitoids, use herbivore-induced plant volatiles and infochemicals produced directly by the herbivore to locate their herbivorous hosts. This process could be interrupted by the presence of herbivores that are not suitable for the development of parasitoid offspring. These non-host herbivores could affect the behaviour of parasitoids both when parasitoids are foraging for host-infested plants by using plant volatiles and when parasitoids are foraging for hosts on the plant by using herbivore infochemicals.

    The aim of this thesis was to study the impact of non-host presence on the parasitoid-host-food plant complex of the parasitoid Cotesia glomerata with its host caterpillar Pieris brassicae and a monoculture of the cultivated plant Brassica oleracea. To study the influence of non-hosts on the plant-volatile-based searching behaviour of the parasitoid, a wind-tunnel set-up in the laboratory was used. In this set-up, the parasitoids were given a choice between two plants or between the leaves of one plant. The plant/leaf on which the parasitoid landed was considered the preferred plant/leaf. A second laboratory set-up was used to study the influence of non-host herbivores on the host-infochemical-based searching behaviour of the parasitoid. In this on-plant experiment, the behaviour of the parasitoid was observed after landing on the plant. The influence of non-hosts on the combination of plant-volatile-based and host-infochemical-based searching, i.e. the total foraging efficiency of the parasitoid, was investigated using an outdoor tent set-up in an agricultural field. In this semi-field experiment, parasitoids were allowed to parasitize their hosts in a non-host environment for one to three days.

    This thesis firstly shows that the feeding guild of non-host herbivores influenced the foraging behaviour of C. glomerata. Leaf-chewing non-hosts negatively impacted the plant-volatile-based searching behaviour of the parasitoid, whereas phloem-feeding non-hosts positively impacted the host-infochemical-based searching. The resulting host-finding efficiency was in general positively affected by phloem-feeding non-hosts. Secondly, the position of host and non-host herbivores on the plant affected the plant-volatile-based and the host-infochemical-based foraging behaviour of the parasitoid, but not the host-finding efficiency. An unnatural distribution of herbivores over the plant (host feeding on old leaf, non-host feeding on young leaf), negatively affected the choice of the parasitoid for a leaf to land on, i.e. the parasitoid more often landed on the non-host infested leaf. Combined feeding by the host and non-host on one leaf positively affected the number of hosts parasitized on that plant compared with the number of hosts parasitized when herbivores were separated. However, the parasitoid was able to compensate for these effects as a result of which the foraging efficiency was unaltered. Thirdly, the density of non-hosts did influence the plant-volatile-based searching of the parasitoid. A high non-host density negatively affected parasitoid preference for host-infested plants. However, the host-infochemical-based foraging and the total foraging efficiency remained unaffected. Fourthly, rather than the species diversity, the species identity of non-host herbivores had an influence on parasitoid host-infochemical-based searching. One of the tested non-host species negatively affected the behaviour of the parasitoid when searching on the plant. However, neither non-host species identity nor diversity affected plant-volatile-based searching of the parasitoid. Fifthly, this thesis investigated if a parasitoid could learn to associate non-host cues with the presence of hosts and if the parasitoid changed the parasitization preference accordingly. After receiving a learning experience, the parasitoid showed an altered landing preference for infested plants according to the learned cues. However, in an outdoor tent set-up, the parasitoid did not show an altered parasitization preference.

    The results of this thesis show that non-host herbivore traits can affect the different phases of parasitoid foraging either positively, negatively or neutrally. The non-host effect on the total foraging efficiency is not necessarily a result of the sum of the effects on the first and the second foraging phase. In fact, the results of two out of three outdoor-tent experiments that investigated the foraging efficiency of the parasitoid showed no non-host effect, while the separate foraging phases were affected by non-host presence.

    It is concluded that the foraging efficiency of the parasitoid C. glomerata when searching for its host P. brassicae is not strongly affected by non-host herbivore presence. The use of herbivore-induced plant volatiles by C. glomerata during this foraging process is not interrupted by non-host herbivores. It is advised to consider all phases of the foraging process in studies of parasitoid foraging behaviour, preferably in one experiment that covers the whole searching process. Altogether, this thesis gives a clear and comprehensive overview of the impact of non-host presence on a parasitoid-host-food plant complex and it thereby contributes to the fundamental knowledge of insect foraging in a multi-herbivore context.

    Push-pull tactics to disrupt the host-seeking behaviour of malaria mosquitoes
    Menger, D.J. - \ 2015
    Wageningen University. Promotor(en): Willem Takken; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462576070 - 173
    culicidae - anopheles - vectoren, ziekten - malaria - gedrag bij zoeken van een gastheer - insectenlokstoffen - insectenafweermiddelen - insectenvallen - vectorbestrijding - culicidae - anopheles - disease vectors - malaria - host-seeking behaviour - insect attractants - insect repellents - insect traps - vector control

    Malaria remains a major health burden, especially in sub-Saharan Africa. The efficacy of the main vector control tools, insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS), is compromised by the development of physiological and behavioural resistance in the target mosquito species and by changes in the species composition of vector populations. These developments underline the need to develop novel vector control approaches which are complementary to insecticide-based methods. In this thesis, the potential of push-pull tactics as a tool to reduce malaria transmission is explored. It is described how the push-pull concept, originally designed for agricultural pest control, may be translated in a system that targets Anopheles mosquitoes. Several novel repellents are identified in the laboratory and a prototype push-pull system is tested in a semi-field setup. The system is improved and evaluated in a malaria endemic field setting and the push-pull approach is compared and combined with the existing practise of eave screening. Based on the experimental results it is concluded that (1) it is possible to reduce house entry of malaria and other mosquitoes using (spatial) repellents and/or attractant-baited traps; (2) the effect of repellents on house entry is larger and more consistent than the effect of attractant-baited traps; (3) the main function of the attractant-baited traps is to deplete mosquito populations through removal trapping; (4) the attractive and repellent components of the push-pull system complement each other and there is no or very little interaction between them; (5) a push-pull system based on repellent and attractive volatiles can be expected to reduce malaria transmission through a strong decrease of the entomological inoculation rate; (6) eave screening is a highly efficient method to reduce house entry of malaria and other mosquitoes and increases outdoor trap catches, while there is little added value in impregnating screening material with a repellent. In the last chapter, the issue of selection for insensitivity to the used compounds is discussed, as well as methods how to manage it. Furthermore, it is described how the principles of behavioural disruption on which push-pull tactics are based make the technique potentially suitable to target a wider selection of arthropod vectors of disease than malaria mosquitoes alone. It is concluded that future vector control strategies will probably consist of the integration of many different approaches, of which push-pull tactics may be one. By integrating different approaches, it will be possible mitigate the development of resistance while targeting vectors in different life stages, uncompromised by changing behavioural patterns and changes in the composition of vector populations. This would require an integrated view on vector control, knowledge on the ecology of vectors and the political will to invest in programmes that focus on long term sustainable control.

