Electroantennographic resolution of pulsed pheromone plumes in two species of moths with bipectinate antennae

Trains of 20-ms-duration pulses of pheromone were deliveredat rates of 1–33 Hz to antennal preparations of malesof Bombyx mori and Lymantria dispar, two moth species with bipectinateantennae. Resolution of rapidly pulsed plumes of pheromone wasnot compromised by a complex antennal morphology or by moderatechanges in wind speed (25–50 cm/s). Fourier analysis ofthe electroantennograms resolved the temporal structure of thesignal at frequencies up to 25 Hz for B. mori and up to 5 Hzfor L. dispar. The ability of these sensory structures to identifythe original (unchanged) frequency of the pulse train is particularlynoteworthy because air is slowed by about an order of magnitudeas it passes through bipectinate antennae. Although an unchangingfrequency in slowed airflow may be counterintuitive, this flowpattern, and its effects on odorant patch shape and spacing,is explained from fluid mechanical principles (i.e., the principleof continuity). An unchanging frequency suggests that as deceleratingair passes through a bipectinate antenna, the slowed patchesof odorant are stretched, thinned, and brought closer togetherby the same factor with which they are slowed.     

Interneurons sensitive to female pheromone in the deutocerebrum of the male silkworm moth, Bombyx mori

ABSTRACT. In response to minute quantities of female sex pheromone, the male silkworm moth, Bombyx mori L., walks upwind to locate the odour source. The axons of antennal receptors specific for the two known components of the pheromone terminate in the deutocerebrum. In this study, single interneurons were recorded extracellularly in the deutocerebrum of the male silkworm moth. Responses were characterized as the antennae were presented with puffs of clean air, or air containing either or both components of the female pheromone, bombykol and bombykal. An apparatus is described which added bombykol or bombykal to a constant air stream flowing over the antenna. Most units (87%) showed qualitatively different responses to bombykol and bombykal. A majority of the pheromone-sensitive units (65%) also showed mechanosensory responses to air puffs. Two units were recorded which were slightly inhibited by either bombykol or bombykal alone, but were excited by a mixture of the two.     

Pheromone-stimulated locomotory and orientation responses in the American cockroach

Males of Periplaneta americana respond to the sex pheromone secreted by females with increased locomotion and positive chemo-orientation. Both sexes orient toward aggregation pheromone without an increase in locomotion. Immediately following removal of one antenna males exhibit ‘circus’ movements, but after 2 days orient toward pheromones; a bi-modal mechanism of chemo-orientation is proposed. Cockroaches turn away from air currents, but orient toward air currents carrying sex or aggregation pheromone, suggesting the possibility of up-wind orientation. Antennae deflect upward and outward when pheromone is first perceived; the head then moves toward the pheromone source. Following removal of one antenna, the pattern of head and antennal movement changes in a manner which enhances the sweeping of the intact antenna.     

Quantitative method for pheromone delivery in studies of sensory adaptation of moth antennae

Abstract.  A pheromone sprayer and an electroantennogram (EAG) are used to study sensory adaptation in the antennae of male obliquebanded leafrollers, Choristoneura rosaceana and oriental fruit moths, Grapholita molesta , to the main pheromone compounds ( Z )-11-tetradecen-1-yl acetate ( Z 11-14:Ac) and ( Z )-8-dodecen-1-yl acetate ( Z 8-12:Ac), respectively. The atomization of 0.125, 0.25, 0.5 or 1 μL ethanol min⁻¹ into the EAG air delivery tube at an airflow rate of 2 L min⁻¹, with resultant concentrations of 6.25, 12.5, 25 or 50 × 10⁻⁵μL ethanol mL air⁻¹, respectively, does not affect the EAG response of C. rosaceana or C. molesta after a 30-min exposure period. The atomization of 0.125 μL min⁻¹ of a solution of 8 mg Z 11-14:Ac mL⁻¹ ethanol into the EAG air delivery tube at an airflow rate of 2 L min⁻¹, with a resultant concentration of 0.5 ng pheromone mL⁻¹ air, reduces the EAG response of C. rosaceana by approximately 70% after a 15-min exposure period. An additional 15 min of exposure to pheromone does not result in increased sensory adaptation. Antennae recover 32% of the lost responsiveness when exposed to pheromone-free air for 15 min. The atomization of 0.125 μL min⁻¹ of a solution of 8 mg Z 8-12:Ac mL⁻¹ ethanol into the EAG air delivery tube at an airflow rate of 2 L min⁻¹, with a resultant concentration of 0.5 ng pheromone mL⁻¹ air, reduces the EAG response of C. molesta antenna by approximately 80% after a 15- or 30-min exposure period. The antennae of this species do not recover responsiveness when exposed to pheromone-free air for 15 min.     

