Arena Assays Research Articles

Plant secondary metabolite has dose‐dependent effects on bumblebees

The presence of secondary metabolites in flower nectar can mediate interactions between plants, pollinators, herbivores, and microbes. Milkweeds range in concentrations of cardenolides in flower nectar from ~ 1 to 100 ng μl–1. Using three different behavioral assays with bumblebees Bombus impatiens, we examined the impacts of the commercially available cardenolide ouabain at the range of concentrations at which cardenolides naturally occur in milkweeds. We show that after four days of exposure bees in consumption assays drank more of a nectar solution with a low ouabain concentration of 10 ng μl–1 than the control sucrose nectar, and over the course of the experiment bees consumed less of the 100 ng μl–1 ouabain solution than the control. Bee activity levels in Petri dish arena assays were not impacted by ouabain consumption, even at the highest concentrations; however, in free‐flying choice assays, bees preferentially visited artificial flowers containing 10 ng μl–1 ouabain more than flowers with sucrose control, or flowers with 100 ng μl–1 of ouabain. We therefore conclude that cardenolides may provide plants advantages to pollination at the low end of the naturally occurring range of concentrations for cardenolides, but may be costly to plants at the high end of the range. This research highlights that secondary metabolites in nectar may be under selection to be maintained at low concentrations, and species with high concentrations of secondary metabolites may be under conflicting selection pressures to maintain high circulating levels to deter herbivores at the cost of reduced pollination.

Collection and Processing of Behavioural Data of the Olive Fruit Fly, Bactrocera oleae, When Exposed to Olive Twigs Treated with Different Commercial Products

The need for the development of sustainable control methods of herbivorous insects implies that new molecules are proposed on the market. Among the different effects the new products may have on the target species, the alteration of insect oviposition behaviour might be considered. At the scope, parallel simple behavioural assays can be conducted in arena. Freely available software can be used to track observed events, but they often need intensive customization to the specific experimental design. Hence, integrating such software with, e.g., R environment, can provide a much more effective protocol development for data collection and analysis. Here we present a dataset and protocol for processing data of the oviposition behaviour of the olive fruit fly, Bactrocera oleae, when exposed to olive twigs treated with different commercial products. Treatments were rock powder, propolis, a mixture of rock powder and propolis, copper oxychloride, copper sulphate, and water as the experimental control. JWatcher was used to simultaneously collect data from 12 arena assays and ad-hoc developed R code was used to process raw data for data analyses. The procedure described here is novel and represents a valuable and transferable protocol to analyse observational events in B. oleae, as well as other biological systems.

Balancing Human and Sea Turtle Safety: Evaluating Long-Wavelength Streetlights as a Coastal Roadway Management Tool

Coastal roadways with tall, full-spectrum streetlights along sea turtle nesting beaches present a challenge for managers seeking to balance protection of sea turtles with public safety. Many communities extinguish these lights during nesting season to avoid impacting nesting and hatchling sea turtles. Long-wavelength light emitting diodes (LEDs) offer an alternative for managers in these communities, but additional information on sea turtle response to these lights is warranted prior to installation. We conducted arena assays on Florida’s west coast to evaluate hatchling orientation when exposed to a shielded, long-wavelength (624 nm) prototype lamp compared to an adjacent beach with the streetlights turned off. We compared orientation in test and control arenas simultaneously over two consecutive nights, recording crawl direction and timing for individual hatchlings. Hatchlings in test and control arenas oriented correctly toward the ocean in all trials, with no differences in hatchling dispersion or circling. Thus, the fully shielded, long-wavelength LED streetlight fixture tested provides an appropriate option to minimize impacts to sea turtles along coastal roadways throughout the Unites States and elsewhere. As such, this alternative solution to extinguishing necessary streetlights can aid coastal managers in concurrently protecting nesting habitat and providing light for public safety.