    Odour-based strategies for surveillance and behavioural disruption of host-seeking malaria and other mosquitoes
    Mweresa, C.K. - \ 2014
    Wageningen University. Promotor(en): Willem Takken; Marcel Dicke; W.R. Mukabana; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789461739247 - 277
    anopheles gambiae - anopheles funestus - anopheles - culicidae - semiochemicals - insectenlokstoffen - lokstoffen - synthetische materialen - geurstoffen - malaria - gedrag bij zoeken van een gastheer - anopheles gambiae - anopheles funestus - anopheles - culicidae - semiochemicals - insect attractants - attractants - synthetic materials - odours - malaria - host-seeking behaviour
    Insect - host interactions : signals, senses, and selection behaviour
    Loon, J.J.A. van - \ 2013
    Wageningen : Wageningen University, Wageningen UR - ISBN 9789461735980 - 28
    insect-plant relaties - chemische ecologie - gedrag bij zoeken van een gastheer - insect plant relations - chemical ecology - host-seeking behaviour
    Unravelling the resistance mechanism of lettuce against Nasonovia ribisnigri
    Broeke, C.J.M. ten - \ 2013
    Wageningen University. Promotor(en): Joop van Loon; Marcel Dicke. - S.l. : s.n. - ISBN 9789461735782 - 228
    lactuca virosa - lactuca sativa - slasoorten - insectenplagen - nasonovia ribisnigri - plaagresistentie - verdedigingsmechanismen - gedrag bij zoeken van een gastheer - insect-plant relaties - lactuca virosa - lactuca sativa - lettuces - insect pests - nasonovia ribisnigri - pest resistance - defence mechanisms - host-seeking behaviour - insect plant relations

    Aphids are serious pests of crop plant species, and host plant resistance is often the most effective and environmentally friendly control strategy to control these pests. One of these aphid pests is the black currant - lettuce aphid, Nasonovia ribisnigri (Mosely), an economically important pest of cultivated lettuce, Lactuca sativa L. Host plant resistance has been used since 1982 to control this aphid species and is mediated by the Nr-gene, originating from wild lettuce Lactuca virosa L. However, this resistance is not effective anymore, since N. ribisnigri aphids virulent to the Nr-resistance have been reported since 2007. The aim of this thesis was to unravel the mechanism of resistance mediated by the Nr-gene against N. ribisnigri, by behavioural studies on the aphids on both resistant and susceptible lettuce, to allow lettuce breeders to accelerate their resistance breeding programmes. Although the exact mechanism of Nr-mediated resistance remains unknown, the data in this thesis provide insight into this mechanism. The active site of the Nr-mediated resistance is mainly located in the phloem and some resistance might be encountered by the aphids along the pathway to the phloem. The inability of the avirulent aphids to feed from the resistant plant could be caused by the failure of aphids to suppress the wound response of the sieve element. The resistance factor(s) are only produced in the shoot, because grafts with resistant shoots and susceptible roots were resistant, whereas grafts with susceptible shoots and resistant roots remained susceptible. An intact vascular system is needed for full resistance, because both detached leaves and leaf disks of resistant lettuce plants were less resistant.

    Variation in virulence was observed among populations of different geographical origin. Aphids from a highly virulent population performed equally well on both resistant and susceptible lettuce plants, whereas semi-virulent aphids performed better on susceptible lettuce plants. Both short-term and long-term virulence loss were observed for virulent aphid populations differing in virulence level, which indicates this virulence is associated with fitness costs. A possible mechanism underlying virulence in N. ribisnigri to the Nr-resistance is the presence of an effector protein in the salivary secretion of the aphids suppressing resistance. Virulent aphids seemed to actively suppress the resistance in lettuce against the avirulent aphids.

    The original donor or the Nr-resistance, L. virosa accession IVT 280, was tested as possible source of new resistance against the virulent biotypes of N. ribisnigri and was foundfully resistant against virulent aphids, and can be exploited as a source of resistance in breeding for new resistance in cultivated lettuce.

    Behavioural effects of fungal infection by Metarhizium anisopliae in adult malaria mosquitoes
    Ondiaka, S.N. - \ 2012
    Wageningen University. Promotor(en): Willem Takken; Marcel Dicke, co-promotor(en): W.R. Mukabana. - S.l. : s.n. - ISBN 9789461732934 - 179
    anopheles gambiae - vectoren, ziekten - malaria - vectorbestrijding - biologische bestrijding - metarhizium anisopliae - diergedrag - paringsgedrag - gedrag bij zoeken van een gastheer - voedingsgedrag - ovipositie - anopheles gambiae - disease vectors - malaria - vector control - biological control - metarhizium anisopliae - animal behaviour - mating behaviour - host-seeking behaviour - feeding behaviour - oviposition

    Malaria remains a major global health problem with the burden of disease greatest in Sub-Saharan Africa. The strategies for malaria control differ throughout the world according to levels of endemicity and the magnitude of disease but the focus remains either to control malaria parasites or vectors. A high degree of drug resistance and the absence of malaria vaccines are a major hindrance to control of the disease. In such circumstances, vector control becomes an alternative and has remained the most effective means to prevent malaria transmission. Contemporary adult mosquito control is almost exclusively based on indoor application of chemical insecticides in the form of indoor residual spraying (IRS) of walls and ceilings and insecticide-impregnated bed nets. However, sustainable use of chemicals is undermined by problems of insecticide resistance in mosquito populations, environmental contamination and risks to human health. Biological control based on fungal pathogens has shown potential to complement existing vector control methods. The entomopathogenic fungi (EPF) Metarhizium anisopliae and Beauveria bassiana have demonstrated ability to infect, kill and reduce the survival of malaria vectors. However, the effect of EPF on the behaviour of malaria vectors has not been fully addressed.

    This thesis was designed to provide baseline information on mosquito-fungus interaction focusing on the efficacy of entomopathogenic fungus M. anisopliae ICIPE 30 on the important life-history behaviours of the African malaria vector Anopheles gambiae Giles sensu stricto under laboratory and semi-field conditions. The information is important to facilitate the further development of malaria vector control based on biological control agents. Host-seeking, sugar-feeding, mating and oviposition were the behaviours investigated. Since mosquito-fungus contact is crucial for infection with EPF, a paper sheet (28.6 × 14.3 cm) lined inside a plastic cylinder (9-cm diameter and 15-cm height) was developed as a cost effective method of infection. Moreover, 0.1 g (approx. 1011 conidia/m2) of dry conidia and 6 hr exposure time sufficient for An. gambiae to pick up large numbers of conidia were established to cause high pathogenicity (Chapter 3). As the impact of EPF on insect behaviour was reported to occur at least three days post-exposure to fungal pathogen (Chapter 2), all experiments were conducted with a special focus on mosquitoes three days post-exposure to fungus. It is, however, important to mention that on average 50% of the mosquitoes died on the third day after fungal exposure (Chapter 3) and only those that survived were used for behavioural assays.

    The host-seeking capability of An. gambiae mosquitoes is an important parameter in the vectorial capacity equation. At short-range (1 m from host) assessment using a dual-choice olfactometer under semi-field conditions, infection with EPF strongly reduced the host-seeking response of mosquitoes, but did not impair their olfactory-based capability to discriminate between hosts (Chapter 4). At medium-range, using experimental cages (3 x 3 x 2 m) under laboratory conditions, fungal infection reduced the host-seeking response and feeding propensity of female An. gambiae mosquitoes (Chapter 7) whereas at long-range (7 m from host) inside a semi-field enclosure, infection with EPF sharply reduced the house-entry response and the hourly human-biting responses of host-seeking mosquitoes indoors and outdoors (Chapter 5). Plant sugar feeding is an important component in the biology of mosquitoes and is the main priority for both sexes at emergence. Infection with fungal pathogen strongly reduced the survival and sugar-feeding propensity of both sexes of the malaria vector An. gambiae but did not affect their potential to feed and digest meals (Chapter 6). Mating behaviour plays a key role in population growth. The activity takes place after sugar feeding and thereafter, the females search for their blood meal host. Infection with M. anisopliae strongly reduced multiple mating propensity and the mating performance of adult male An. gambiae mosquitoes in a large arena such as a screenhouse. Although this resulted in a reduction in the number of females inseminated, it facilitated the transfer of fungal conidia to conspecific healthy females during mating (Chapter 8). Finally, after blood meal intake, the females prepare to lay eggs. Infection with M. anisopliae reduced the oviposition propensity of female An. gambiae mosquitoes although the number of eggs laid remained unaffected (Chapter 7).

