桔小实蝇雄成虫联系性嗅觉学习

【目的】为了探究桔小实蝇 Bactrocera dorsalis (Hendel)雄成虫的嗅觉学习能力。【方法】本研究采用经典性嗅觉条件反射训练法（classical olfactory conditioning）在室内对固定的羽化后14-17日龄的桔小实蝇雄成虫进行气味与食物的联合学习训练, 即薄荷精油和10%蔗糖溶液联合的奖赏性训练（appetitive conditioning）以及甲基丁香酚(methyl eugenol, ME)和饱和盐溶液联合的惩罚性训练（aversive conditioning）,并以伸喙反射行为（proboscis extension reflex, PER）作为学习与否的判定标准。【结果】经过奖赏性训练后,桔小实蝇雄成虫对薄荷精油的伸喙反射率可从0%增加至68%;而经过惩罚性训练后,桔小实蝇对甲基丁香酚的伸喙反射率可从100%降低至36.54%,且这种伸喙反射率的变化是通过气味条件刺激（conditioned stimulus）和食物非条件刺激（unconditioned stimulus）的对称性联合而产生的。【结论】结果表明,桔小实蝇雄性成虫具有较强的联系性嗅觉学习能力,并且两种刺激的联合是形成学习记忆的必要条件。     

Behavioral responses of female oriental fruit flies to the odor of papayas at three ripeness stages in a laboratory flight tunnel (Diptera: Tephritidae)

Behavioral responses of adult female oriental fruit flies, Dacus dorsalisHendel, to the odor of papayas from three ripeness classes were studied using a threechoice flight tunnel bioassay. Laboratoryreared flies were allowed to respond freely to any of three papaya odors (mature green, colorbreak to one-fourth ripe, and one-half to full ripe) emanating from identical (spherical) fruit models. Five behaviors were measured in assessing the fly's relative attraction to the odors (number of landings), arrestment (total fly seconds on sphere), fly-fly interactions on the fruit model (maximum and modal fly density), and acceptance for oviposition (total eggs laid). Females showed no significant difference in total fly landings based on all age classes combined. Significant differences were noted among age classes. Females spent more total time on the sphere and showed a higher maximum density and modal fly density to ripe fruit than to green fruit odors. Ovipositional acceptance of fruit models based on the total number of eggs laid in a sphere was greater in response to the ripefruit odor than to the other two odor classes. Olfactorystimulated behavioral responses of females to the odor of ripe papayas were significantly different from the other ripeness classes for all behaviors at 8 days postemergence and then declined in 11-day-old flies. Behavioral responses were greater during the afternoon than in the morning. Observations of wild oriental fruit flies to papayas in the field indicated a preference for residing on riper fruit. The results of this study are discussed with regard to the role of olfactory inputs generated by the odor of ripening fruit on female attraction and oviposition behavior resulting in infestation of papayas by oriental fruit fly.     

Visual and olfactory stimuli and fruit maturity affect trap captures of Oriental fruit flies (Diptera: Tephritidae)

An effective lure-and-kill trap is a potentially important instrument in monitoring and controlling oriental fruit flies, Bactrocera dorsalis (Hendel). A number of experiments were performed in an orchard of commercial guava, Psydium guajava L., to determine how fly captures are affected by combining visual and olfactory stimuli, and by the timing of trap deployment relative to host phenology. Baiting sticky Ladd traps with hydrolyzed liquid protein significantly increased the number of captured flies. Mostly male flies were caught in the absence of mature guava fruit, whereas mostly female flies were caught when ripe fruit was abundant. These results suggest that an effective oriental fruit fly trap should include both visual and olfactory lures, and that proper timing of trap deployment can be an important factor in monitoring female abundance in oriental fruit fly populations.     

