Résultat de la recherche
A standardised in vivo and in vitro test method for evaluating tick repellents
The threat of transmission of Lyme borelliosis and tick-borne encephalitis by ixodid ticks has resulted in an increasing number of tick repellents coming onto the market. To allow proper evaluation of the efficacy of different types of compounds and their formulations, there is a need for standardised methods for testing ticks repellents. Ticks show a marked negative geotactic response following contact with a potential host, i.e., they climb up in order to locate attachment and feeding sites, whereas exposing ticks to repellents induces positive geotaxis, i.e., ticks walk downwards or drop off the treated host or substrate. We describe here complementary tests that employ these geotactic responses to evaluate repellents: one in vitro on a warm glass plate and the other on the lower human leg (shin). The compounds tested were DEET, EBAAP, icaridin, capric acid, lauric acid, geraniol, citriodiol, citronella essential oil and lavender essential oil, all non-proprietary ingredients of widely distributed tick repellent formulations.
In controls on both the warm glass plate and the human leg, the majority of Ixodes ricinus nymphs walk upwards. By contrast, in both the in vitro and in vivo tests, effective doses of repellents cause ticks to either walk downwards or fall off the substrates, termed here “affected ticks”. The ED75 values for affected ticks on the human leg indicate that the test products can be divided into three groups: (1) icaridin, EBAAP, DEET and capric acid with values between 0.013 and 0.020 mg/cm2, (2) citriodiol and lauric acid with values between 0.035 and 0.058 mg/cm2, and (3) geraniol, citronella oil and lavender essential oil with values between 0.131 and 1.58 mg/cm2. The latter three products can be considered as less effective repellents. The tests on the warm glass plate resulted in very similar efficacy rankings for the products tested in vivo, and the ticks’ behavioural responses also corresponded closely to those observed on the treated human leg. The ED75 values on the glass plate ranged from half to one sixth needed on the leg. The warm glass plate test thus provides a reliable alternative to human subjects for an initial evaluation of new repellents, and is particularly appropriate for testing products with still to be determined human toxicity and dermatological effects.
In vitro feeding assays for hard ticks
Prevention of tick bites and transmission of tick-borne pathogens requires the use of molecules that target physiological processes crucial to both tick and pathogen survival. These molecules are best tested in standardized in vitro assays. Because hard ticks require several days to feed to repletion, the development of in vitro feeding assays for these species is challenging. A standard and easily automated feeding assay has been developed for the tick Ixodes ricinus that involves feeding on blood through a membrane that mimics the elasticity of skin. The system can be adapted to feed other hard tick species in vitro. This assay permits, among others, investigations on the role of tick endosymbionts on tick survival, the identification of potential vaccine candidates and drugs, and the application of genomic tools in vitro, including RNA interference experiments.
In vitro assays for repellents and deterrents for ticks: differing effects of products when tested with attractant or arrestment stimuli
Most in vivo and in vitro tests with repellents or deterrents against ticks have not considered which sensory channel is being targeted. We have recorded the responses of two hard tick species (Acari: Ixodidae) in vitro to determine if such products can disrupt the perception of an attractant in a repellent assay or the perception of an arrestment stimulus in a deterrent assay. Ethyl butylacetylaminopropionate (EBAAP), N,N-diethyl-methyl-benzamide (deet), permethrin and indalone were chosen to test their capacity to inhibit the attraction of Amblyomma variegatum Fabricius to its aggregation-attachment pheromone. Vapours of each test product plus those from a synthetic blend of the pheromone were delivered to the walking tick in an air stream on a locomotion compensator. Neither EBAAP, deet, permethrin nor indalone could inhibit attraction of A. variegatum even when each of the test products was delivered at 106 times the pheromone. Indalone did decrease the attraction of A. variegatum to the pheromone and induced repulsion of A. variegatum when presented on its own in the air stream. The effect of permethrin, a sodium channel blocker, was also tested in a deterrent assay measuring the arrestment of Ixodes ricinus (L.) adults on its own faeces and faecal constituents. Permethrin deterred arrestment at doses of 670 fg/cm2 to 67 ng/cm2, i.e. at levels five times lower than the dose of chemostimuli present in the arrestment stimulus. This sensitivity to permethrin suggests that it acts via the contact chemoreception channel.