Predator kairomones elicit bold, exploratory behaviours in juvenile bluegill, Lepomis macrochirus

Behavioural responses of animals are often placed conceptually along some axis characterized by the extremes, e.g. bold versus shy or timid. Ecological and evolutionary pressures can be associated with increased frequencies of different behavioural patterns that are also subject to intrinsic factors such as differing metabolic requirements or cognitive abilities. We exposed individual wild-caught juvenile bluegill, Lepomis macrochirus, to kairomones from predatory northern pike, Esox lucius, or to lake water controls during a Z-maze arena assay commonly used to assess relative levels of boldness and exploratory behaviours. Fish were subsequently lethally sampled for liver and brain mass to obtain estimates of relative body condition (hepato-somatic index, HSI) and cognitive capacity (brain-somatic index, BSI), as well as the state of the gallbladder, to test for relationships between these indices and observed boldness-related behaviours. Using principal components analysis (PCA) we identified a boldness–exploration syndrome in bluegill independent of chemical treatment and body metrics where bolder fish emerged from a start chamber in less time, travelled farther through the maze and crossed more lines than less bold fish. Bluegill exposed to kairomones were bolder on average than their counterparts in the control treatment independent of body metrics, suggesting that elevated risk levels indicated by predator kairomones can elicit behavioural responses consistent with boldness, exploration and risk taking. The role of relative cognitive ability in the expression of boldness and exploration is equivocal and may be resolved through repeated assays on individuals at different risk levels.

Natural insensitivity and the effects of concentration on the repellency and survival of American dog ticks (Dermacentor variabilis) by DEET.

N,N-Diethyl-m-toluamide (DEET) is by far the most used repellent worldwide. When applied topically to the skin, the active ingredient has been shown to provide protection from a variety of hematophagous insects, including mosquitoes and flies. DEET's effectiveness against ticks is influenced by a variety of factors (e.g., duration and concentration of application, drying time, route of exposure, tick species and developmental stage), and may differ from insects due to their unique chemosensory system that primarily involves the Haller's organ. We therefore used several approaches to investigate DEET's efficacy to repel Dermacentor variabilis at different concentrations (5, 30 or 75%), as well as explore its toxicological properties and natural variability in DEET insensitivity across populations from Manitoba, Canada. Climbing bioassays indicated that higher concentrations of DEET were more effective at repelling D. variabilis, and that ticks from some sampling localities were more sensitive to lower concentrations than others. Petri dish arena assays revealed ticks exposed to high concentrations of the repellent lose their ability to discriminate lower concentrations, perhaps due to overstimulation or habituation. Finally, our tactile assays demonstrated reduced tick survival after contact with high DEET concentrations, with mortality occurring more rapidly with increased concentration. Dermacentor variabilis from these tactile assays displayed a multitude of physiological and neurological symptoms, such as 'hot foot' and various bodily secretions. Overall, our study shows a strong association between repellency, concentration and the acaricidal effects of DEET on D. variabilis.

Ritualized aggressive behavior reveals distinct social structures in native and introduced range tawny crazy ants.

How workers within an ant colony perceive and enforce colony boundaries is a defining biological feature of an ant species. Ants fall along a spectrum of social organizations ranging from single-queen, single nest societies to species with multi-queen societies in which workers exhibit colony-specific, altruistic behaviors towards non-nestmate workers from distant locations. Defining where an ant species falls along this spectrum is critical for understanding its basic ecology. Herein we quantify queen numbers, describe intraspecific aggression, and characterize the distribution of colony sizes for tawny crazy ant (Nylanderia fulva) populations in native range areas in South America as well as in their introduced range in the Southeastern United States. In both ranges, multi-queen nests are common. In the introduced range, aggressive behaviors are absent at all spatial scales tested, indicating that within the population in the Southeastern United States N. fulva is unicolonial. However, this contrasts strongly with intraspecific aggression in its South American native range. In the native range, intraspecific aggression between ants from different nests is common and ritualized. Aggression is typically one-sided and follows a stereotyped sequence of escalating behaviors that stops before actual fighting occurs. Spatial patterns of non-aggressive nest aggregation and the transitivity of non-aggressive interactions demonstrate that results of neutral arena assays usefully delineate colony boundaries. In the native range, both the spatial extent of colonies and the average number of queens encountered per nest differ between sites. This intercontinental comparison presents the first description of intraspecific aggressive behavior for this invasive ant and characterizes the variation in colony organization in the native-range, a pre-requisite to a full understanding of the origins of unicoloniality in its introduced range.