Olfactory conditioning with single chemicals in the German Cockroach, Blattella germanica (Dictyoptera: Blattellidae)

Many insects find resources by means of the olfactory cues of general odors after learning. To evaluate behavioral responses to the odor of a particular chemical after learning with reward or punishment quantitatively, we developed a standardized odor-training method in the German cockroach, Blattella germanica (Linnaeus), an important urban pest species. A classical olfactory conditioning procedure for a preference test was modified to become applicable to a single odor, by which a (?)-menthol or vanillin odor was independently associated with sucrose (reward) or sodium chloride solution (punishment). The strength of the association with the odor was evaluated with the increase or decrease in visit frequencies to the odor source after olfactory conditioning. The frequency increased after (?)-menthol was presented with a reward, while it did not change with the rewarded vanillin odor. With both odors, the frequency decreased significantly after training with a punishment. These results indicate that cockroaches learn a single compound odor presented as a conditioned stimulus, although the association of the odor with a reward or punishment depends on the chemical. This olfactory conditioning method can not only facilitate the analysis of cockroach behavior elicited by a learned single chemical odor, but also quantify the potential attractiveness or repellency of the chemical after learning.     

Conditioning odor-shock associations in the black blowfly,Phormia regina

Discriminative classical conditioning of an olfactory avoidance response was demonstrated in the blowfly, Phormia regina.Learning indices were calculated as the fraction of flies avoiding the CS+ (conditioned stimulus paired with electric shock reinforcement) minus the fraction of flies avoiding the CS-(conditioned stimulus not paired with electric shock), averaged over two different groups of flies, in which reciprocal odors were used as the CS+. Avoidance responses to both odors presented simultaneously at a T-maze choice point yielded mean learning indices of zero for naive flies or pseudoconditioned (shock alone) or sensitized (odors alone) controls. In contrast, pairing an odor with electric shock produced a mean learning index significantly greater than zero. These results are similar to those reported for Drosophila melanogaster.Blow flies, however, showed much lower levels of associative learning than fruit flies.     

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.     

Associative learning and memory in Drosophila: beyond olfactory conditioning

The associative learning abilities of the fruit fly, Drosophila melanogaster, have been demonstrated in both classical and operant conditioning paradigms. Efforts to identify the neural pathways and cellular mechanisms of learning have focused largely on olfactory classical conditioning. Results derived from various genetic and molecular manipulations provide considerable evidence that this form of associative learning depends critically on neural activity and cAMP signaling in brain neuropil structures called mushroom bodies. Three other behavioral learning paradigms in Drosophila serve as the main subject of this review. These are (1) visual and motor learning of flies tethered in a flight simulator, (2) a form of spatial learning that is independent of visual and olfactory cues, and (3) experience-dependent changes in male courtship behavior. The present evidence suggests that at least some of these modes of learning are independent of mushroom bodies. Applying targeted genetic manipulations to these behavioral paradigms should allow for a more comprehensive understanding of neural mechanisms responsible for diverse forms of associative learning and memory.     

Attraction of Bactrocera dorsalis (Diptera: Tephritidae) and nontarget insects to methyl eugenol bucket traps with different preservative fluids on Oahu Island, Hawaiian Islands

Attraction of oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), and nontarget insects to preservative fluids ethylene glycol antifreeze, propylene glycol antifreeze, or mineral oil in bucket traps that contained captured decaying male oriental fruit flies, a male lure (methyl eugenol), and a toxicant (DDVP vapor insecticidal strip) were compared with dry control traps. Significantly (P < 0.05) greater numbers of B. dorsalis were captured in propylene glycol antifreeze traps than in other attractant trap types. Among attractant trap types with lowest negative impacts on nontarget insects, control traps captured significantly lower numbers of three species and one morphospecies of scavenger flies, one species of plant-feeding fly, and one species each of sweet-and lipid-feeding ants. Mineral oil traps captured significantly lower numbers of two species of scavengers flies and one morphospecies of plant-feeding fly, and one species of sweet-feeding ant. Because of the fragile nature of endemic Hawaiian insect fauna, the propylene glycol antifreeze bucket trap is best suited for use in environments (e.g., non-native habitats) where endemic species are known to be absent and mineral oil traps are more suited for minimizing insect captures in environmentally sensitive habitats.     

