In vitro acaricidal activity of essential oils and their binary mixtures against ixodes scapularis (Acari: Ixodidae).

Ixodes scapularis ticks are vectors of infectious agents that cause illness in humans, including Lyme disease. Recent years have seen a surge in tick-borne diseases (TBD) resulting in a high demand for tick management products. Plants offer a valuable source of active compounds for the development of novel, eco-friendly tick control products, reducing potential risks to human and animal health. Essential oils (EOs) have emerged as potential acaricides and repellents against ticks providing an alternative to synthetic chemicals and aiding in the prevention of TBD by lowering the risk of tick bites. We investigated the acaricidal activity of EOs from lemongrass (Cymbopogon citratus), geranium (Pelargonium x asperum), savory thyme (Thymus saturejoides), and white thyme (Thymus zygis) on I. scapularis. The interactions (i.e., synergistic, antagonistic, or additive) of their binary mixtures were also evaluated. EO samples were analyzed via gas chromatography-mass spectrometry to determine their chemical composition. The adult immersion test was used to determine the lethal concentration (LC50) of each EO alone and in mixtures. Quantitative assessment of synergistic, additive, or antagonistic effect of the binary mixtures was performed by calculating the combination index. Strong acaricidal activity was recorded for savory thyme and white thyme EOs, with LC50 values of 28.0 and 11.0 µg/µL, respectively. The LC50 of lemongrass and geranium EOs were 49.0 and 39.7 µg/µL, respectively. Among the tested EOs, savory thyme and white thyme had a strong acaricidal effect on I. scapularis, which might be linked to the presence of carvacrol (26.05 % ± 0.38) and thymol (53.6 % ± 2.31), main components present in savory thyme and white thyme EOs, respectively. The tick killing efficacy of lemongrass and geranium EOs was lower when mixed than when used separately (LC50 of 65.3 µg/µL). The same happened with savory thyme and white thyme EOs, except at 9.75 µg/µL where they had a synergistic effect (LC50 of 58.3 µg/µL). Lemongrass and savory thyme EOs had a synergistic effect at low concentrations, and an antagonistic effect at higher concentrations (LC50 of 95.4 µg/µL). Lemongrass and white thyme EOs had a synergistic effect against ticks from 15 to 120 µg/µL (LC50 of 18.5 µg/µL) similar to white thyme EO. Geranium and savory thyme EOs had an antagonistic effect at all concentrations, with an LC50 of 66.8 µg/µL. Geranium and white thyme EOs also had an antagonistic effect, except at 12.7 µg/µL where they had a synergistic effect (LC50 of 66.8 µg/µL). The interaction observed when combining selected essential oils suggests promising potential for developing acaricidal formulations aimed at controlling ticks and curbing the transmission of tick-borne disease agents.

Soil and foliar application of rock dust as natural control agent for two-spotted spider mites on tomato plants.

Mineral-based products represent a valid alternative to synthetic pesticides in integrated pest management. We investigated the effects of a novel granite dust product as an agent for controlling two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), on tomato plants (Solanum lycopersicum L.). Two-choice tests for repellency and repulsiveness, and no-choice bioassays with different type of applications (soil, foliar, and soil-foliar) were used in order to evaluate performance and action of the product. Evaluation of epidermal micromorphology and mesophyll structure of treated plants and elemental analyses of leaves were performed. In repulsiveness experiments, almost all dust treatments significantly inhibited mites from migrating to and/or settling on the treated leaf. In repellency experiments, foliar and soil dust treatments were not significantly different from control. Significant mortality was observed for all dust treatments in two-choice and in no-choice bioassays, suggesting mites are susceptible to rock dust by contact, and by indirect interaction through the feeding on plants subjected to soil application of rock dust. Leaf epidermal micromorphology and mesophyll structure of treated plants showed structural variation due to mineral accumulation, which was also confirmed by elemental analyses of leaves. These results demonstrate for the first time that granite rock dust interacts with two-spotted spider mites by modifying pest behavior and via acaricidal action, providing more insights in understanding the mechanism of this novel natural product as pest management tool.

Effects of Nosema apis, N. ceranae, and coinfections on honey bee (Apis mellifera) learning and memory.

Western honey bees (Apis mellifera) face an increasing number of challenges that in recent years have led to significant economic effects on apiculture, with attendant consequences for agriculture. Nosemosis is a fungal infection of honey bees caused by either Nosema apis or N. ceranae. The putative greater virulence of N. ceranae has spurred interest in understanding how it differs from N. apis. Little is known of effects of N. apis or N. ceranae on honey bee learning and memory. Following a Pavlovian model that relies on the proboscis extension reflex, we compared acquisition learning and long-term memory recall of uninfected (control) honey bees versus those inoculated with N. apis, N. ceranae, or both. We also tested whether spore intensity was associated with variation in learning and memory. Neither learning nor memory differed among treatments. There was no evidence of a relationship between spore intensity and learning, and only limited evidence of a negative effect on memory; this occurred only in the co-inoculation treatment. Our results suggest that if Nosema spp. are contributing to unusually high colony losses in recent years, the mechanism by which they may affect honey bees is probably not related to effects on learning or memory, at least as assessed by the proboscis extension reflex.

Fate of dermally applied miticides fluvalinate and amitraz within honey bee (Hymenoptera: Apidae) bodies.