    In conclusion, these findings demonstrate that the entomopathogenic fungus M. anisopliae alters the major life history behaviours of An. gambiae mosquitoes. This is possible because the fungus strongly impairs flight performance of mosquitoes that makes the insect less able to fly and engage in host-seeking, sugar-feeding, mating and oviposition behaviours. The high mortalities observed in the early days of infection prior to conducting behavioural assays, mortalities observed while conducting behavioural assays and a reduction in behavioural response of M. anisopliae-infected mosquitoes collectively are likely to have a significant impact in suppressing a vector population. The susceptibility of male mosquitoes to fungal conidia opens a new strategy for mosquito vector control. Overall, this thesis has demonstrated that EPF may be a good complement to other mosquito vector control tools for the reduction of mosquito bites, and transmission of malaria and other mosquito-borne diseases.

    The role of skin microbiota in the attractiveness of humans to the malaria mosquito Anopheles gambiae Giles
    Verhulst, N.O. - \ 2010
    Wageningen University. Promotor(en): Willem Takken; Marcel Dicke. - S.l. : s.n. - ISBN 9789085858300 - 236
    anopheles gambiae - mens - geurstoffen - bacteriën - lokstoffen - gedrag bij zoeken van een gastheer - vluchtige verbindingen - in vitro - in vivo experimenten - anopheles gambiae - man - odours - bacteria - attractants - host-seeking behaviour - volatile compounds - in vitro - in vivo experimentation
    Malaria is one of the most serious infectious diseases in the world. The African mosquito Anopheles gambiae sensu stricto (henceforth termed An. gambiae) is highly competent for malaria parasites and preferably feeds on humans inside houses, which make it one of the most effective vectors of the disease. Human body odours are presumably the most important cues that enable An. gambiae to find its host. Knowledge on the odours that mediate the host-seeking behaviour of malaria vectors is expected to contribute to novel intervention methods for disease control.
    The skin microbiota plays an important role in the production of human body odours and the human microbial and chemical signature displays a qualitative and quantitative correlation. Several studies have indicated a possible role of skin bacteria in the production of volatiles attractive to mosquitoes. The principle goal of this thesis was to understand the role of the human skin microbiota in mosquito-host interactions and to identify which compounds produced by these micro-organisms are involved in the attraction of An. gambiae to humans.
    Skin bacteria isolated from human feet and grown in vitro on agar plates attracted An. gambiae and this attraction was affected by incubation time and dilution of the skin microbiota. Semi-field experiments showed similar results and field experiments in Kenya suggested that skin bacterial volatiles also attract other disease vectors. Volatiles produced by five bacterial species common on the human skin showed that volatile blends produced by some species, including Staphylococcus epidermidis, were more attractive than blends produced by other species. Volatiles produced by Pseudomonas aeruginosa did not affect the behaviour of An. gambiae.
    Analysis of the volatiles produced by human skin microbiota grown in vitro led to the identification of 16 compounds, the majority of which had an effect on An. gambiae behaviour. 3-Methyl-1-butanol enhanced the attractiveness of a synthetic blend by a factor of three, and could be used to increase mosquito trap catches for monitoring or vector control purposes. 2-Phenylethanol decreased mosquito catches of a synthetic blend and may act as a spatial repellent.
    In order to examine the interaction between the microbiota on the skin and human attractiveness to mosquitoes, skin emanation and skin microbiota samples were taken from 48 individuals. The skin emanations from the individuals varied significantly in attractiveness to An. gambiae and several compounds originating from the skin were associated with individuals that were highly attractive or less attractive to mosquitoes. Individuals with a higher abundance of bacteria on their skin were more attractive to An. gambiae, whereas individuals with a higher diversity of skin microbiota were less attractive. Staphylococcus spp. were associated with individuals that were highly attractive and Pseudomonas spp. with individuals that were less attractive to mosquitoes. Human Leukocyte Antigens (HLA) genes are considered to influence the human body odour profile and HLA profile analysis of the 48 human individuals indicated that these genes may also affect the attractiveness of humans to mosquitoes.
    The studies described in this thesis show that volatiles produced by the human skin microbiota play an important role in the host-seeking behaviour of An. gambiae and the abundance and composition of the skin microbiota determine an individual’s attractiveness to mosquitoes. Optimised blends of the compounds identified can be used in push-pull strategies for the manipulation of mosquitoes, thereby reducing the number of malaria mosquitoes, the human-biting frequency, and the intensity of Plasmodium transmission. Research on the role of skin microbiota in the host-seeking behaviour and host preference of biting insects may lead to a better understanding of vector-host interactions and contribute to the fight against vector-borne diseases.

    Sensory and behavioural responses of the malaria mosquito Anopheles gambiae to human odours
    Qiu, Y.T. - \ 2005
    Wageningen University. Promotor(en): Joop van Lenteren, co-promotor(en): Joop van Loon; Willem Takken. - [S.l.] : S.n. - ISBN 9789085042921 - 210
    anopheles gambiae - culicidae - diergedrag - geurstoffen - reuk - mens - malaria - lokstoffen - gedrag bij zoeken van een gastheer - anopheles gambiae - culicidae - animal behaviour - odours - smell - man - malaria - attractants - host-seeking behaviour
    Malaria is one of the most serious human diseases, affecting between 300 and 600 million people per year and killing, on average, two children per minute. In tropicalAfricathe mosquito Anopheles gambiae Giles sensu stricto is responsible for much of the transmission of malaria parasites between humans. This mosquito species preferably feeds on human blood, rests inside human houses and breeds close to human dwellings, making it an effective malaria vector. The major cues guiding Anopheles gambiae females to their human hosts are volatiles emanating from the human body. The main aim of the present thesis was to investigate the chemical components in human emanations that play a role in the host-seeking behaviour of this mosquito species and how these human odours are perceived by the olfactory system of the mosquito. The knowledge obtained can be applied in developing odour-baited traps that can be used to protect humans from being bitten by mosquitoes or to decrease the chance of being bitten by mosquitoes and to provide an alternative for the traditional but questionable "human landing" method in the investigation of mosquito population size.

    Glass beads to which skin emanations from human hands had been transferred elicited a level of attraction similar to a human hand ( Chapter 2 ). The attractiveness of these handled glass beads faded away four hours after transfer onto the beads. The headspace of handled glass beads elicited a dose-dependent EAG response. Glass beads provided a suitable neutral substrate for the transfer of human odour to enable the investigation of behavioural and electrophysiological activities of An. gambiae exposed to these odours and to allow chemical analysis of the human skin emanations by gas-chromatography-mass spectrometry performed in a twin-project.

    To study the chemical basis for the inter-individual differences in human attractiveness to mosquitoes , emanations from 27 human individuals, collected on glass beads, were tested against ammonia in a dual-choice olfactometer to establish the degree of attractiveness to An. gambiae ( Chapter 3 ). There were clear differences in the trap entry response as well as in the attractiveness relative to that of ammonia between the skin emanations of different volunteers. Consistency of the differences was observed when emanations of the three most and the three least attractive volunteers were tested pair-wise. Emanations from males elicited a higher trap entry response than those from females. Odours of younger volunteers significantly raised the trap entry response and were preferred over odours from older volunteers. Electroantennogram responses to skin emanations from volunteers with different behavioural attractiveness were not in all cases positively related to the behavioural response level, suggesting the involvement of repellent components.

