An inexpensive electronic device for measuring electroantennogram responses to sex pheromone components with a voltmeter

ABSTRACT. An inexpensive, portable device which allows measurement of electroantennogram (EAG) maximum amplitudes with a voltmeter instead of an oscilloscope is described. Potential advantages of the device include more rapid recording, low-cost construction of additional EAG recording systems for a laboratory with several users, and portability allowing studies in the field and occasional bench top use. The device is intended for use in conventional EAG recording, where EAG signals with rapid depolarizations are being measured, and where the maximum amplitude of the EAG signal is the only parameter of interest.     

Improvement of signal-to-noise ratio in electroantennogram responses using multiple insect antennae

Using an array of insect antennae connected in series or in parallel, electroantennogram (EAG) responses and noise levels were investigated in an attempt to improve signal-to-noise (S/N) ratio and sensitivity. Both the EAG response amplitude and noise level increased when the antennae of male Helicoverpa zea (Lepidoptera: Noctuidae) were connected in series. Due to lower relative increase in noise level than EAG amplitude as the number of antennae increased, the S/N ratio was also significantly improved by the serial connection. As a result the sensitivity of EAG was improved by the serial connection, which showed ca. ten-fold improvement in the threshold detection levels compared with a single antenna when four antennae were connected in series. In contrast to the serial connection, there were no differences in EAG amplitudes and overall noise levels when different numbers of antennae were connected in parallel. When only large-amplitude noise was taken into account, however, the S/N ratio was somewhat improved by the parallel connection. The frequency of overall noise remained at the same level both in the serial and in the parallel connection. However, the frequency of the large-amplitude noise increased in serial connection but decreased in parallel connection. The present study clearly indicates that both the sensitivity and S/N ratio of the EAG biosensor could be significantly improved by using the multiple antennal connections.     

An improved aphid electroantennogram

Excised antennae have been used to record aphid electroantennograms (EAGs) but these preparations have small, rapidly declining responses and a short usable life. An improved EAG technique is described and evaluated using alate virginoparae of the black bean aphid, Aphis fabae, and a series of plant volatiles. EAG recording with fine-tip glass electrodes inserted into the surface of the intact antenna of a whole insect preparation gave stable EAG peaks of a few mV and EAG peak amplitude decreased by only 22% over an 8 hour recording period. The EAG responses showed typical dose-dependent characteristics. There was variation in EAGs from different preparations, so normalisation of the EAG responses against a standard stimulus is still necessary. The thirty compounds tested elicited an EAG response profile largely similar to that reported previously from excised antennae. EAG responses recorded with the electrode at three different positions, between the 5th-6th (A), 4th-5th (B), and 3rd-4th (C) antennal segments, were smaller when recorded closer to the head. Position B produced larger EAG responses than those at C although there was no olfactory receptor between position B and C.     

Effects of Trifluoromethyl Ketones and Related Compounds on the EAG and Behavioural Responses to Pheromones in Male Moths

Trifluoromethyl ketones (TFMKs) and analogues affect pheromonedetection and behaviour in male moths. 3-Octylthio-1, 1, 1-trifluoropropan-2-one(OTFP), one of the most effective antiesterase agents, decreasedthe EAG amplitude and increased the repolarization time in spodopteralittoralis. It also modified EAG responses of Marnestra brassicaeand Heliothis zea to their pheromones and analogues, containingan acetate, an alcohol or an aldehyde function. In addition,OTFP also reduced the amplitude of the EAG response to linalool,a monoterpenic alcohol, but not its kinetics. The responsesof male S. littoralis to the pheromone in the wind tunnel weresignificantly reduced after pre-exposure to vapours of OTFP.Comparison of the activity of other TFMKs and analogues withthat of OTFP revealed a good agreement on EAG and behaviour.The effects of TFMKs on the EAG kinetics are discussed consideringthe hypothesis of an inhibition of the pheromone deactivationin the antenna. Chem. Senses 22: 407–416, 1997.     