Comparison of Palaeobotanical Observations with Experimental Data on the Leaf Anatomy of Durmast Oak [Quercus petraea(Fagaceae)] in Response to Environmental Change

To test whether stomatal density measurements on oak leaf remainsare reliable tools for assessing palaeoatmospheric carbon dioxideconcentration [CO₂], under changing Late Miocene palaeoenvironmentalconditions, young seedings of oak (Quercus petraea,Liebl.) weregrown at elevatedvs.ambient atmospheric [CO₂] and at high humiditycombined with an increased air temperature. The leaf anatomyof the young oaks was compared with that of fossil leaves ofthe same species. In the experiments, stomatal density and stomatalindex were significantly decreased at elevated [CO₂] in comparisonto ambient [CO₂]. Elevated [CO₂] induced leaf cell expansionand reduced the intercellular air space by 35%. Leaf cell sizeor length were also stimulated at high air humidity and temperature.Regardless of a temperate or subtropical palaeoclimate, leafcell size in fossil oak was not enhanced, since neither epidermalcell density nor length of the stomatal apparatus changed. Theabsence of these effects may be attributed to the phenologicalresponse of trees to climatic changes that balanced temporalchanges in environmental variables to maintain leaf growth underoptimal and stable conditions.Quercus petraea,which evolvedunder recurring depletions in the palaeoatmospheric [CO₂], maypossess sufficient phenotypic plasticity to alter stomatal frequencyin hypostomatous leaves allowing high maximum stomatal conductanceand high assimilation rates during these phases of low [CO₂].Copyright1998 Annals of Botany Company Atmospheric CO₂, high humidity, elevated temperature,Quercus petraea,durmast oak, Late Miocene, palaeoclimates, leaf anatomy, stomatal density, stomatal index     

Suction sampling technique for obtaining standardized cytologic specimens from the oral mucosa

To enhance the value of exfoliative cytology for the study of the oral mucosa, a simple apparatus was developed to permit adequate sampling of a specific site so that samples collected on different occasions could be compared. The device essentially consists of a collecting cup connected to a blood collection evacuation system. The collecting cup is a modified female half of a stainless-steel filter holder supporting a 13-mm-diameter cellulose filter of 0.05-microns pore size. Suction pressure is applied by means of a 10-mL glass tube of premeasured vacuum. After positioning the collecting cup on the selected site on the buccal mucosa, the vacuum (440 mm Hg) is applied for five seconds. The mucosa is drawn in against the filter, producing a monolayered imprint of cells. This sample may be disengaged from the filter by agitation into a solution; this allows quantitative cytologic studies, such as the measurement of cell numbers by an electronic counter or the estimation of the areas of cells and nuclei by computer-aided image analysis of Cytospin preparations. Five separate samplings from each of three test subjects produced a harvest of 3,000 to 7,000 epithelial cells per sample; the cellular areas ranged from 784 to 1,052 sq microns while the nuclear areas ranged from 18.4 to 21.8 sq microns.     