Classical reward conditioning in Drosophila melanogaster

Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.     

Olfactory Deficits in an Alpha-Synuclein Fly Model of Parkinson’s Disease

Parkinson’s disease (PD) is the most common motor neurodegenerative disorder. Olfactory dysfunction is a prevalent feature of PD. It often precedes motor symptoms by several years and is used in assisting PD diagnosis. However, the cellular and molecular bases of olfactory dysfunction in PD are not known. The fruit fly Drosophila melanogaster, expressing human alpha-synuclein protein or its mutant, A30P, captures several hallmarks of PD and has been successfully used to model PD in numerous studies. First, we report olfactory deficits in fly expressing A30P (A30P), showing deficits in two out of three olfactory modalities, tested – olfactory acuity and odor discrimination. The remaining third modality is odor identification/naming. Second, oxidative stress is an important environmental risk factor of PD. We show that oxidative stress exacerbated the two affected olfactory modalities in younger A30P flies. Third, different olfactory receptor neurons are activated differentially by different odors in flies. In a separate experiment, we show that the odor discrimination deficit in A30P flies is general and not restricted to a specific class of chemical structure. Lastly, by restricting A30P expression to dopamine, serotonin or olfactory receptor neurons, we show that A30P expression in dopamine neurons is necessary for development of both acuity and discrimination deficits, while serotonin and olfactory receptor neurons appeared not involved. Our data demonstrate olfactory deficits in a synuclein fly PD model for exploring olfactory pathology and physiology, and for monitoring PD progression and treatment.     

The Effect of Sucrose Concentration on Olfactory-Based Associative Learning in Culex quinquefasciatus Say (Diptera: Culicidae)

A classical conditioning approach was used to examine the ability of Culex quinquefasciatus Say (Diptera: Culicidae) to associate odor with a sugar-meal of varying quality. The objectives of this study were to investigate the impact of different sucrose concentrations (5 %, 10 %, or 50 %) on positive response to conditioning and to examine sucrose concentration preference following exposure to a 10 % solution. Mosquitoes conditioned in conjunction with all three sucrose concentrations showed evidence of learning; including the concentration of the conditioning stimulus, and the sex of the mosquito. Using colored solutions to determine feeding patterns of experienced mosquitoes indicated male mosquitoes showed no preference but females showed a preference for 10 % over 5 % sucrose but not between 10 % and 50 % sucrose solutions.     

Neural mechanisms of context-dependent processing of CO2 avoidance behavior in fruit flies

The fruit fly, Drosophila melanogaster, innately avoids even low levels of CO₂. CO₂ is part of the so-called Drosophila stress odor produced by stressed flies, but also a byproduct of fermenting fruit, a main food source, making the strong avoidance behavior somewhat surprising. Therefore, we addressed whether feeding states might influence the fly’s behavior and processing of CO₂. In a recent report, we showed that this innate behavior is differentially processed and modified according to the feeding state of the fly. Interestingly, we found that hungry flies require the function of the mushroom body, a higher brain center required for olfactory learning and memory, but thought to be dispensable for innate olfactory behaviors. In addition, we anatomically and functionally characterized a novel bilateral projection neuron connecting the CO₂ sensory input to the mushroom body. This neuron was essential for processing of CO₂ in the starved fly but not in the fed fly. In this Extra View article, we provide evidence for the potential involvement of the neuromodulator dopamine in state-dependent CO₂ avoidance behavior. Taken together, our work demonstrates that CO₂ avoidance behavior is mediated by alternative neural pathways in a context-dependent manner. Furthermore, it shows that the mushroom body is not only involved in processing of learned olfactory behavior, as previously suggested, but also in context-dependent innate olfaction.     