    Stockings worn by humans were previously shown to be highly attractive to females of An. gambiae . The headspace of nylon stockings was collected and analysed with gas chromatography coupled on-line to electroantennography (EAG). EAG responses were detected consistently at 23 retention times, and 14 compounds that elicited such EAG responses were tentatively identified. These compounds, however, were not of typically human origin ( Chapter 4 ).

    Ammonia, L-lactic acid and a mixture of carboxylic acids were previously found attractive to An. gambiae. These compounds are all present in human skin odours, therefore a mixture of these components was studied in a dual-choice olfactometer ( Chapter 5 ). Ammonia was an attractant on itself, whereas lactic acid alone was not attractive. Carboxylic acids, offered as a mixture of 12 compounds, were repellent at the concentration tested. The addition of ammonia to the carboxylic acid mixture overruled the repellent effect of the latter. Combining ammonia with either lactic acid or the carboxylic acid mixture did not enhance the attractiveness of ammonia alone. However, a synergistic effect was found when ammonia, lactic acid and the carboxylic acids were applied as a blend.

    Human odour compounds that elicited electrophysiological or behavioural responses were tested in combination with ammonia + L-lactic acid against ammonia alone ( Chapter 6 ). The results showed that C3-C8 and C14 carboxylic acids augmented the attractiveness of ammonia + lactic acid at certain concentrations, whereas alcoholsketones4-ethylphenol and indole only reduced the attractiveness at the concentrations tested. For some compounds, no effect was found at any of the concentrations tested.

    Based on the behavioural and electrophysiological findings, a field study in The Gambia (West Africa) was carried out to investigate the efficiency of mosquito traps baited with synthetic odour blends or human odour ( Chapter 7 ). This study showed that odours released from counterflow geometry (CFG) traps baited with up to nine compounds that were mixed during release were in many cases more attractive than odours from a tent occupied by a human. Carbon dioxide substantially increased the catch of the CFG traps for all mosquito species. CFG traps baited with the mixture of ammonia + lactic acid + 3-methyl butanoic acid + CO 2 resulted in the highest catches for most mosquito groups; the mixture is considered to be a promising candidate odour blend in the control of nuisance mosquitoes. Experiments with traps indoors showed that one odour mixture, consisting of ammonia + lactic acid + CO 2 + geranyl acetone + indole + 4-ethyl phenol was more attractive for An. gambiae than the control odour; this mixture holds promise for further experiments under conditions of higher An. gambiae abundance and for implementation in vector control programs.

    Using a single sensillum recording method, an electrophysiological study on the olfactory neuron responses of female An. gambiae mosquitoes was undertaken ( Chapter 8 ). Six functional types of sensilla trichodea and five functional types of sensilla basiconica (grooved peg sensilla) were identified. "Generalist" ORNs that are tuned to a broad range of odours were found in sensilla trichodea subtype E, whereas "moderate specialist" ORNs that are tuned to a narrow range of odours were found in subtype C and grooved peg sensilla, with two "extreme specialist" ORNs tuned to only one odour. There was overlap in response spectra between sensilla trichodea E and C or grooved peg sensilla, but no overlap was found between sensilla trichodea C and grooved peg sensilla except that both responded to ammonia. Neurons associated with the same sensillum tended to respond to similar odour stimuli but with different sensitivities. Neurons in grooved peg sensilla were tuned to more polar compounds including the important behavioural attractant ammonia and its synergist lactic acid, responses to which were only found in grooved peg sensilla. Phenols were among the most effective stimulants for several neuron types belonging to different functional classes. Across-fibre patterning is the most plausible coding principle operating in the olfactory system of this mosquito species.

    After a blood meal, female mosquitoes minimise host seeking activity and rest during egg maturation. To investigate whether the sensitivity of olfactory neurons changed after a blood meal and whether these changes correlate with the observed behavioural change, we compared the responses of ORNs in sensilla trichodea and grooved peg sensilla 2 - 24 h post blood meal with that of mosquitoes that had not fed on blood ( Chapter 9 ). Three instead of two functional types of sensilla trichodea E were found following a blood meal. A functional type that had not been detected in mosquitoes deprived of blood was found repeatedly. The most responsive neuron of the "new" functional type of sensillum showed a high sensitivity to indole. This neuron was also highly responsive to C6-9 carboxylic acids and moderately responsive to the human-specific odour compounds 7-octenoic acid and 3-methyl-2-hexenoic acid. These results indicate that changes in sensitivity and response profile of ORNs as a result of a blood meal are involved in modulating behaviour of An. gambiae females.

    The main conclusions from this thesis can be summarised as follows. This thesis provides additional evidence that chemical cues play a substantial role in the host attraction of An. gambiae (Chapter 2) and that skin emanations alone contribute significantly to inter-individual differences in attractiveness of humans to mosquitoes (Chapter3). The GC-EAG method can be used in the detection of kairomones used by An. gambiae , but a suitable substrate for collecting odours is essential (Chapter 4). Synergism was demonstrated to operate between ammonia, lactic acid and a mixture of carboxylic acids in attracting females of An. gambiae (Chapter 5) and olfactometric studies demonstrated the dose-dependent effects of human odour components to An. gambiae in addition to ammonia and lactic acid (Chapter 6). The results of our field study provided evidence that mosquito traps baited with synthetic mixtures were superior to those baited with a human being, suggesting great potential of these traps in future malaria control programs (Chapter 7). Based on the response to several compounds, olfactory receptor neurones were classified into functional groups, providing fundamental information for future studies of these neurons (Chapter 8). Qualitative and quantitative changes were found in olfactory neuron responsiveness before and after a blood meal, suggesting the involvement of the peripheral nervous system in the modulation of mosquito behaviour observed in different physiological stages (Chapter 9).

    How to behave? : evolution of host-handling behaviour in the whitefly parasitoid Encarsia formosa
    Burger, J.M.S. - \ 2002
    Wageningen University. Promotor(en): J.C. van Lenteren; L.E.M. Vet; L. Hemerik. - S.l. : S.n. - ISBN 9789058087430 - 194
    encarsia formosa - organismen ingezet bij biologische bestrijding - insectenplagen - parasitoïden - gedrag bij zoeken van een gastheer - voedingsgedrag - evolutie - aleyrodidae - encarsia formosa - biological control agents - insect pests - aleyrodidae - parasitoids - host-seeking behaviour - feeding behaviour - evolution

    The main aim of evolutionary ecology is to explain the adaptation of form, function and behaviour of organisms to their environment. In this thesis, I studied host-handling behaviour of the whitefly parasitoid Encarsia formosa form such an evolutionary point of view. This parasitoid is applied as biological control agent against whitefly pests in greenhouses. Previous studies revealed mechanistic explanations for the parasitoid's behaviour. Here I focussed on functional explanations for the parasitoid's decision to reject, feed upon or parasitise a host. Since host feeding yields nutrients for egg maturation but destroys an opportunity to oviposit, the decision between host feeding and oviposition reflects a life-history trade-off between current and future reproduction.

    First, manipulation experiments were conducted to seek for evolutionary benefits of destructive host feeding over non-destructive feeding on host-derived honeydew. The first experiments suggested that honeydew could be an advantageous alternative to host feeding. Honeydew did have a positive effect on egg load and estimated number of eggs matured within 20 and 48 hours, whereas a host feeding did not. Host feeding did not positively affect survival when honeydew was supplied. In the second experiments the observation period was extended. Even in the presence of honeydew, host feeding did have a positive effect on the number of ovipositions per hour of foraging per host-feeding attempt, without affecting parasitoid survival or egg volume.