Lemon Terpenes Influence Behavior of the African Citrus Triozid Trioza erytreae (Hemiptera: Triozidae)

The African citrus triozid, Trioza erytreae Del Guercio (Hemiptera: Triozidae) is one of the primary vectors of the bacterium Candidatus Liberibacter spp. which causes citrus greening, a disease of global economic importance in citrus production. Despite its economic importance, little is known about its chemical ecology. Here, we used behavioral assays and chemical analysis to study the chemical basis of interaction between T. erytreae and one of its preferred host plants, Citrus jambhiri. In dual choice Y-tube olfactometer assays, lemon leaf odors attracted females but not males compared to plain air or solvent controls. However, in a petri dish arena assay, both sexes were arrested by lemon leaf odors. Coupled gas chromatography-mass spectrometry (GC/MS) analysis revealed quantitative differences in the odors of flushing and mature leaves, dominated by terpenes. Twenty-six terpenes were identified and quantified. In Petri dish arena assays, synthetic blends of the most abundant terpenes mimicking lemon flushing leaf odors elicited varying behavioral responses from both sexes of T. erytreae. A nine-component blend and a blend of the three most abundant terpenes; limonene, sabinene and β-ocimene arrested both sexes of T. erytreae. In contrast, a six-component blend lacking in these three components elicited an avoidance response in both sexes. Furthermore, both sexes of T. erytreae preferred the three-component synthetic blend to lemon crude volatile extract. These results suggest that lemon terpenes might be used in the management of T. erytreae.

Development of pull and push–pull systems for management of lesser mealworm, Alphitobius diaperinus, in poultry houses using alarm and aggregation pheromones

The lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae), is the most important insect pest affecting poultry production around the world, with all life stages being susceptible to infection by bacteria, viruses and fungi. Control of A. diaperinus in poultry houses using intensive insecticide application is not effective due to the cryptic behaviour of this pest. Here, we evaluated the potential of recently identified A. diaperinus alarm (1,4-benzoquinone, 2-methyl-1,4-benzoquinone and 2-ethyl-1,4-benzoquinone) and aggregation [(R)-limonene, 2-nonanone, (E)-ocimene, (S)-linalool, (R)-daucene and (E,E)-α-farnesene] pheromones as tools for the management of this pest in poultry houses in Brazil. Laboratory arena assays with synthetic alarm pheromone confirmed A. diaperinus repellency. In an initial field assay, traps baited with synthetic aggregation pheromone captured significantly more insects than control traps. In further field assays that compared a pull (aggregation pheromone) and a push-pull (simultaneous alarm/aggregation pheromone deployment) system, a higher number of A. diaperinus were captured in aggregation pheromone-baited traps in the push-pull system. Our results suggest that alarm and aggregation pheromones can be deployed in poultry houses to trap significant numbers of adult A. diaperinus. Studies are underway to determine the potential for using these components as part of an integrated A. diaperinus management strategy. © 2018 Society of Chemical Industry.

Sea-finding in marine turtle hatchlings: What is an appropriate exclusion zone to limit disruptive impacts of industrial light at night?

Artificial light is increasingly being recognized as a globally-significant ecological threat, but appropriate management has lagged behind that of other environmental pollutants. Industrial developments associated with the extraction of natural resources generate large amounts of artificial light. Marine turtles are particularly vulnerable to disruption from artificial light, thus effective management of lighting is critical in areas where industrial developments occur close to nesting habitat. Given the complexity of managing lighting in industry, ensuring an adequate lighting exclusion zone between the development and the beach may be the most effective strategy for limiting impacts, yet there appears to have been little focus on clearly delineating a distance which constitutes an ‘adequate’ buffer. Using arena assays, we assessed flatback turtle (Natator depressus) and green turtle (Chelonia mydas) hatchling sea-finding ability in response to three standard industrial light sources (high pressure sodium (HPSV), metal halide (MH) and fluorescent white (FW)), positioned at distances of 100, 200, 500 and 800m. Sea-finding in both species was disrupted by all three light types when lights were positioned 200m or closer, but not when lights were positioned ≥500m away. However, when shielding the lights so that light glow, but not the luminaire itself, was visible from the arena, the observed sea-finding disruption was considerably reduced. Given that facilities are typically lit by numerous luminaires, our findings demonstrate that future industrial developments should be separated from nearby nesting beaches by a buffer of at least 1.5km, as previously theorized, with all installed lighting appropriately shaded. Such measures will help minimize lighting impacts on marine turtles as extractive resource operations continue to encroach on nesting beaches around the world.