Quantitative measurement of general odorant using electroantennogram of male silkworm moth,Bombyx mori

The investigation of electroantennogram (EAG) using insect antennae has been primarily focused on the measurement of insect pheromone. Insect has highly specialized olfactory receptors inside their antennae. In this paper, EAG was applied to detect general odorants and the feasibility of this system for the olfactory biosensor was investigated. Electroantennogram measurement was carried out using the antennae of male silkworm moth,Bombyx mori, and ammonia gas as the model odorant. EAG parameters including peak amplitude, decay, and level were analyzed for the quantitative measurement. The peak amplitude increased linearly with the ammonia concentration and the reproducible electrical signals were generated at least for 2 hrs after the antenna was cut off from the silkworm moth.     

Electroantennogram response gradient on the antenna of the European corn borer, Ostrinia nubilalis

The electroantennogram (EAG) response of male moths of the European corn borer (Ostrinia nubilalis) to their two pheromone components, (Z)- and (E)-11-tetradecenyl acetates, were studied. The EAG amplitude of the fast initial phase was approximately proportional to the antennal length. Local EAG responses were obtained along the length of the antenna with multiple electrodes. Response-interaction studies indicated a unique spread of the EAG response in a proximal direction in the antenna. A possible mechanism of the EAG summation was considered. The EAG response characteristic was similar for both (Z)- and (E)-11-tetradecenyl acetate along the length of the antenna.     

美洲大蠊对多种化合物的触角嗅觉电位反应

用多种化合物的气味制激美洲大蠊(Periplaneza americana)触角,诱导、记录触角电位(EAG)反应.试验了各类化合物154种,其中53%能诱发出明显的EAG反应.绝大多数EAG反应是负相慢波,波幅多数为0.5—2mV,时程1.5—3秒.苯等少数化合物能诱发正相EAG反应.剂量-反应曲线大致呈“S”形,还可见适应现象.萜类化合物和挥发性植物油类诱发EAG反应的作用最强,其次是酮、醇、酯、胺类化合物.化合物的含氧功能基团和空间构型与EAG反应的诱发有一定的关系.可能是同一种化合物对不同种的感受器都可能诱发EAG反应.一般性化合物诱发雌、雄、成、幼虫EAG反应的差异不大,但雌虫类提取物仅对雄性成虫触角诱发较强的EAG反应.一些已知的驱、诱、杀虫剂也能诱发EAG反应,但仅表现为对一般化合物的反应特征.     

Mechanism of insect sex-pheromone sensory transduction: Role of adenyl cyclase

1. A microperfusion technique was devised for insect antennae.2. Amputated antennae of silkmoths were perfused with: c-AMP, dibutyryl c-AMP, dibutyryl c-GMP, c-UMP, c-IMP, 5′-AMP, ADP, ATP, adenosine, caffeine, theophylline, sodium citrate.3. Olfactory responses to Antheraea pernyi sex-pheromone were measured electrophysiologically by the EAG method.4. Pronounced enhancement of EAG amplitude was found with dibutyryl c-AMP, and with phosphodiesterase inhibitors.5. A model depicting a role for adenyl cyclase in insect olfactory transduction is presented and discussed.     

Measuring activity in olfactory receptor neurons in Drosophila: Focus on spike amplitude

Olfactory responses at the receptor level have been thoroughly described in Drosophila melanogaster by electrophysiological methods. Single sensilla recordings (SSRs) measure neuronal activity in intact individuals in response to odors. For sensilla that contain more than one olfactory receptor neuron (ORN), their different spontaneous spike amplitudes can distinguish each signal under resting conditions. However, activity is mainly described by spike frequency.Some reports on ORN response dynamics studied two components in the olfactory responses of ORNs: a fast component that is reflected by the spike frequency and a slow component that is observed in the LFP (local field potential, the single sensillum counterpart of the electroantennogram, EAG). However, no apparent correlation was found between the two elements.In this report, we show that odorant stimulation produces two different effects in the fast component, affecting spike frequency and spike amplitude. Spike amplitude clearly diminishes at the beginning of a response, but it recovers more slowly than spike frequency after stimulus cessation, suggesting that ORNs return to resting conditions long after they recover a normal spontaneous spike frequency. Moreover, spike amplitude recovery follows the same kinetics as the slow voltage component measured by the LFP, suggesting that both measures are connected.These results were obtained in ab2 and ab3 sensilla in response to two odors at different concentrations. Both spike amplitude and LFP kinetics depend on odorant, concentration and neuron, suggesting that like the EAG they may reflect olfactory information.     