Standardizing Visual Control Devices for Tsetse Flies: West African Species Glossina tachinoides, G. palpalis gambiensis and G. morsitans submorsitans
Here we describe field trials designed to standardize tools for the control of Glossina tachinoides, G. palpalis gambiensis and G. morsitans submorsitans submorsitans in West Africa based on existing trap/target/bait technology. Blue and black biconical and monoconical traps and 1 m2 targets were made in either phthalogen blue cotton, phthalogen blue cotton/polyester or turquoise blue polyester/viscose (all with a peak reflectance between 450–480 nm) and a black polyester. Because targets were covered in adhesive film, they proved to be significantly better trapping devices than either of the two trap types for all three species (up to 14 times more for Glossina tachinoides, 10 times more for G. palpalis gambiensis, and 6.5 times for G. morsitans submorsitans). The relative performance of the devices in the three blue cloths tested was the same when unbaited or baited with a mixture of phenols, 1-octen-3-ol and acetone. Since insecticide-impregnated devices act via contact with flies, we enumerated which device (traps or targets) served as the best object for flies to land on by also covering the cloth parts of traps with adhesive film. Despite the fact that the biconical trap proved to be the best landing device for the three species, the difference over the target (20–30%) was not significant. This experiment also allowed an estimation of trap efficiency, i.e. the proportion of flies landing on a trap that are caught in its cage. A low overall efficiency of the biconical or monoconical traps of between 11–24% was recorded for all three species. These results show that targets can be used as practical devices for population suppression of the three species studied. Biconical traps can be used for population monitoring, but a correction factor of 5–10 fold needs to be applied to captures to compensate for the poor trapping efficiency of this device for the three species.
The Tick Blood Meal: From a Living Animalor from a Silicone Membrane ?
An artificial feeding unit with a reinforced silicone membrane to replace host skin provides ticks with a perch over blood with a tick attachment rate of 75-100%. Some 5 mg of an acaricide like fipronil is sufficient to establish survival curves over different doses down to ppb levels in blood. This in vitro feeding assay for hard ticks is more advantageous than in vivotrials on animals.
Behavioural and chemoreceptor cell responses of the tick, Ixodes ricinus, to its own faeces and faecal constituents
Ticks are ectoparasites of vertebrates and utilize a variety ofinfochemicals for host finding and acceptance as well as for intraspecific aggregation and mating responses. Individual male and female Ixodes ricinus, the vector of Lyme disease in Europe, readily arrest onfilter paper strips contaminated with their own faeces. I. ricinus also responds, but to a lesser degree, tofaeces-contaminated papers enclosed in metal mesh envelopes, i.e. without directly contacting the faeces, suggesting a role for volatiles in the arrestment response. The faecal constituents guanine, xanthine, uric acid and 8-azaguanine (a bacterial breakdown product of guanine) also caused arrestment of individual I.ricinus males and females. However, mixtures of these products induced arrestment of I. ricinus at doses one hundred fold lower than the lowest active dose of any of them tested singly. Saline extracts of faeces activated receptor cells in terminal pore sensilla on the first legtarsi of I. ricinus. One cell in these sensilla responded in a similar dose dependent manner to guanine and 8-azaguanine, whereas a second cell was more sensitive to lower doses of 8-azaguanine. The response threshold approached 100 fM for both cells. These findings suggest that faeces and faecal breakdown products are implicated in aggregation responses of I. ricinus. This may account for the clumped distribution of this ectoparasite on the ground and contribute to the high proportion of mated individuals recorded prior to host colonization.
The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes
Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.
An in vitro feeding assay to test acaricides for control of hard ticks
Animal husbandry could not be practised over large areas of the planet without acaricides. The prevention of tick bite and the transmission of diseases requires the use of pesticides, but this contributes to the development of tick resistance against acaricides. This drives the quest for new molecules that target physiological processes crucial to tick survival. In vivo trials involve multiple repetitions because of inherent variations between host animals, requiring large amounts of test products and ticks. An in vitro alternative should permit the testing of the ability of a product to restrict attachment and feeding by ticks at precise doses. In this paper an in vitro feeding system is described where the European tick Ixodes ricinus L. feeds on blood through a cellulose rayon-reinforced silicone membrane. The membrane Shore hardness is modified to imitate the elastic retraction forces of skin that ensure the closing of tick penetration sites on the membrane to prevent bleeding. Tick attachment (75-100%) is achieved by adding chemical and mechanical stimuli to the membrane. Survival curves for different doses of fipronil and ivermectin tested with the method showed highly reproducible acaricide effects within 5-7 days. Significant effects are recorded down to ppb levels in blood. Standardised tests can be made with blood from the same donor animal or culture medium under the membrane.
The Dynamics of an Avoidance Behavior by Ixodid Ticks to Liquid Water
Life stages of different tick species avoid walking on a wet surface surrounding a dry patch by systematically returning to the dry each time they contact the wet surface beyond the border with the tip of a first leg tarsus. Sequential analysis of the border behaviors shows that repetitive contact with the water increases the probability of walks astride the border. Ticks accept this unilateral contact with the water for longer intervals and eventually walk on to the wet surface after a combination of a short patch walk followed by a border walk which is longer than the foregoing ones. Staying time on a small circular patch is shorter than on a large one, arising probably from faster adaptation of peripheral receptors following a higher frequency of border contacts. However, an equal number of border reactions on patches of different sizes and shapes suggests that a counter in the CNS may also influence dry patch departure.