Novel object exploration in the C58/J mouse model of autistic-like behavior

Mouse models of autistic like behaviors are a valuable tool to use when studying the causes, symptoms, and potential treatments for autism. The inbred C58/J strain is a strain of interest for this model and has previously been shown to possess face validity for some of the core traits of autism, including low social behavior and elevated motor stereotypies. Higher order repetitive behaviors have not been extensively studied in this strain, or in mice in general. In this study, we looked for evidence of higher-order repetitive behaviors in the C58/J strain using a novel object assay. This assay utilized a mouse's natural exploratory behavior among unfamiliar objects to identify potential sequencing patterns in motor activity. The motor stereotypies displayed by the C58/J strain during testing were consistent with past studies. The C58/J strain also displayed a high preference for a single object in the round arena assays and the females demonstrating elevated sequencing patterns in the round arena. Although the C58/J strain did not show pervasive evidence of higher-order repetitive behaviors across all measures, there was evidence of higher order repetitive behaviors in certain situations. This study further demonstrates the potential of the C58/J mouse strains as a model for lower-order and potentially, higher-order repetitive behaviors. This study also demonstrates that the shape of the novel object arena can change the behavior displayed by the test animals. Further studies utilizing the C58/J strain and further validation of the novel object assay are warranted.

Use of a Highly Sensitive Immunomarking System to Characterize Face Fly (Diptera: Muscidae) Dispersal From Cow Pats

We tested an immunomarking system that used egg white as marker and enzyme-linked immunosorbent assay as a detection assay to characterize face fly (Musca autumnalis DeGeer) dispersal from cow pats in a pastured beef cattle operation. In microcage assays, adult flies acquired marker after contact with cow pats that were treated with marker and field aged up to 11 d. In arena assays on sprayed full-size cow pats, 77% of eclosed face flies acquired the marker. In a field-marking study, four applications of egg white marker were applied on freshly deposited cow pats over a summer at two peripheral paddocks to a main grazing pasture of ≍50 head of beef cattle. Of the 663 face flies captured, 108 were positive for the egg white marker (16.3%). Of the marked flies, ≍ twofold more male than female flies were captured. Sex-specific dispersal distances were roughly equal up to 450 m, with 11% of female flies dispersing >450 m. Dispersal capability of face flies is discussed in relation to efficacy of rotational grazing and other IPM strategies.

Homing trajectories and initial orientation in a Neotropical territorial frog, Allobates femoralis (Dendrobatidae)

IntroductionThe ability to relocate home or breeding sites after experimental removal has been observed in several amphibians and the sensory basis of this behavior has been studied in some temperate-region species. However, the actual return trajectories have rarely been quantified in these studies and it remains unknown how different cues guide the homing behavior. Dendrobatidae (dart-poison frogs) exhibit some of the most complex spatial behaviors among amphibians, such as territoriality and tadpole transport. Recent data showed that Allobates femoralis, a frog with paternal tadpole transport, successfully returns to the home territories after experimental translocations of up to 400 m. In the present study, we used harmonic direction finding to obtain homing trajectories. Additionally, we quantified the initial orientation of individuals, translocated 10 m to 105 m, in an arena assay.ResultsTracking experiments revealed that homing trajectories are characterized by long periods of immobility (up to several days) and short periods (several hours) of rapid movement, closely fitting a straight line towards the home territory. In the arena assay, the frogs showed significant homeward orientation for translocation distances of 35 m to 70 m but not for longer and shorter distances.ConclusionsOur results describe a very accurate homing behavior in male A. femoralis. The straightness of trajectories and initial homeward orientation suggest integration of learned landmarks providing a map position for translocated individuals. Future research should focus on the role of learning in homing behavior and the exact nature of cues being used.