Varroa mites, Varroa destructor Anderson & Trueman, are economically important pests of honey bees. Varroa mites are principally controlled within honey bee colonies using miticides. However, despite their importance in managing mite populations for apiculture, potential effects of miticides on honey bees are poorly understood. Using gas chromatography-flame ionization detection, we investigated concentrations, over variable time frames and within different body regions, of two commonly used miticides, tau-fluvalinate and amitraz, after dermal exposure to honey bees. We also quantified mortality of honey bees exposed to each miticide at both a low and high dose. Significant differences were observed in distributions of miticides among body regions. Within honey bee body parts, tau-fluvalinate was more readily absorbed and decreased in concentration more rapidly than amitraz. Mortality increased with higher dosages of miticides, and at higher dosages mortality was greater from fluvalinate than from amitraz. For individual honey bees, our results for rate of breakdown suggest that fluvalinate may be the preferred miticide for apiculturists, whereas our mortality results suggest that amitraz may be preferable. Either choice must be weighed against geographic variation in varroa resistance to each pesticide and attendant costs of parasitism.

Effects of fluvalinate on honey bee learning, memory, responsiveness to sucrose, and survival.

Contaminants can affect organisms' behaviour and, as a consequence, survival. Tau-fluvalinate (hereafter fluvalinate) is the active ingredient in a pesticide commonly used in North America to control Varroa destructor mites in honey bee (Apis mellifera) colonies. Fluvalinate's effects on honey bees are not well known. Honey bee cognitive and neural function can be assessed using the proboscis extension reflex (PER), which applies Pavlovian conditioning techniques. This study used PER to evaluate effects of fluvalinate on honey bee acquisition learning, (long-term) memory recall, responsiveness to sucrose, and mortality. We also evaluated how exclusion criteria for honey bees that did not exhibit PER during training and memory trials affected interpretation of results. Fluvalinate was administered both orally and dermally at high and low doses to mimic routes by which honey bees are exposed. We found negative effects of fluvalinate on honey bee learning, memory, responsiveness to sucrose, and survival, especially in high oral doses. We also found significant consequences to interpretation of results using different exclusion criteria. For example, almost 50% of individuals that failed to show evidence of learning subsequently showed evidence of memory. The latter results have important implications regarding traditional assessment of PER-based learning and memory; the former results suggest that evaluation of honey bee exposure to fluvalinate and attendant consequences warrants further investigation.

The proboscis extension reflex to evaluate learning and memory in honeybees (Apis mellifera): some caveats.

The proboscis extension reflex (PER) is widely used in a classical conditioning (Pavlovian) context to evaluate learning and memory of a variety of insect species. The literature is particularly prodigious for honeybees (Apis mellifera) with more than a thousand publications. Imagination appears to be the only limit to the types of challenges to which researchers subject honeybees, including all the sensory modalities and a broad diversity of environmental treatments. Accordingly, some remarkable insights have been achieved using PER. However, there are several challenges to evaluating the PER literature that warrant a careful and thorough review. We assess here variation in methods that makes interpretation of studies, even those researching the same question, tenuous. We suggest that the numerous variables that might influence experimental outcomes from PER be thoroughly detailed by researchers. Moreover, the influence of individual variables on results needs to carefully evaluated, as well as among two or more variables. Our intent is to encourage investigation of the influence of numerous variables on PER results.

Hairpencil volatiles influence interspecific courtship and mating between two related moth species.

Reproductive isolation between sympatric, closely related species can be accomplished through a variety of pre-zygotic isolating mechanisms, including courtship-signaling behavior that involves pheromones. In the moths Heliothis virescens and H. subflexa, males display abdominal hairpencils (HP), which release volatile chemicals during courtship. In this study, we demonstrated that HP volatiles released by male H. subflexa function to improve mating success with conspecific females. Interspecific mating experiments were conducted to determine any influence of HP volatiles on species isolation. Female H. virescens and H. subflexa were observed during courtship with males of the other species, following either sham-operation or ablation of HPs, both with and without concurrent presentation of HP volatiles. Mating success was improved by co-presentation of HP extract from males of the same species during courtship. Ablation of HPs improved mating between H. subflexa females and H. virescens males. During interspecific matings, male H. virescens attempted copulation less frequently in the presence of H. virescens HP extract, though H. subflexa males were not affected by the presence of H. subflexa HP extract. This suggests that HP volatiles produced by males of each species may inhibit mating between species through effects on males (H. virescens) and females (H. subflexa).

Effects of cold immobilization and recovery period on honeybee learning, memory, and responsiveness to sucrose.

In addition to human error and variation in laboratory conditions, there are numerous factors that can complicate comparisons among studies. Furthermore, differences in how experimental methods are executed can make it difficult to distinguish between effects of focal versus extraneous variables. Insect neural function is commonly evaluated using Pavlovian conditioning techniques; learning and memory in many species can be assessed using the proboscis extension reflex (PER). However, there are significant inconsistencies in methods used to immobilize insects prior to PER tests. We compared responses of honeybees immobilized in a refrigerator, on ice, and in a freezer, and evaluated influence of recovery interval before testing. Ice-chilling weakly decreased learning (response to an originally neutral odor) more so than refrigeration or freezing, but not 24-h recall of odor. We found no significant differences in responsiveness to sucrose relative to cooling method, but responsiveness was significantly lower among honeybees left to recover for only 0.75h versus 1.5 or 3h. Finally, we observed increased responsiveness to sucrose and geraniol between June and August. Our results suggest that inconsistencies in cold immobilization methods could confound interpretation and comparison of results from a large body of work on honeybee learning and memory.