Activation of the ambusher tick Rhipicephalus microplus (Acari: Ixodidae) exposed to different stimuli

The present study aimed to evaluate the behaviour of larvae of Rhipicephalus microplus exposed to different stimuli. A Y-olfactometer was positioned vertically and R. microplus larvae were exposed to environmental air, CO₂ alone, N,N-diethyl-3-methylbenzamide (DEET) alone, and CO₂ combined with the repellents DEET and (E)-2-octenal. Tests were also conducted with the olfactometer positioned horizontally; in this case, however, only CO₂ was tested. In all tests conducted with the Y-olfactometer positioned vertically, CO₂ activated R. microplus larvae even in the presence of DEET and (E)-2-octenal, although activation was lower when these repellents were used. In the absence of CO₂, larval behaviour against DEET was similar to that of the larvae in the control group. In the tests performed with the olfactometer positioned horizontally, the larvae had no significant response to the presence of CO₂. The larvae were not attracted to or repelled by any compound tested in either the vertical or horizontal position of the olfactometer. The lack of horizontal displacement, attraction or repellence may have been a result of the ambush behaviour of this tick species. However, when larvae were exposed to stimuli and the olfactometer was positioned vertically, the interference of attractant and repellent stimuli in larval behaviour was assessed.     

Substituted phenols as repellents for male Costelytra zealandica (Goleoptera: Scarabaeidae) in mating flights

Methyl‐ and halogen‐substituted phenols were found to be strongly repellent to male Costelytra zealandica (White) beetles in mating flights. Phenols substituted in the 2‐position were stronger repellents than those substituted in the 3‐ or 4‐position. Some of these compounds reduced the effectiveness of a potent sex attractant source by > 90 %.     

Plant‐based compounds with potential as push‐pull stimuli to manage behavior of leaf‐cutting ants

Leaf‐cutting ants are a serious pest of young forestry plantations. Currently, the main control method is the use of broad‐spectrum insecticides, which have a negative effect on non‐target organisms and the environment. In this work, plant‐based compounds were evaluated in laboratory assays with Acromyrmex ambiguus Emery (Hymenoptera: Formicidae) for their potential use as repellent and attractant stimuli to be used in a push‐pull strategy. Farnesol, a sesquiterpene present in many essential oils, was tested as a repellent at doses of 10, 50, and 100 mg. Its distance of action was studied by comparing the repellent effect of farnesol in a situation in which ants had to touch the farnesol in order to reach the food source in comparison to when ants could reach the food source without getting into direct contact with it. Different parts of the orange fruit (pulp and peel) were evaluated and compared as attractants, given that citrus‐based baits are among the most popular attractants used. Results from laboratory bioassays indicated that farnesol is repellent at doses of 50 mg and acts upon contact or at a very short distance. Furthermore, orange pulp was more attractive than the peel, and volatile compounds were highly responsible for the attraction. When both stimuli were tested simultaneously in a laboratory experiment, repellency of farnesol was enhanced in the presence of orange pulp odor. When tested in a field push‐pull experiment, the results also showed a good repellent effect of farnesol as well as an attractant effect of the orange pulp. These results encourage long‐term studies with these substances in a field setting and suggest that repellents can be enhanced by the use of attractants to manage leaf‐cutting ants behavior.     

Role of the response oscillator in inverse responses of Halobacterium halobium to weak light stimuli.

Under certain conditions Halobacterium halobium organisms respond to a weak attractant light stimulus with a repellent response and to a weak repellent stimulus with an attractant response. The appearance of inverse responses depends on the stimulus strength, on the interval length between spontaneous reversals, and on the moment of stimulation during the interval. Although the cells are absolutely refractory to repellent stimuli for 500 ms after a reversal, repellent responses can be evoked even during that period if they are inverse responses to weak attractant stimuli. Simultaneous attractant and repellent stimuli cancel each other even when one of them leads to an inverse response, indicating that normal cellular signals occur at the site of signal integration. We postulate that the inverse responses are caused by certain properties of a cellular oscillator for which we previously postulated a role in response regulation and sensory control in halobacteria (A. Schimz and E. Hildebrand, Nature [London] 317:641-643, 1985).     