Crossmodal visual input for odor tracking during fly flight

Flies generate robust and high-performance olfactory and visual behaviors. Adult fruit flies can distinguish small differences in odor concentration across antennae separated by less than 1 mm [1], and a single olfactory sensory neuron is sufficient for near-normal gradient tracking in larvae [2]. During flight a male housefly chasing a female executes a corrective turn within 40 ms after a course deviation by its target [3]. The challenges imposed by flying apparently benefit from the tight integration of unimodal sensory cues. Crossmodal interactions reduce the discrimination threshold for unimodal memory retrieval by enhancing stimulus salience [4], and dynamic crossmodal processing is required for odor search during free flight because animals fail to locate an odor source in the absence of rich visual feedback [5]. The visual requirements for odor localization are unknown. We tethered a hungry fly in a magnetic field, allowing it to yaw freely, presented odor plumes, and examined how visual cues influence odor tracking. We show that flies are unable to use a small-field object or landmark to assist plume tracking, whereas odor activates wide-field optomotor course control to enable accurate orientation toward an attractive food odor.     

Classical conditioning to visual stimuli in the housefly, Musca domestica

In the housefly, Musca domestica, classical conditioning to monochromatic light was demonstrated by using a proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response. Sequential and temporal relationships between the conditioned stimulus (CS) and unconditioned stimulus (UCS) to form conditioning were studied; conditioning occurred most readily when the CS was presented before the UCS without an interstimulus interval. Spectral response curves, which were obtained by examining the responsiveness of flies conditioned for one colour to other colours, were divided into two groups; one was that of 462 nm-conditioned flies and the other those of 516 nm-, 579 nm-, and 642 nm-conditioned flies.     

Operant conditioning in Drosophila melanogaster wild-type and learning mutants with defects in the cyclic AMP metabolism

Wild-type Drosophila melanogaster and the learning mutants dunce, amnesiac and rutabaga, were tested using a new operant conditioning paradigm for single flies. All strains are able to learn to different extents, but no evidence of memory was found in the mutants amnesiac and rutabaga, while dunce has a reduced but extended memory. The relationship between this characteristic and cAMP levels are discussed. The three mutants have previously been shown, using classical conditioning paradigms to be deficient in olfactory learning and/or memory, and show reduced visual learning. The variability of the response of the mutants in the different paradigms is discussed in relation to the generality of the Aplysia model of the cellular mechanism underlying learning. In the operant conditioning paradigm described here, 93% of the wild-type flies learned to criterion. The performance of individual flies was consistent.     

Classical olfactory conditioning in the cockroach Periplaneta americana

We established a classical conditioning procedure for the cockroach, Periplaneta americana, by which odors were associated with reward or punishment. Cockroaches underwent differential conditioning trials in which peppermint odor was associated with sucrose solution and vanilla odor was associated with saline solution. Odor preference of cockroaches was tested by allowing them to choose between peppermint and vanilla sources. Cockroaches that had undergone one set of differential conditioning trials exhibited a significantly greater preference for peppermint odor than did untrained cockroaches. Memory formed by three sets of differential conditioning trials, with an inter-trial interval of 5 min, was retained at least 4 days after conditioning. This conditioning procedure was effective even for cockroaches that had been harnessed in plastic tubes. This study shows, for the first time in hemimetaborous insects, that both freely moving and harnessed insects are capable of forming olfactory memory by classical conditioning procedure. This procedure may be useful for future electrophysiological and pharmacological studies aimed at elucidation of neural mechanisms underlying olfactory learning and memory.     

Olfactory interference during inhibitory backward pairing in honey bees

Background

Restrained worker honey bees are a valuable model for studying the behavioral and neural bases of olfactory plasticity. The proboscis extension response (PER; the proboscis is the mouthpart of honey bees) is released in response to sucrose stimulation. If sucrose stimulation is preceded one or a few times by an odor (forward pairing), the bee will form a memory for this association, and subsequent presentations of the odor alone are sufficient to elicit the PER. However, backward pairing between the two stimuli (sucrose, then odor) has not been studied to any great extent in bees, although the vertebrate literature indicates that it elicits a form of inhibitory plasticity.