    To understand the conditions under which E. formosa has evolved, fieldwork was carried out in Costa Rica to quantify natural whitefly densities and distributions. The number of hosts on the lower side of a leaflet of an average plant within an average spot along an average transect could be described by a Poisson distribution with mean and variance equal to 0.241. This mean was most variable at plant level. Spatial dependence between numbers of whiteflies on leaves was detected within individual plants and within a 100-m transect. Thus, host density in the field was low compared with pest densities, but aggregation occurred at several spatial scales.

    A dynamic state-variable model was developed to predict optimal host-handling decisions, which maximise lifetime reproductive success, in relation to host density and parasitoid's life expectancy. Random decisions resulted in only 35 to 60 % of the lifetime reproductive success from optimal decisions. Host feeding was predicted to be maladaptive at presumable field conditions of low average host density and short parasitoid's life expectancy. Nutrients from the immature stage should be sufficient to prevent egg limitation. Both host density and parasitoid's life expectancy had a positive effect on the optimal host-feeding ratio. Explaining evolution of host-feeding behaviour under natural conditions may require incorporation of variation in host density, incorporation of parasitised host types or field data showing that parasitoid's life expectancy in the field is longer than assumed.

    In a semi-field set-up, parasitoids were allowed to forage at field host density in either a uniform or an aggregated host distribution. Contrary to predictions by the optimal-foraging model, parasitoids did host feed, i.e. upon about 11 % of the accepted hosts, even when host aggregation was ignored. In the model, host encounter rate was underestimated from host density. Nevertheless, there was also a host distribution by time interaction on host-feeding tendency. Host encounter decreased the leaving tendency on an average leaflet when time since latest host encounter was short, but increased the leaving tendency when time since latest host encounter was long, independent of host distribution. This suggested that parasitoids can forage efficiently at different host distributions.

    The value of future reproduction and the decision whether to host feed or parasitise strongly depends on the parasitoid's life expectancy. I hypothesised that a parasitoid's life expectancy in the laboratory is an overestimate of a parasitoid's life expectancy in the field. Several age determination methods were tested, using pteridine, wing fray and relative residual longevity. Unfortunately, none proved a useful technique to estimate the age of a field-caught specimen.

    The main conclusion is that host-handling behaviour in the whitefly parasitoid Encarsia formosa may have evolved as an adaptation to a spatially and possibly temporally heterogeneous environment. To truly understand the evolution of host-handling behaviour, future studies should focus on physiological, biochemical and genetic mechanisms; the effect of spatial and temporal heterogeneity in host availability on the risk of egg limitation; and the parasitoid's life expectancy in the field.

    Biocontrol in store: spatial and behavioural aspects of foraging by Uscana lariophaga, egg parasitoid of Callosobruchus maculatus, in stored cowpea
    Stolk, C. - \ 2002
    Wageningen University. Promotor(en): J.C. van Lenteren; A. van Huis; W. van der Werf. - S.l. : S.n. - ISBN 9789058087416 - 160
    vignabonen - callosobruchus maculatus - voorraadplagen - biologische bestrijding - vigna unguiculata - trichogrammatidae - parasitoïden - voedingsgedrag - gedrag bij zoeken van een gastheer - cowpeas - vigna unguiculata - callosobruchus maculatus - stored products pests - trichogrammatidae - parasitoids - feeding behaviour - host-seeking behaviour - biological control

    Cowpea ( Vigna unguiculata Walpers), an important crop for West African subsistence farmers, is often infested in storage by the bruchid beetle Callosobruchus maculatus Fabricius. The indigenous egg parasitoid Uscana lariophaga Steffan (Hym.: Trichogrammatidae) is responsible for substantial mortality of C. maculatus eggs and might therefore be used in a conservation strategy of biological control. This thesis focuses on foraging behaviour of U. lariophaga females in a spatial context. In stored cowpea, the bruchid oviposits in clusters. Uscana lariophaga is well adapted to such clusters, since it shows a strong arrestment response after an encounter with an unparasitized host. Previous investigations had already shown attraction of the parasitoid to host-related odours; it is now shown that directed search probably occurs at a short distance (4-6 beans) from the host patch. The probability that a host patch in stored cowpea is found decreases rapidly with increasing distance between the host patch and the site of release of the parasitoid. The 'critical distance' within which the host patch is quickly found by the parasitoid increases if more searching time is allowed. If an experienced parasitoid arrives in a host patch and encounters parasitized hosts, it is likely to superparasitize, but it will stop superparasitizing as soon as an unparasitized host has been encountered in the same patch. Superparasitism by experienced females is not due to failure in host discrimination, as appears from the fact that females adapt the sex ratio of their offspring during superparasitism. If no or few hosts are available, the parasitoid lives shorter than when many hosts are available. This reduced longevity at low host densities may be due to an increased walking activity at low host densities. Finally, the potential of a simulation model of U. lariophaga behaviour is shown, and consequences of behaviour for the prospects of biological control are discussed.

    Differential attractiveness of humans to the African malaria vector Anopheles gambiae Giles : effects of host characteristics and parasite infection
    Mukabana, W.R. - \ 2002
    Wageningen University. Promotor(en): J.C. van Lenteren; W. Takken; B.G.J. Knols. - S.l. : S.n. - ISBN 9789058087546 - 142
    anopheles gambiae - culicidae - gedrag bij zoeken van een gastheer - gastheerpreferenties - mens - reuk - gastheer parasiet relaties - plasmodium falciparum - malaria - anopheles gambiae - culicidae - host-seeking behaviour - host preferences - man - smell - host parasite relationships - plasmodium falciparum - malaria

    The results of a series of studies designed to understand the principal factors that determine the differential attractiveness of humans to the malaria vector Anopheles gambiae are described in this thesis. Specific attention was paid to the role of body emanations and infection (of humans) with the malaria parasite Plasmodium falciparum . The main findings of these studies are summarised in the following sections.

    Differential attractiveness of humans to Anopheles gambiae ( Chapter 1 )

    Although it has frequently been reported that human beings differ in their degree of attractiveness to mosquitoes, the principal causes that make certain individuals to be preferred more than others are not well known. This gap in knowledge has hindered the understanding of the transmission dynamics of malaria and other mosquito-borne diseases. From an epidemiological point of view, high malaria transmission rates are expected if mosquito vectors preferentially select infective humans for a blood meal, become infected, survive long enough for the parasites to develop to infective stages and proceed to bite uninfected individuals selectively. In terms of fitness, mosquito vectors would be better off if they select hosts that (1) are docile and less defensive so as to minimise feeding-associated risks of mortality, (2) have blood of a high nutritional value, (3) are free of (gametocyte) parasites, healthy and (4) have no anti-mosquito immunity. The answers to these epidemiological and fitness factors are still lacking. Furthermore, since host seeking is odour-mediated, the identification of chemical compounds responsible for attracting mosquitoes to their vertebrate hosts would help in developing traps that are useful for vector surveillance and control. Research in this area is rapidly advancing but has not produced tools for field application. It is felt that more research effort is needed so that new approaches towards understanding and combating disease vectors can be developed.