Identification of electrophysiologically-active compounds for the malaria mosquito, Anopheles gambiae, in human sweat extracts

Abstract. Human sweat samples were chemically fractionated into acid and non-acid components. The most abundant volatile compounds present in the fractions were identified by linked gas chromatography mass spectrometry. The acid fractions were found to be composed of a range of twenty aliphatic and three aromatic carboxylic acids ranging, on average, from 0.02 to 20 ig per ml of sweat sampled. Non-acid fractions were found to contain: 6-methyl-5-hepten-2-one, l-octen-3-ol, decanal, benzyl alcohol, dimethylsulphone, phenylethanol, phenol and 4-mefhylphenol, collectively amounting to 0.1 and 3 |ig per ml of sweat. The major component of sweat was found to be L-lactic acid which constituted from 1 to 5 mg/ml.
Using the intact antennae of the anthropophilic malaria vector mosquito Anopheles gambiae Giles, the peripheral olfactory activities of compounds identified in the sweat fractions were investigated by electroantennography (EAG). Short-chain saturated carboxylic acids, methanoic, ethanoic, propanoic, butanoic, pentanoic and hexanoic acids were found to elicit significantly larger EAG responses than longer chain saturated carboxylic acids from female An.gambiae. For a given dose the largest amplitude EAG response was elicited by methanoic acid. Pentanoic acid elicited larger EAG responses than either butanoic or hexanoic acids. Two non-acidic compounds, l-octen-3-ol and 4-methylphenol, were found to elicit significant dose-dependent EAG responses from female An.gambiae. 1 -Octen-3-ol elicited larger EAG responses than 4-methylphenol for a given dose, but both compounds elicited smaller EAG responses than the same dose of C_]-C₆straight-chain aliphatic carboxylic acids. The possible behavioural significance of the EAG-active compounds identified in human sweat samples is discussed.     

Mechanistic insight into human ether-à-go-go-related gene (hERG) K+ channel deactivation gating from the solution structure of the EAG domain

Human ether-à-go-go-related gene (hERG) K(+) channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2-26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1-26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26-135 form a Per-Arnt-Sim domain, but a structure for NT1-26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1-26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1-26 domain. We propose a model in which NT1-26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.     

Circadian rhythm in olfactory response in the antennae controlled by the optic lobe in the cockroach

The olfactory response in antennae of the cockroach, Leucophaea maderae, was investigated by measuring electroantennograms (EAGs) in restrained animals. The amplitude of the EAG response to pulses of ethyl acetate, octanol, or fenchone, exhibited a robust, light entrained, circadian rhythm that persisted at least 14 days in constant darkness. Dilution-response curves measured at the peak and trough of the rhythm indicated there was a 10-fold change in sensitivity. The EAG rhythm was abolished by severing the optic tracts, while entrainment was abolished by ablation of the compound eyes. The results indicate that the circadian system modulates olfactory sensitivity in the antennae and that the rhythm is driven by a circadian pacemaker in the optic lobes that is entrained by photoreceptors in the compound eyes.     

棉铃虫蛾对寄主植物挥发油的嗅觉电生理及行为反应

本项研究以Tenax TA、Tenax GR为吸附剂,正己烷为洗脱剂,得到了玉米雄穗、玉米花丝、棉花花、棉花顶尖、棉蕾、芹菜、花生叶及花的挥发油。触角电位(EAG)测试结果表明,参试寄主植物及器官的挥发油均有一定的EAG活性,EAG的反应幅度为0.1～1.2mV,雌性棉铃虫Helicoverpa armigera对其产卵寄主或器官的EAG反应在1～10日龄内有随着日龄增加而增大的趋势,交配蛾对棉花花、棉花嫩尖、芹菜、棉蕾的挥发油的EAG反应显著高于处女蛾,雌蛾在经过花生、玉米花丝及棉花顶尖挥发油处理的滤纸上落卵量显著高于对照,表明这几种挥发油中含有可刺激棉铃虫选择其产卵的它感信息化合物。剂量反应曲线表明,交配可显著降低雌蛾对棉花嫩尖和棉花花挥发油的敏感阈值。四臂嗅觉仪测试棉铃虫对不同寄主植物挥发油的行为反应表明,棉蕾、棉花叶、玉米花丝、花生的挥发油均具有-定的诱蛾活性,选择系数分别为0.78、0.61、0.49、0.31。     