Methodology/Principal Findings

If hungry bees are fed with sucrose, they will release a long lasting PER; however, this PER can be interrupted if an odor is presented 15 seconds (but not 7 or 30 seconds) after the sucrose (backward pairing). We refer to this previously unreported process as olfactory interference. Bees receiving this 15 second backward pairing show reduced performance after a subsequent single forward pairing (excitatory conditioning) trial. Analysis of the results supported a relationship between olfactory interference and a form of backward pairing-induced inhibitory learning/memory. Injecting the drug cimetidine into the deutocerebrum impaired olfactory interference.

Conclusions/Significance

Olfactory interference depends on the associative link between odor and PER, rather than between odor and sucrose. Furthermore, pairing an odor with sucrose can lead either to association of this odor to PER or to the inhibition of PER by this odor. Olfactory interference may provide insight into processes that gate how excitatory and inhibitory memories for odor-PER associations are formed.     

桔小实蝇和番石榴实蝇对6种寄主果实的产卵选择适应性

植食性昆虫对寄主植物的选择适应性是研究昆虫和植物协同进化关系的核心内容之一。评估寄主植物对植食性昆虫种群的适合度,需要综合分析昆虫对寄主的产卵选择性和寄主对昆虫的取食适合性。以桔小实蝇和番石榴实蝇为研究对象,分别测定了这两种实蝇对6种寄主果实:番石榴、香蕉、杨桃、木瓜、甜橙、番茄的产卵选择性以及幼虫取食后对其生长发育的影响。寄主产卵选择性实验分别采用完整寄主果实直接供试产卵和块状寄主果实气味引诱产卵两种处理方式;在生长发育适应性实验中,以幼虫和蛹的存活和生长发育等相关参数作为评价指标。实验结果表明,寄主的供试方式不同,两种实蝇的选择性均有明显差异;对寄主气味选择性强的寄主更适合于两种实蝇后代的生长发育。两种实蝇对6种寄主果实的产卵选择性和后代发育适合性两者相关性不显著,与许多文献报道单一地采用发育适合性(如发育历期、存活率或蛹重等)作为评价寄主选择性的结果不一致。两种实蝇之间对6种寄主果实的产卵选择和幼虫取食适合性既具相似性也具差异性,表明这两种实蝇在寄主生态位上既有重叠性又有分化性。     

Reversing Stimulus Timing in Visual Conditioning Leads to Memories with Opposite Valence in Drosophila

Animals need to associate different environmental stimuli with each other regardless of whether they temporally overlap or not. Drosophila melanogaster displays olfactory trace conditioning, where an odor is followed by electric shock reinforcement after a temporal gap, leading to conditioned odor avoidance. Reversing the stimulus timing in olfactory conditioning results in the reversal of memory valence such that an odor that follows shock is later on approached (i.e. relief conditioning). Here, we explored the effects of stimulus timing on memory in another sensory modality, using a visual conditioning paradigm. We found that flies form visual memories of opposite valence depending on stimulus timing and can associate a visual stimulus with reinforcement despite being presented with a temporal gap. These results suggest that associative memories with non-overlapping stimuli and the effect of stimulus timing on memory valence are shared across sensory modalities.     

Effect of Flumethrin on Survival and Olfactory Learning in Honeybees

Flumethrin has been widely used as an acaricide for the control of Varroa mites in commercial honeybee keeping throughout the world for many years. Here we test the mortality of the Asian honeybee Apis cerana cerana after treatment with flumethrin. We also ask (1) how bees react to the odor of flumethrin, (2) whether its odor induces an innate avoidance response, (3) whether its taste transmits an aversive reinforcing component in olfactory learning, and (4) whether its odor or taste can be associated with reward in classical conditioning. Our results show that flumethrin has a negative effect on Apis ceranàs lifespan, induces an innate avoidance response, acts as a punishing reinforcer in olfactory learning, and interferes with the association of an appetitive conditioned stimulus. Furthermore flumethrin uptake within the colony reduces olfactory learning over an extended period of time.