    Characterisation of mosquito blood meals using DNA markers (Chapters 2 & 3)

    The analysis of arthropod blood meals using molecular genetic markers was reviewed and forensic techniques borrowed from the Federal Bureau of Investigation (FBI), USA, used to evaluate the effect of blood meal size and extent of digestion on the ability to identify human DNA extracted from blood meals of Anopheles gambiae . The review recommended that proper and appropriate storage, determination of the concentration of host DNA and collection within few hours after ingestion are important parameters for improving the success of identifying blood meal sources of field-collected mosquitoes. Further, microsatellite markers were highlighted as being more appropriate than minisatellites in analysing blood meals that have been highly degraded e.g. through prolonged digestion. Also, mitochondrial DNA targets were recommended to be better than nuclear DNA targets for analysing blood meals that have been highly degraded. Blood meal size and (microsatellite) locus (analysed) were shown not to affect the success of amplifying human DNA extracted from blood meals of An. gambiae after having been digested for 0, 8, 16, 24 and 32 hours. However, a significant negative relationship between the time since ingestion and the success probability of obtaining positive PCR reactions among blood meals digested for between eight and 32 hours was demonstrated. There was no significant difference in the success probability of amplifying human DNA from blood meals of mosquitoes killed at zero and 8 hours after ingestion. The research demonstrated that not the quality of ingested blood, but the time since ingestion determined the success of blood meal analysis.

    Host characteristics and differential attractiveness of humans to An. gambiae (Chapter 4, 5 & 6)

    A tent olfactometer that accommodates complete humans as sources of host-seeking stimuli was designed, developed and tested. The olfactometer was used to study (1) differential attractiveness of humans to host-seeking An. gambiae and (2) how the differences, so elicited, are affected by human breath, body odour, heat and moisture. Nine human subjects were successfully ranked for their attractiveness to the mosquitoes based on (mosquito) responses to their complete body emanations encompassing body odour, heat and moisture. The nine subjects were classified into least (3 persons), medium (4 persons) and most attractive groups (2 persons). Breath was shown to reduce mosquito responses, whereas body odour was highly attractive. Breath was also shown to be an important contributor to between-person differences in relative attractiveness to An. gambiae . Whereas differential attractiveness of two human subjects for the mosquitoes could be demonstrated based on their total body emanations (breath plus body odour), the attractiveness of the two subjects did not differ significantly based on body odour alone. Body odour from either individual was consistently more attractive than total emanations from the other. The same results were obtained with another pair of individuals. It was concluded that breath, although known to contain attractive semiochemicals like carbon dioxide, may also contain compounds that inhibit attraction and may thus serve as an important contributor to between-person differences in relative attractiveness to this important malaria vector. The inhibitory effect of breath was postulated to be allomonal as it benefits the emitter (human being) but does not harm the recipient (mosquito vector). Body heat and moisture were shown to have significant effects on the attraction of An. gambiae to humans. In general, An. gambiae was more attracted to the individual whose body emanations were warmer but less moist than those of an opposing counterpart, in choice experiments. It was concluded that body heat and moisture influence host-selection by An. gambiae at short range and that their effect is probably achieved through interaction with breath components.

    Parasite infection and differential attractiveness of humans to An. gambiae (Chapter 7 & 8)

    A rare window of opportunity allowed for the investigation of the effect of clinical symptoms and parasitaemia due to Plasmodium falciparum on variability in human attractiveness to the malaria mosquito An. gambiae using the tent olfactometer previously developed. The relative attractiveness of an individual who was always more attractive than a specific counterpart (when both were uninfected) was suppressed when he (the 'putatively' more attractive individual) displayed clinical symptoms (fever and profuse sweating) of malaria caused by P. falciparum . This finding provided both new and alternative evidence, within the intricate web of Anopheles-Homo-Plasmodium interactions, that the malaria parasite P. falciparum influences the olfactory signals produced by human hosts. Field studies, in which a twin pair of male humans was recruited as a follow-up to the olfactometer study, found that being positive with malaria parasites (trophozoites and possibly gametocytes of P. falciparum ) is associated with higher numbers of attracted mosquitoes ( An. gambiaesensu lato ). This effect was not a consequence of the time of the year and the higher number of mosquitoes present in that period. It was recommended that further investigations be carried out. If these results are confirmed to be true, this work will constitute the first evidence that P. falciparum is capable of enhancing its own transmission by manipulating the physico-chemical characteristics of its vertebrate hosts in such a way that infected individuals are preferentially selected as blood meal sources by host-seeking malaria vectors.

    What questions remain unanswered (Chapter 9)

    Much as this thesis has provided some answers to why humans differ in their degrees of attractiveness to mosquitoes ( An. gambiae ), many more questions remain unanswered. The key questions in point include the following: (1) How can we tell whether one is more or less attractive to host-seeking mosquitoes? (2) How do parasites affect the host-seeking behaviour of their insect vectors? and (3) How do evolutionary processes related to host-seeking behaviour affect the fitness of mosquito vectors? The answers to these questions and those to a myriad of others are still waiting to be resolved.

    Human skin emanations in the host-seeking behaviour of the malaria mosquito Anopheles gambiae
    Braks, M. - \ 1999
    Agricultural University. Promotor(en): J.C. van Lenteren; W. Takken. - S.l. : S.n. - ISBN 9789058081414 - 122
    anopheles gambiae - culicidae - gedrag bij zoeken van een gastheer - zweet - huid - mens - anopheles gambiae - culicidae - host-seeking behaviour - sweat - skin - man

    Malaria is an infectious disease caused by a parasite ( Plasmodium spp.) that is transmitted between human individuals by mosquitoes, belonging to the order of insects, Diptera, family of Culicidae (mosquitoes) and genus of Anopheles (malaria mosquitoes). Mosquitoes feed on humans (and other animals) because they need blood for their reproduction. Like most other haematophagous insects, only the female mosquitoes bite and use the protein-rich blood meal for egg development. Whilst feeding on a person infected with malaria, the mosquito can be pick up the parasites from the blood stream. After a developmental period in the mosquito, the parasites can be transmitted to another person when the mosquito takes a next blood meal. Thus, malaria transmission depends largely on the characteristics of the mosquito population. Knowledge about the ecology, behaviour, infection level and size of the mosquito population is essential for the development, implementation and evaluation of control programs. Development of an adequate trapping device for monitoring the mosquito population is of high priority for ecological and epidemiological studies.

    Malaria is one of the most important human parasitic infectious diseases and one third of the world population is under threat of the disease. Most victims are found in the sub-Saharan countries of Africa. The Afrotropical malaria mosquito, Anopheles gambiae sensu stricto, is the most important vector since it strongly prefers to feed on humans. Like most anopheline species, An. gambiae s.s. is nocturnal and its host-seeking behaviour is mainly odour-mediated. Consequently, odour-baited traps are considered as possible monitoring devices. However, despite the important role of this mosquito in malaria transmission, knowledge regarding host odour components (or kairomones) that bring about the attraction to humans is limited. For the development of odour-baited traps, attractive host odours need to be identified. In this thesis a behavioural ecological investigation to the source, identification and production of kairomones for An. gambiae (henceforth simply termed 'malaria mosquitoes') is described.

    Source of kairomones for malaria mosquitoes

    Since the beginning of the century it has been recognised that malaria mosquitoes utilise host odours in their host-seeking behaviour. The source of these olfactory stimuli is expired air, the skin or both. Carbon dioxide present in expired air is an important kairomone for many haematophagous insects. For this reason carbon dioxide is often used in odour-baited traps. From field research (Chapter 2) we learnt that malaria mosquitoes can find their host in the absence of breath, and, thus, the presence of carbon dioxide is not compulsory for finding a host. This suggests that volatiles from the skin of the host also play a role in the attraction of malaria mosquitoes. The addition of skin volatiles to a carbon dioxide baited trap will probably bring about higher trap catches. Moreover, for logistic reasons, an odour-baited trap without presence of carbon dioxide is preferable. Carbon dioxide is highly volatile and can be delivered only by gas cylinders or dry ice (= frozen carbon dioxide), which is impractical in the African field situation. The composition of body odour is complex: more than 300 components have been identified. However, a synthetic blend of the complete human odour has not yet been synthesised. For this reason, the identification of some important components that attract malaria mosquitoes was initiated. A prerequisite for the identification was the entrapment of natural skin emanations separate from the skin. Sweat appeared to be an attractive complex olfactory stimulus since it is not artificial but rather true to nature (Chapter 4 and 5) in the bioassays in the laboratory and it forms the 'heart' of the thesis.