Electrophysiological basis for the behavioural response of male and female Trichoplusia ni to synthetic female pheromone

Electroantennogram (EAG) studies demonstrated that antennae of both male and female Trichoplusia ni have: (1) receptor-neurones sensitive to female pheromone, (2) a low response threshold, (3) an identical mean-percentage EAG curve over a broad concentration range of pheromone, and (4) a similar absolute recovery interval from adaptation to pheromonal stimulation. These factors suggest that antennae of male and female T. ni have homologous and homogeneous acceptor sites for the female pheromone. Pheromonal stimulation of female antennae elicited EAGs with only 25% of the amplitude of those elicited in males.     

人尿及汗液中几种化合物对黄脊竹蝗的引诱活性

黄脊竹蝗Ceracris kiangsu Tsai成虫对发酵人尿及汗液有明显的趋向行为。利用昆虫触角EAG反应和林间诱蝗试验对氯化钠、碳酸氢氨及乙醇3种主要的人尿、汗液化合物的诱蝗活性进行测定。结果表明:3种物质均能明显激发黄脊竹蝗雌、雄成虫的触角电位反应,激发活性的强弱次序为乙醇>碳酸氢氨>氯化钠。3种化合物对雌蝗触角的刺激作用强于雄蝗。在林间,氯化钠、碳酸氢氨及乙醇及其混合物对竹蝗的引诱效果不明显,化合物浓度及药剂种类是影响竹蝗引诱量的因子之一。     

Flavonoid regulation of EAG1 channels

The voltage-gated, K⁺-selective ether á go-go 1 (EAG1) channel is expressed throughout the brain where it is thought to regulate neuronal excitability. Besides its normal physiological role in the brain, EAG1 is abnormally expressed in several cancer cell types and promotes tumor progression. Like all other channels in the KCNH family, EAG1 channels have a large intracellular carboxy-terminal region that shares structural similarity with cyclic nucleotide–binding homology domains (CNBHDs). EAG1 channels, however, are not regulated by the direct binding of cyclic nucleotides and have no known endogenous ligands. In a screen of biological metabolites, we have now identified four flavonoids as potentiators of EAG1 channels: fisetin, quercetin, luteolin, and kaempferol. These four flavonoids shifted the voltage dependence of activation toward more hyperpolarizing potentials and slowed channel deactivation. All four flavonoids regulated channel gating with half-maximal concentrations of 2–8 µM. The potentiation of gating did not require the amino-terminal or post-CNBHD regions of EAG1 channels. However, in fluorescence resonance energy transfer and anisotropy-based binding assays, flavonoids bound to the purified CNBHD of EAG1 channels. The CNBHD of KCNH channels contains an intrinsic ligand, a conserved stretch of residues that occupy the cyclic nucleotide–binding pocket. Mutations of the intrinsic ligand in EAG1 (Y699A) potentiated gating similar to flavonoids, and flavonoids did not further potentiate EAG1-Y699A channels. Furthermore, the Y699A mutant CNBHD bound to flavonoids with higher affinity than wild-type CNBHD. These results suggest that the flavonoids identified here potentiated EAG1 channels by binding to the CNBHD, possibly by displacing their intrinsic ligand. EAG1 channels should be considered as a possible target for the physiological effects of flavonoids.     