Negative chemotaxis in Dictyostelium and Polysphondylium

A method has been described to measure negative chemotaxis in the cellular slime molds directly and to purify the repellents. Conclusive evidence is given that negative chemotaxis exists in the cellular slime molds and that it occurs generally in Dictyostelium and Polysphondylium. Amoebae respond shortly after their exposure to repellents, which are secreted by vegetative and not by preaggregative cells. The amoebae are sensitive to repellents in both development stages and contain enzyme(s) to inactivate them. Cross reactions of different species indicate that there is more than one repellent, although it cannot be excluded that the variability in response depends on the balancing effect of attractants and repellents.     

The genetics of chemotaxis in Drosophila melanogaster: Selection for repellent insensitivity

Summary In a Y-unit maze wild-type flies of Drosophila melanogaster were tested for their chemotactic behavior reactions to insect repellents. Selection over 12 generations in two parallel experiments yielded two insensitive lines. Crosses indicated that the genes that were responsible for insensitivity were at least in part dominant. Lines selected for insensitivity to one repellent were also insensitive to a second repellent.     

Sensory adaptation mutants of E. coli.

The ability of E. coli to adapt to constant levels of attractant and repellent chemicals was studied by examining the patterns of flagellar movement in cells subjected to abrupt concentration changes. Wild-type bacteria exhibited transient responses to such stimuli, in support of previous findings. Nonchemotactic mutants of the cheX class responded to both attractants and repellents, but were unable to terminate these behavioral changes as long as the stimulating chemical was present. The sensory adaptation defect of cheX strains may be due to an inability to methylate several cytoplasmic membrane proteins that initiate changes in flagellar movement in response to chemoreceptor signals. Based on these results, possible mechanisms of stimulus transduction and sensory adaptation during chemotaxis are discussed.     

High‐throughput screening method for evaluating spatial repellency and vapour toxicity to mosquitoes

Spatial repellents are an essential tool for personal protection against mosquitoes that bite and transmit disease pathogens to humans. Current repellent screening methods, such as olfactometers and alternative choice tests, are complex systems that require a relatively large quantity of compound (mg). The present study validates a high‐throughput spatial repellent screening method using a glass tube that has the ends covered with netting, in addition to treated filters and plastic end caps. The apparatus occupies relatively little space, is easy to decontaminate, and requires small amounts of compound (μg). In a horizontal tube orientation, DEET (N,N‐diethyl‐meta‐toluamide), citronella oil and IR3535 had 1 h half repellent concentration (EC₅₀) values of 32, 32 and 298 μg/cm², respectively, against the Orlando strain of Aedes aegypti (L.) (Diptera: Culicidae). Vertical tube orientation increased EC₅₀ values by approximately two‐fold, except IR3535, which remained essentially unchanged. Transfluthrin showed concentration‐dependent spatial repellency (1 h EC₅₀ = 0.5 μg/cm²) without any knockdown, although only in vertical tubes. Transfluthrin showed 50% knockdown in 1 h at 0.5 μg/cm² and 50% mortality at 0.15 μg/cm² in horizontal tubes. In conclusion, this high‐throughput screening method is useful for assessing vapour toxicity and the spatial repellency of candidate molecules prior to semi‐field and field studies.     

Plants cultivated in Choco,Colombia, as source of repellents against Tribolium castaneum (Herbst)

Essential oils (EOs) of eight plants collected in Choco, Colombia, including Piper divaricatum, P. pseudolanceifolium, P. confertinodum, P. diazanum, Ocimum campechianum, Siparuna conica, Mikania micrantha and Hedychium coronarium, were analyzed by gas chromatography–mass spectrometry and tested as repellents against Tribolium castaneum, using the area preference method, after 2 and 4 h exposure. The main components found in EOs were methyl eugenol, trans-β-cariophyllene, methyl eugenol, α-pinene, δ-cadinene, γ-elemene, α-pinene and 1,8-cineol, for O. campechianum, P. pseudolanceifolium, P. divaricatum, P. confertinodum, P. diazanum, S. conica, M. micrantha, and H. coronarium, respectively. Best repellent activities were observed for oils from O. campechianum and P. pseudolanceifolium with mean repellent concentration (RC₅₀) values of 0.00006 and 0.0001 μL/cm² after 2 h, and 0.00003 and 0.0001 μL/cm² after 4 h, respectively; whereas the least potent was that from M. micrantha, with RC₅₀ values of 0.074 and 0.040 μL/cm² at 2 and 4 h exposure times, respectively. Based on average percentage repellence, oils from P. pseudolanceifolium and O. campechianum were classified as Class IV repellents and were better than the commercial repellent IR3535, classified as Class II. These data evidence the Choco region as an important source of natural repellents with promising commercial opportunities.     