Aggregation Behavior of the Southern Chinch Bug (Hemiptera: Blissidae)

ABSTRACT The southern chinch bug, Blissus insuhris Barber, forms dense, multigenerational aggregations in St. Augustinegrass lawns leading to grass death from sap feeding. We conducted laboratory bioassays to better understand the signals responsible for the formation and maintenance of southern chinch bug aggregations. In small arena assays, chinch bugs demonstrated a stronger aggregation response over time and aggregated more often on or beneath St. Augustinegrass leaf blades than on or under artificial leaf-like shelters constructed from white or green paper. In Y-tube olfactometer assays, bugs of different age and sex were attracted to volatiles from mixed-sex chinch bug aggregations and showed particular attraction to groups of adult female chinch bugs. Adult males and nymphs were also attracted to adult males. Nymphs were attracted to nymphs and were also more attracted to aggregation volatiles when they could see bugs in the arm of the Y-tube. Adult males were more attracted to short-winged than lo...

Magnetic compass orientation in two strictly subterranean rodents: learned or species-specific innate directional preference?

Evidence for magnetoreception in mammals remains limited. Magnetic compass orientation or magnetic alignment has been conclusively demonstrated in only a handful of mammalian species. The functional properties and underlying mechanisms have been most thoroughly characterized in Ansell's mole-rat, Fukomys anselli, which is the species of choice due to its spontaneous drive to construct nests in the southeastern sector of a circular arena using the magnetic field azimuth as the primary orientation cue. Because of the remarkable consistency between experiments, it is generally believed that this directional preference is innate. To test the hypothesis that spontaneous southeastern directional preference is a shared, ancestral feature of all African mole-rats (Bathyergidae, Rodentia), we employed the same arena assay to study magnetic orientation in two other mole-rat species, the social giant mole-rat, Fukomys mechowii, and the solitary silvery mole-rat, Heliophobius argenteocinereus. Both species exhibited spontaneous western directional preference and deflected their directional preference according to shifts in the direction of magnetic north, clearly indicating that they were deriving directional information from the magnetic field. Because all of the experiments were performed in total darkness, our results strongly suggest that all African mole-rats use a light-independent magnetic compass for near-space orientation. However, the spontaneous directional preference is not common and may be either innate (but species-specific) or learned. We propose an experiment that should be performed to distinguish between these two alternatives.

Experience-based behavioral and chemosensory changes in the generalist insect herbivore Helicoverpa armigera exposed to two deterrent plant chemicals.

Behavioral and electrophysiological responses of larvae of the polyphagous moth species Helicoverpa armigera to two plant-derived allelochemicals were studied, both in larvae that had been reared on a diet devoid of these compounds and in larvae previously exposed to these compounds. In dual-choice cotton leaf disk and pepper fruit disk arena assays, caterpillars reared on a normal artificial diet were strongly deterred by strychnine and strophanthin-K. However, caterpillars reared on an artificial diet containing strychnine were insensitive to strychnine and strophanthin-K. Similarly, caterpillars reared on an artificial diet containing strophanthin-K were also desensitized to both deterrent chemicals. Electrophysiological tests revealed that the deterrent-sensitive neurons in taste sensilla on the maxillae of caterpillars reared on each deterrent-containing diet displayed reduced sensitivity to the two chemicals compared with the caterpillars reared on normal diets. We conclude that the experience-dependent behavioral plasticity was partly based on the reduced sensitivity of taste receptor neurons and that the desensitization of taste receptor neurons contributed to the cross-habituation to the two chemicals.

Molecular analysis of flies selected for aggressive behavior

Aggressive behavior is pervasive throughout the animal kingdom, and yet very little is known about its molecular underpinnings. To address this problem, we have developed a population-based selection procedure to increase aggression in Drosophila melanogaster. We measured changes in aggressive behavior in the selected subpopulations with a new two-male arena assay. In only ten generations of selection, the aggressive lines became markedly more aggressive than the neutral lines. After 21 generations, the fighting index increased more than 30-fold. Using microarray analysis, we identified genes with differing expression levels in the aggressive and neutral lines as candidates for this strong behavioral selection response. We tested a small set of these genes through mutant analysis and found that one significantly increased fighting frequency. These results suggest that selection for increases in aggression can be used to molecularly dissect this behavior.