    The identification of kairomones for malaria mosquitoes

    Sweat was collected from the foreheads of a number of volunteers, who performed exercises on a hometrainer in a warm and humid room. The behavioural response of the malaria mosquitoes to this fresh sweat was rather variable; they were attracted to some fresh sweat samples (Chapter 8 and 9) but not to others (Chapter 5 and 7). However, the response of the mosquitoes to sweat that had been incubated for two days at body temperature was stable, and all incubated sweat samples were attractive to the mosquitoes. It appeared that the incubation released volatile components that were attractive to mosquitoes. Sweat is basically a watery solution of lactic acid, urea and ammonia. After incubation the lactic acid and urea concentration had decreased and the ammonia concentration showed a distinct increase (Chapter 8 and 9). For this reason ammonia was tested in the bioassay. For the first time, malaria mosquitoes were attracted to a single component other than carbon dioxide, namely ammonia. Lactic acid is an essential kairomone for another mosquito species, the yellow fever mosquito Aedes aegypti . However, the selective removal of lactic acid from the sweat did not affect the reaction of malaria mosquitoes. Therefore, we conclude that lactic acid is not an essential component of attractive odour blends for malaria mosquitoes. Urea was not tested, as it is not volatile. The fact that attraction was sometimes found to the fresh sweat with a rather low concentration of ammonia indicates that components other than ammonia also play a role in the host-seeking behaviour of malaria mosquitoes. The identity of these components needs further exploration.

    The production of kairomones for malaria mosquitoes

    The skin of humans (and other animals) forms a good habitat for some microorganisms (bacteria and fungi), together called the skin microflora. During the collection of sweat samples, microorganisms are taken up with the sweat. An exponential growth of microorganisms in the sweat samples is found during incubation (Chapter 4, 6 and 7). Sweat constituents are broken down into more volatile components by the growing microorganismal population and this appears to bring about the enhancement of the attractiveness of sweat to malaria mosquitoes (Chapter 9). Such processes also probably play a role in the production of kairomones on the skin. However, this needs further exploration.

    Conclusions

    Kairomones for malaria mosquitoes originate from the human skin, in addition to carbon dioxide from exhaled air. Microorganisms of the skin flora play an important role in the production of kairomones for malaria mosquitoes An. gambiae s.s.. Ammonia is one of the components responsible for the attraction of malaria mosquitoes to sweat.

    Odour - mediated host - seeking behaviour of the Afro-tropical malaria vector Anopheles gambiae Giles
    Knols, B.G.J. - \ 1996
    Agricultural University. Promotor(en): J.C. van Lenteren; W. Takken. - S.l. : Knols - ISBN 9789054854876 - 213
    Culicidae - Anopheles - malaria - diergedrag - gedrag bij zoeken van een gastheer - nematocera - Culicidae - Anopheles - malaria - animal behaviour - host-seeking behaviour - nematocera

    Malaria remains the single most important parasitic disease of man in tropical regions of the world. It is estimated that 40% of the world's population, in 102 countries, is at risk from the disease. Some 100-200 million cases occur annually worldwide, of which 90 million in Africa, with 1-2 million deaths.

    Efforts to control malaria by chemoprophylactic and/or curative drugs are seriously jeopardized due to widespread parasite resistance, and Anopheles mosquito vectors are rapidly developing physiological resistance against insecticides commonly used for indoor spraying or impregnation of bed nets. Trials with the malaria vaccine SPf66 have shown a 31% morbidity reduction in Tanzania but hardly had an effect on disease in The Gambia. The deteriorating malaria situation in the world thus urges the development of new control methods which need to be environmentally safe, socio-economically acceptable, and applicable for use within primary health care systems.

    The African mosquito Anopheles gambiae sensu stricto Giles, is a prime vector of malaria due to its distinct preference for biting man (anthropophily). It generally feeds indoors (endophagy) between midnight and dawn and rests indoors after feeding for the first 1-2 days of its gonotrophic cycle (endophily). High survival rates and (seasonal) occurrence in high densities further increase its vectorial capacity. DDT resistance has been reported in several countries and other control measures such as environmental management are not possible due to its breeding in a wide variety of (sunlit) water bodies. Exactly how a female An. gambiae s.s. exploits human-related cues to orient itself while locating a host at night remains largely unknown. Field studies on closely related sibling species have shown that olfactory cues evoke behavioural responses from mosquitoes over considerable distances, but the identity of these kairomones has not been established. Furthering the knowledge of this process might ultimately result in the development of new control strategies analogous to those for tsetse flies. The host-seeking behaviour of tsetse flies has received considerable interest over the last two decades and has resulted in bait systems now widely applied for monitoring and control of trypanosomiasis in several African countries.

    The goal of the work presented in this thesis was to gain more insight in the chemical ecology of An. gambiae s.s. and the resulting interactions with its human host. This encompassed identification of kairomones and the development of tools to study their effect on mosquito behaviour in the laboratory and field. The work was carried out in collaboration with research groups in the United Kingdom, Italy and Tanzania within a European Community funded project titled: 'Behavioural studies on malaria vectors'.

    Haematophagy, disease transmission, and factors affecting host-seeking by mosquitoes(Chapter 1)

    The two possible evolutionary routes leading to haematophagy are described, these being the prolonged association with vertebrates and morphological pre-adaptation for piercing the skin surface. Three associations, depending on the time spent on the host, exist between vertebrates and haematophagous Diptera and pathogen transmission is suggested to benefit most in association with temporary ectoparasites. The host-seeking process, defined as any kind of behaviour that increases the chance to encounter a suitable host followed by blood-feeding, is categorised into two classes, namely 'appetitive search' behaviour prior to contacting host-related cues and activation/orientation upon perceiving these. Three classes of cues produced by hosts are defined namely olfactory, physical and visual cues. The relative importance of cues belonging to different classes depends on the behavioural ecology of the insect, and is affected by factors such as circadian rhythm and host range. Carbon dioxide, expired by all vertebrates, has been incriminated to affect the host- seeking process of all Dipteran blood-feeders. It is thought that this chemical dominated the olfactory-mediated part of host-seeking during the evolution of haematophagy. Specialism towards feeding on specific hosts required olfactory responses to odours other than carbon dioxide typifying the identity of those hosts. To date, only few of these compounds have been identified and for mosquitoes none of these are used on a large scale in the field.

    An. gambiae s.s. belongs to a complex of six sibling species with large variations in host-specificity and feeding habits. Host-specificity is genetically determined and differential responses to olfactory cues from different hosts have been observed in the field. Although carbon dioxide has been found to elicit responses from the various siblings, this chemical obviously can not be responsible for these differential responses. Odours other than carbon dioxide must influence this host-selection process and identification of kairomones for the highly anthropophilic An. gambiae s.s. is addressed in subsequent chapters of this thesis.