Insensitivity to the Spatial Repellent Action of Transfluthrin in Aedes aegypti: A Heritable Trait Associated with Decreased Insecticide Susceptibility

Background

New vector control paradigms expanding the use of spatial repellents are promising, but there are many gaps in our knowledge about how repellents work and how their long-term use might affect vector populations over time. Reported here are findings from a series of in vitro studies that investigated the plasticity and heritability of spatial repellent (SR) behaviors in Aedes aegypti exposed to airborne transfluthrin, including results that indicate a possible link between repellent insensitivity and insecticide resistance.

Methodology/principal findings

A dual-choice chamber system was used to observe directional flight behaviors in Aedes aegypti mosquitoes exposed to passively emanating transfluthrin vapors (1.35 mg/m³). Individual SR responder and SR non-responder mosquitoes were identified, collected and maintained separately according to their observed phenotype. Subsequent testing included re-evaluation of behavioral responses in some mosquito cohorts as well as testing the progeny of selectively bred responder and non-responder mosquito strains through nine generations. At baseline (F₀ generation), transfluthrin actively repelled mosquitoes in the assay system. F₀ mosquitoes repelled upon initial exposure to transfluthrin vapors were no more likely to be repelled again by subsequent exposure 24h later, but repelled mosquitoes allowed to rest for 48h were subsequently repelled at a higher proportion than was observed at baseline. Selective breeding of SR responders for nine generations did not change the proportion of mosquitoes repelled in any generation. However, selective breeding of SR non-responders did produce, after four generations, a strain of mosquitoes that was insensitive to the SR activity of transfluthrin. Compared to the SR responder strain, the SR insensitive strain also demonstrated decreased susceptibility to transfluthrin toxicity in CDC bottle bioassays and a higher frequency of the V1016I^kdr mutation.

Conclusions/significance

SR responses to volatile transfluthrin are complex behaviors with multiple determinants in Ae. aegypti. Results indicate a role for neurotoxic irritation of mosquitoes by sub-lethal doses of airborne chemical as a mechanism by which transfluthrin can produce SR behaviors in mosquitoes. Accordingly, how prolonged exposure to sub-lethal doses of volatile pyrethroids might impact insecticide resistance in natural vector populations, and how already resistant populations might respond to a given repellent in the field, are important considerations that warrant further monitoring and study. Results also highlight the critical need to develop new repellent active ingredients with novel mechanisms of action.     

A novel test system for detection of tick repellents

A bioassay was developed to examine the response of ticks towards potential repellents that may protect vertebrates against tick bites. Such tick repellents must be effective despite the unavoidable presence of various attractive host-derived stimuli. Therefore, a moving-object-bioassay (MO-bioassay) was developed that mimicks body warmth and movement of vertebrates by a rotating and heated drum. Compounds which were tested for their effects on ticks were applied onto a small elevated area of the drum. Ticks were allowed to approach the drum by walking on a glass rod which ended 1 mm away from the local elevation. Ticks could cling to this elevation that intermittently passed by, whereas the remaining drum surface was too far away from the tip of the rod to be contacted by the ticks.Without the presence of any repellents, 85.5% of 600 hungry, field-collected Ixodes ricinus nymphs moved to the heated, rotating drum within 2 min. Further experiments with unfed I. ricinus nymphs were performed to test whether one established and two potential tick repellents elicit an avoidance reaction in the ticks despite the proven attractiveness of the drum. Freshly applied DEET (N,N-diethyl-m-toluamide) at a concentration of 0.11 mg cm^–2 proved active as a repellent in the MO-bioassay over a distance of a few mm as well as by direct contact. A similar repellent effect was observed with (–)-myrtenal at 1 mg cm^–2, but not at 0.1 mg cm^–2, indicating that this terpenaldehyde is a weaker repellent for I. ricinus nymphs than DEET. No repellent effect was observed with camphor (0.1 mg cm^–2).The MO-bioassay thus is a rapid, simple and low-cost test method allowing the investigation of tick host-contact behaviour as well as the screening of candidate repellents which are either perceived as volatiles or via contact chemoreception.     