Autodetection and chemistry of female and male pheromone in both sexes of the tiger moth Panaxia quadripunctaria

Female moths of Panaxia quadripunctaria PODA (Lepidoptera, Arctiidae), produce (Z,Z)-6,9-heneicosadiene (I) as the major and (Z,Z)-6,9-eicosadiene (II) as the minor component of a putative pheromone. Related compounds occur in trace amounts. The abdominal scent glands contain 5–10 μg of (I) and 50–100 ng of (II). Recordings of electroantennogram (EAG) responses to (I), (II), and to female glands are of equal amplitude in both sexes. Females are thus capable of pheromone autodetection in contrast to the majority of moths where females are considered to be anosmic for their own attractant. The EAG threshold to (I) was below 1 ng at the odour source. The odour of the male scent gland (corema) elicited significant EAGs in both sexes. The chemical contents of coremata varied with the provenience of the moths. A variety of ethyl esters was always found, yet hydroxydanaidal (up to 20 μg/corema) and traces of danaidal, only in some samples. All these scents might be components of a male pheromone. Peculiar scent scales on the coremata are exposed during the extrusion. Antennae of both sexes have similar inventories of trichoid sensilla. Accepted: 11 July 1997     

粘虫雄蛾触角对其性信息素的电生理反应

用自行组装的触角电位仪,测定了粘虫Mythimna separata雄蛾蛾龄对标准化合物顺-11-十六碳烯醛(Z11-16：A1d)、顺-9-十六碳烯醛(Z9-16:Ald)和十六碳醛(16：A1d)的EAG反应的影响,分析了雄蛾触角对3种标准化合物的剂量-反应关系,发现粘虫雄蛾对Zll-16,Ald和Z9-16：Ald的剂量-反应曲线呈现出典型的“S”型,并且反应阐值较低。而对16：A1d几乎没有反应。最为重要的是检测了粘虫雄蛾对雌蛾腺体提取物的EAG反应,反应值的大小与样品中所提取的雌蛾腺体数目成正比。通过检测粘虫雄蛾对羽化不同天数,以及同一天羽化、在暗期不同时辰提取的雌蛾腺体提取物的EAG反应,证实了粘虫雄蛾的反应曲线与雌蛾释放性信息素的时辰节律呈正相关。还比较了烟青虫和粘虫雄蛾对粘虫雌蛾腺体提取物的EAG反应,间接证实了粘虫雌蛾腺体提取物中可能含有次要组分Z9-16;A1d。     

Electroantennogram responses of the stable fly, Stomoxys calcitrans, to carbon dioxide and other odours

ABSTRACT. Electroantennograms (EAGs) were recorded from Stomoxys calcitrans (L.) in response to increasing concentrations of carbon dioxide in an airstream. The magnitude of the EAG increases logarithmically from +0.023% carbon dioxide up to approximately +2.0% where a maximum is reached. Flies deprived of food for 48–50 h are more responsive to small increases in the carbon dioxide concentration than those deprived of food for only 20–23 h. It is concluded that the sensitivity of carbon dioxide receptors on the antennae of S. calcitrans increases as hunger develops. EAGs were also recorded in response to cattle odour, odour from fresh cattle faeces, expired human breath, acetone, and l-octen-3-ol. Acetic acid vapour causes a reversal of the usual EAG response indicating inhibition.     

An electroantennogram apparatus for testing the activity of semiochemicals on the olive beetle, Phloeotribus scarabaeoides (Coleoptera: Scolytidae): first recordings of the response to ethylene

Abstract. An electroantennogram (EAG) apparatus for testing the activity of pheromones of the olive beetle, Phloeotribus scarabaeoides , is described. This inexpensive device is suitable for whole antennal recording from scolytids. The modification of the amplifier and sample injection system, described previously, eliminate parasitic noise and improve the recording of the EAG signal.
Results from previous studies revealed significant beetle attraction to a source releasing ethylene. Serial solutions of 2-chloro-ethyl phosphonic acid were tested, and 10% acetic acid served as a standard.
A significant variation in amplitude of depolarization was found among the different concentrations of 2-chloro-ethyl phosphonic acid tested. Maximum depolarization was elicited at a concentration of 0.1% in both males and females. In general, no statistical differences in responses were found between males and females, but females showed a significantly lower threshold of response.