    Selection of biting sites by mosquitoes on man: a new approach to kairomone identification(Chapters 2 & 3)

    A preliminary study on the selection of biting sites by haematophagous Diptera showed that only few species display a random biting pattern on their hosts. The vast majority preferentially bites specific parts of the body, and these preferences have been attributed to factors intrinsic to the behaviour of the insect (e.g. visual responses to extremities of hosts) or to the behaviour of the host (e.g. defensive behaviour). By studying the selection of biting sites by five mosquito species on the same volunteer under identical experimental circumstances it was observed that a) different species prefer different parts of the body, b) that for at least three of these species this process was influenced by odour emanating from the preferred biting region and c) that these preferences might not only reveal the origin of chemical cues involved in hostseeking, but that the biting pattern can provide information on the range of hosts fed upon. An. gambiae s.s. showed a strong preference for biting the legs and foot region (76% of all bites) and alteration of the odour from this region, by washing the feet with a bactericidal soap, significantly altered this preference (to 36%). Likewise, the significant preference of An. atroparvus and An. albimanus for biting the face (50 and 49% of all bites respectively) was influenced by breath since the pattern of biting changed dramatically after breath was removed from the host (to 19 and 20% respectively).

    Studying the selection of biting sites by mosquitoes was proven to be a new method to reveal the origin of olfactory cues influencing their host-seeking behaviour.

    Odour-mediated host-seeking behaviour and kairomone identification: a laboratory approach(Chapter 4,5 & 6)

    At the onset of the present study it was realised that a reliable laboratory bioassay was essential to facilitate rapid screening of behavioural effects of (human) odours on An. gambiae s. s.. The then existing windtunnel, which was constructed to study responses of individual females, was accordingly modified and resulted in a system closely resembling conventional dual-port olfactometers used for screening behavioural effects of odours on the yellow fever mosquito Aedes aegypti . Using this system, test odours were classified as 'attractive' whenever trap catches were significantly higher (using chi-squared) then those of control traps. Carbon dioxide at a human equivalent (4.5% in clean air) caught 4 times more An. gambiae s.s. than control traps baited with clean air only.

    Results from chapters 2 & 3 showed that foot odour played a role in the selection of biting sites for An. gambiae s.s. and breath influenced this process for An. atroparvus/ An. albimanus. Since carbon dioxide at a human equivalent was clearly 'attractive' it was assessed whether whole human breath was similarly attractive. It was shown that human breath (of three volunteers) was not attractive when tested against the carbon dioxide concentration (3.30-3.65%) of that breath only. When breath of the same volunteers (with carbon dioxide concentrations between 3.28-3.80%) was tested against clean air this was also not attractive, nor was carbon dioxide at a concentration of 3.56%. The difference in response to this latter concentration of carbon dioxide and the highly 'attractive' 4. 5% concentration was attributed to the dilution of the gas to levels which did no longer result in catches significantly different from those in the control traps.

    The headspace of Limburger cheese, to the human nose reminiscent of foot odour, was significantly attractive to An. gambiae s.s. from East and West Africa. GC and GC-MS analyses of the acid fractions of Limburger cheese, human sweat and toe scrapings showed strong similarities in their aliphatic fatty acid content and composition. The fact that An. gambiae s.s. responded to odours of bacterial origin (produced by Brevibacterium linens on the cheese) strongly suggests that the human skin microflora is (at least in part) responsible for the production of kairomones for this species. Nineteen saturated and unsaturated fatty acids, ranging in carbon chain length from C 2 -C 18 were identified in the acid fraction of the cheese, whereas twelve of these were present in its headspace. In the windtunnel bioassay, the undiluted acid extract was repellent, but became significantly attractive at concentrations of 5*10 -4to 10 -7. A synthetic blend of the twelve acids identified in the headspace was significantly attractive at a concentration of 10 -8. Electroantennographic studies showed significant and reproducible responses to (saturated) cheese headspace, the synthetic mixture, and all individual acids but hexadecanoic acid. These fatty acids form the first group of kairomones identified for An. gambiae s.s.

    Host attractiveness and odour-mediated host-seeking behaviour of mosquitoes: a field approach(Chapter 7,8,9 & 10)

    The study of the effects of kairomones on mosquito behaviour in Tanzania was initially hampered by the absence of sampling devices. Standard CDC light traps, commonly used for sampling malaria vectors indoors, were not applicable for outdoor use. Tents ('mobile huts) were modified for sampling mosquitoes by fitting exit traps and constructing artificial eaves for mosquito entry. These tents could be positioned at various sites, in randomized block designs, and could be washed which make them ideal for studying the attractiveness of different odours. Initially it was assessed whether three isolated individuals varied in their attractiveness to An. gambiae s.l., An. funestus, Culex quinquefasciatus and Mansonia spp. , which for all but the latter species was the case. These differences were attributed to the odour of the volunteers since this was the only variation factor not eliminated in the experimental setup. Also, this experiment showed for the first time that isolated individuals vary in their attractiveness to mosquitoes.

    In addition to these results it was found that different mosquito species show preferences for different individuals within a group. Catches of An. funestus and An. squamosus were inversely correlated for two individuals of the group. Furthermore, a negative correlation between group size and individual catch was observed, which suggests that mosquito sampling is more efficient if individuals are not grouped.

    The tents were also used to assess the relative importance of the physical presence of man versus his odour only in the number of mosquitoes attracted (and caught). This was done by positioning a man inside a bed net or underground in a pit from which odours were pumped into the bed net. Both treatments caught equal numbers of An. gambiae s.l. and An. funestus, which indicates that human odour is the main factor in attracting these species to a bait inside a house. When carbon dioxide (at a human equivalent) was released inside a bed net the catches were 9 and 27% of those by man for An. gambiae s.l. and An. funestus respectively. A five-fold increase in carbon dioxide dose increased the catch of the latter species to 69% but those of An. gambiae s.l. remained the same. The relative contribution of carbon dioxide to the overall attractiveness of man to An. gambiae s.l. was thus limited and underlined the importance of other human odours in host-seeking by this species.

    Electric nets (a grid of wires electrocuting insects when they contact them) have played a paramount role in the study of tsetse fly behaviour, and it was studied whether similar sampling systems were applicable for outdoor use, and more specifically to study responses to odours and analyse flight behaviour. Various designs were tested, and when baited with carbon dioxide, sampled all mosquito species present in the study area. Furthermore it was shown that primarily zoophilic mosquito species were attracted to human breath or carbon dioxide, but that the anthropophilic species (An. gambiae s.1. and An. funestus) hardly responded to these baits. Whole human odour baited nets caught higher numbers of these latter species, thus confirming the results from the tent experiments. A large cylindrical net was used to assess the flight direction of mosquitoes upon contacting carbon dioxide and showed that they engage in positive anemotaxis. The overall efficiency (number caught/number approached) of a rectangular electric net (28 x 40 cm, wires at 4 mm), when baited with carbon dioxide, was 42%. This work showed that electric nets can be used for outdoor sampling of mosquitoes, for analysing responses towards laboratory identified kairomones, and for the analysis of mosquito flight behaviour.

    Odour-mediated host-seeking behaviour of An. gambiae s.s. in relation to its human host: a case for adding the skin microflora and Plasodium parasites as interactants(Chapter 11)

    The general discussion reviews the results of the research in a broader context. The preceding chapters suggested that olfactory cues influencing the host- seeking behaviour of An. gambiae s.s. originate from the human skin. Their production is (at least partially) the result of the metabolic activity of the skin microflora. It is argued that the influence of the skin microflora on the interaction between man and mosquito is of such importance that this justifies the recognition of the skin microflora as a separate entity. In addition to this it is hypothesized that the influence of the Plasmodium malaria parasite may affect the production of infochemicals mediating the interaction between man and mosquito, or, alternatively, is responsible for the production of these allomones. It is concluded that the study of odour-mediated host-seeking behaviour of An.gambiae s.s. will benefit from analysing the interactions between man and mosquito within a multipartite, rather than the conventional bitrophic (man- mosquito) context.

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