Methylation of chemotaxis-specific proteins in Escherichia coli cells permeable to S-adenosylmethionine

Using a modification of the EGTA treatment of Oishi and Smith [Oishi, M., & Smith, C. L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3569], Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis. The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells. Addition of S-adenosyl-L-[methyl-3H]methionine to the permeable cells specifically labels two proteins, MCP I and MCP II. Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar. The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level. Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine.     

Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae

The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (a_w) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1–osr11 (osmotic stress response). All mutations were recessive and showed a clear 2⁺ : 2^– segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae. Received: 5 January 1998 / Accepted: 24 March 1998     

Repellents for Escherichia coli operate neither by changing membrane fluidity nor by being sensed by periplasmic receptors during chemotaxis

A long-standing question in bacterial chemotaxis is whether repellents are sensed by receptors or whether they change a general membrane property such as the membrane fluidity and this change, in turn, is sensed by the chemotaxis system. This study addressed this question. The effects of common repellents on the membrane fluidity of Escherichia coli were measured by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene in liposomes made of lipids extracted from the bacteria and in membrane vesicles. Glycerol, indole, and L-leucine had no significant effect on the membrane fluidity. NiSO4 decreased the membrane fluidity but only at concentrations much higher than those which elicit a repellent response in intact bacteria. This indicated that these repellents are not sensed by modulating the membrane fluidity. Aliphatic alcohols, on the other hand, fluidized the membrane, but the concentrations that elicited a repellent response were not equally effective in fluidizing the membrane. The response of intact bacteria to alcohols was monitored in various chemotaxis mutants and found to be missing in mutants lacking all the four methyl-accepting chemotaxis proteins (MCPs) or the cytoplasmic che gene products. The presence of any single MCP was sufficient for the expression of a repellent response. It is concluded (i) that the repellent response to aliphatic alcohols can be mediated by any MCP and (ii) that although an increase in membrane fluidity may take part in a repellent response, it is not the only mechanism by which aliphatic alcohols, or at least some of them, are effective as repellents. To determine whether any of the E. coli repellents are sensed by periplasmic receptors, the effects of repellents from various classes on periplasm-void cells were examined. The responses to all the repellents tested (sodium benzoate, indole, L-leucine, and NiSO4) were retained in these cells. In a control experiment, the response of the attractant maltose, whose receptor is periplasmic, was lost. This indicates that these repellents are not sensed by periplasmic receptors. In view of this finding and the involvement of the MCPs in repellent sensing, it is proposed that the MCPs themselves are low-affinity receptors for the repellents.     

Chemorepellents in Paramecium and Tetrahymena

Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t-maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.     

A model of excitation and adaptation in bacterial chemotaxis.

We present a model of the chemotactic mechanism of Escherichia coli that exhibits both initial excitation and eventual complete adaptation to any and all levels of stimulus ("exact" adaptation). In setting up the reaction network, we use only known interactions and experimentally determined cytosolic concentrations. Whenever possible, rate coefficients are first assigned experimentally measured values; second, we permit some variation in these rate coefficients by using a multiple-well optimization technique and incremental adjustment to obtain values that are sufficient to engender initial response to stimuli (excitation) and an eventual return of behavior to baseline (adaptation). The predictions of the model are similar to the observed behavior of wild-type bacteria in regard to the time scale of excitation in the presence of both attractant and repellent. The model predicts a weaker response to attractant than that observed experimentally, and the time scale of adaptation does not depend as strongly upon stimulant concentration as does that for wild-type bacteria. The mechanism responsible for long-term adaptation is local rather than global: on addition of a repellent or attractant, the receptor types not sensitive to that attractant or repellent do not change their average methylation level in the long term, although transient changes do occur. By carrying out a phenomenological simulation of bacterial chemotaxis, we find that the model is insufficiently sensitive to effect taxis in a gradient of attractant. However, by arbitrarily increasing the sensitivity of the motor to the tumble effector (phosphorylated CheY), we can obtain chemotactic behavior.     