Predation capacity of two predatory laelapid mites on soil‐dwelling thrips stages

AbstractThe life cycle of the Western Flower Thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), one of the most important glasshouse pests, includes a soil passage composed of three instars that deserve more attention in terms of biocontrol strategies. It has been repeatedly reported that two polyphagous predatory mites, Stratiolaelaps miles (Berlese) and Hypoaspis (Geolaelaps) aculeifer (Canestrini) (Acari: Laelapidae), also prey on these thrips stages, in addition to several other soil inhabiting prey species. However, the potential thrips consumption rates have never been quantified for these predatory mites. Therefore, an arena experiment was carried out to investigate the potential predation rates of the two mites on second instar larvae, prepupae, and pupae of F. occidentalis. In addition, the fecundity on the thrips diet was assessed and compared to oviposition rate on a nematode prey. All thrips instars were accepted as prey by each mite species. Females of H. aculeifer preyed on 3.5 (± 0.5) thrips instars and laid 2.5 (± 0.87) eggs per day, whereas females of S. miles preyed on 1.64 (± 0.3) thrips and laid 0.8 (± 0.53) eggs. Males of both species killed 0.6 (± 0.3) thrips per day. The fitness of the two predatory mites on F. occidentalis as prey and their suitability as biocontrol agents are elucidated. Reasons for reduced thrips control in the soil environment, in contrast to the results obtained in arena assays are discussed.

Effects of [His 7]-corazonin on the phase state of isolated-reared (solitarious) desert locusts, Schistocerca gregaria

[His 7]-corazonin is a neuropeptide produced in the pars lateralis of the brain. It is stored in the corpora cardiaca and probably released from there. The only well-documented effect in locusts is increased melanization of the cuticle. We investigated whether this hormone might also be causally related to changes in behavior and morphometrics that, like melanization, occur during crowding-induced gregarization. Solitary fourth-instar nymphs of Schistocerca gregaria (Forsk.) were injected thrice with 1 nmol [His 7]-corazonin. After molting to the 5th stadium their behavioral phase state was measured in an arena assay and analyzed using multiple logistic regression analysis. The hormone was found not to induce behavioral phase changes. Upon reaching adulthood, morphometrics shifts occurred towards values typical for crowd-reared and regregarized animals. Our results thus indicate that [His 7]-corazonin is not involved in behavioral gregarization but may participate in morphometrical phase change.

Host location and host discrimination behavior of Telenomus isis, an egg parasitoid of the African cereal stem borer Sesamia calamistis.

In the Republic of Benin, the scelionid egg parasitoid Telenomus isis (Polaszek) is one of the most important control factors of the noctuid maize stem borer Sesamia calamistis. In the present study, the role of various sources of contact kairomones (male or virgin or mated female moths) and of the moth's oviposition substrate (leaf sheath versus filter paper: host plant species) in host location and oviposition behavior of T. isis was investigated in Munger cells, open arenas, and/or Petri dish assays. Furthermore, its ability to distinguish between unparasitized eggs and eggs parasitized by a conspecific female or by the trichogrammatid Lathromeris ovicida was studied. In the Munger cell experiment, T. isis spent more time in moths' odor fields than in the control. There was no difference between virgin and mated females. In the open arena assay, traces left by both the male and female moths acted as contact cues, which elicited an arrestment response in the parasitoid. The residence and patch retention time in the arena with virgin or mated females of S. calamistis was about 4.8 times as long as that with males. The presence of maize leaf sheaths stimulated the oviposition behavior of T. isis when compared to eggs offered on filter paper. During the first 6 hr, more eggs were parasitized on maize leaves, although there was no difference in the final number of offspring between the two substrates. In addition, if eggs of S. calamistis were offered together with different host plant species or alone, maize and sorghum were both more attractive than millet or the egg alone and equally attractive between themselves, indicating that the plant tissue influences host finding of T. isis. Both T. isis and L. ovicida recognized markings of conspecific females, and intraspecific superparasitism was therefore low. Interspecific superparasitism was more than three times higher for L. ovicida than for T. isis, indicating that only T. isis was able to recognize the marking of the other species and tried to avoid superparasitism. Emergence of parasitoids from multiparasitized eggs generally was in favor of L. ovicida regardless of species order.