Optimization of pyrethroid and repellent on fabrics against Stegomyia albopicta (=Aedes albopictus) using a microencapsulation technique

A new approach employing a combination of pyrethroid and repellent is proposed to improve the protective efficacy of conventional pyrethroid‐treated fabrics against mosquito vectors. In this context, the insecticidal and repellent efficacies of commonly used pyrethroids and repellents were evaluated by cone tests and arm‐in‐cage tests against Stegomyia albopicta (=Aedes albopictus) (Diptera: Culicidae). At concentrations of LD₅₀ (estimated for pyrethroid) or ED₅₀ (estimated for repellent), respectively, the knock‐down effects of the pyrethroids or repellents were further compared. The results obtained indicated that deltamethrin and DEET were relatively more effective and thus these were selected for further study. Synergistic interaction was observed between deltamethrin and DEET at the ratios of 5 : 1, 2 : 1, 1 : 1 and 1 : 2 (but not 1 : 5). An optimal mixing ratio of 7 : 5 was then microencapsulated and adhered to fabrics using a fixing agent. Fabrics impregnated by microencapsulated mixtures gained extended washing durability compared with those treated with a conventional dipping method. Results indicated that this approach represents a promising method for the future impregnation of bednet, curtain and combat uniform materials.     

Efficacy and safety of catnip (Nepeta cataria) as a novel filth fly repellent*

Catnip (Nepeta cataria) is known for its pseudo‐narcotic effects on cats. Recently, it has been reported as an effective mosquito repellent against several Aedes and Culex species, both topically and spatially. Our laboratory bioassays showed that catnip essential oil (at a dosage of 20 mg) resulted in average repellency rates of 96% against stable flies, Stomoxys calcitrans (L.) and 79% against houseflies, Musca domestica (L.), respectively. This finding suggested that the application of repellent could be used as part of filth fly management. Further evaluations of catnip oil toxicity were conducted to provide a broad‐spectrum safety profile of catnip oil use as a potential biting and nuisance insect repellent in urban settings. Acute oral, dermal, inhalation, primary dermal and eye irritation toxicity tests were performed. The acute oral LD₅₀ of catnip oil was found to be 3160 mg/kg body weight (BW) and 2710 mg/kg BW in female and male rats, respectively. The acute dermal LD₅₀ was > 5000 mg/kg BW. The acute inhalation LD₅₀ was observed to be > 10 000 mg/m³. Primary skin irritation tested on New Zealand white rabbits showed that catnip oil is a moderate irritant. Catnip oil was classified as practically non‐irritating to the eye. In comparison with other U.S. Environmental Protection Agency‐approved mosquito repellents (DEET, picaridin and p‐menthane‐3,8‐diol), catnip oil can be considered as a relatively safe repellent, which may cause minor skin irritation.     

The palatability of undyed carrot surface-coated with repellents,or dyed blue or green to a terrestrial macroinvertebrate

ABSTRACT

Birds are at risk of being poisoned during pest control operations not only through eating toxic baits, but potentially by preying on invertebrates that have, themselves, consumed the toxic baits. Blue baits coated with anthraquinone and/or mint-scented repellents are avoided by some bird species compared with green baits coated with cinnamon oil; however, data on invertebrate avoidance patterns have not been explored. In our first experiment, we measured consumption rates in a large invertebrate, the Auckland tree wētā (Hemideina thoracica) for carrot that had been surface-coated with three repellent formulations: anthraquinone (0.8?g?kg^?1) (a secondary repellent); anthraquinone (0.8?g?kg^?1) and pennyroyal oil (a mint-scented product which acts as a primary repellent) (0.5?g?kg^?1); and cinnamon oil (0.15?g?kg^?1) (a primary repellent), over 4 days. In a second experiment, we tested whether tree wētā preferred carrot dyed either blue or green over 2 days. Tree wētā ate similar quantities of carrot from all three of the repellent formulations tested, but consumption of all three formulations was significantly lower than the control from the third day of the experiment. Tree wētā ate less blue-dyed carrot than green-dyed carrot on both days of the second experiment, but differences were not significant (day 1, P=?0.057; day 2, P?=?0.145). Our findings complement the results of previous studies on some bird species. Together they show the potential of surface-coating baits with anthraquinone and/or mint oil and dyeing them blue in pest control operations to reduce non-target avian by-kill, while not increasing the risk of secondary poisoning of insectivorous species. Our results also indicate that cinnamon oil can be used not only as a toxin mask but also as a repellent to wētā.