Neurophysiological responses of pheromone-sensitive receptor neurons on the antenna of Trichoplusia ni (Hubner) to pulsed and continuous stimulation regimens

Both the frequency and the temporal pattern of action potentialproduction in an insect olfactory receptor neuron are stronglyaffected by odorant composition and the time course over whichstimulus concentration varies. To investigate the temporal characteristicsof the neurophysiological responses of these neurons, we deviseda stimulus delivery system that allows us to repeatedly presentwell-mixed, constant concentration odor pulses with relativelysharp onsets and offsets. Here we compare neurophysiologicalresponses to several different stimulation regimens, includingpulses of different durations and repetition rates. During stimulationwith high concentrations of pheromone, the temporal patternof neural activity from olfactory receptor neurons on the antennaof Trichoplusia ni (Hübner) is characterized by an initialphasic period (100–200 ms), followed by a tonic periodwhich is typically maintained for the remaining duration ofthe stimulus. Different olfactory receptor neurons appear tovary among themselves in the relative distribution between thephasic and tonic portions of the overall discharge. During stimulationregimens involving rapid repeated pulses of odorants, a portionof the phasic response levels is preserved during each pulse.Consequently, T. ni males probably detect much of the fluctuationin concentration of pheromone that may normally occur downwindfrom the site of pheromone release.     

Organic electrospun nanofibers as vehicles toward intelligent pheromone dispensers: characterization by laboratory investigations

Organic nanofibers have a history of technical application in various independent fields, including medical technology, filtration technology, and applications of pharmaceuticals via inhalation into the lungs. Very recently, in a joint effort with polymer chemists, agricultural applications have been added to this list of priorities. The aim is finding novel approaches to insect control. Pheromones, dispensed in a quantifiable way, are being used here in disrupting the mating communication between male and female pest insects, e.g. the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae), where current dispenser technology does not fully meet the high expectations of growers and environmentalists with respect to longevity of constant release, self decomposition, mechanical distribution, renewability as well as sustainability of resources. The methodology of electrospinning is exhaustively covered by Greiner and Wendorff (2007), with technical details reported by Hellmann et al. (2009), Hein et al. (2011), and Hummel et al. (2010). Wind tunnel studies were run within a tunnel with adjustable laminar flow and 0.5 m/sec air velocity. Mass losses of the electrospun fiber bundles were determined with a sensitive analytical balance 2-3 times per week and recorded as time vs. mass change. CLSA experiments were performed with a self developed glass apparatus (Lindner, 2010) based on various suggestions of previous authors. Microgram quantities of volatile pheromone (E,Z)-7,9-Dodecadienylacetate were absorbed on a filter of rigorously purified charcoal and desorbed by repeated micro extraction with a suitable solvent mixture. Aliquots of the solution were subjected to temperature programmed capillary GLC. Retention times were used for identification, whereas the area covered by the pheromone peak originating from a FID detector signal was integrated and compared with a carefully calibrated standard peak. Since these signals were usually in the low nanogram range, several replications were averaged for statistical improvement. - Thermogravimetric analysis between ambient temperature and 500 degrees C provided a series of degradation curves where the diagram contained information on the evaporation of pheromone alone, polymer fiber alone and pheromone included in the fiber.- Microscopic investigations resulted in pictures of nanofibers from which the overall morphology and the fiber dimensions could be quantified. Organic nanofibers loaded with the grapevine moth pheromone have been well characterized by 5 different lab methods, followed by field bioassays reported elsewhere in these communications volumes (HUMMEL et al., 2011). This comprehensive analytical approach to fiber characterization is new and will be further refined. The federal agency JKI Berlin subjected the pheromone loaded organic fibers to various independent toxicological and ecotoxicological tests and found no adverse side effects.     

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.     

An improved electroantennogram apparatus with a new automatic sample injection system

ABSTRACT. An improved electroantennogram (EAG) apparatus with a new automatic sample injection system, based on a cyclic timer connected to a 12VD.C. electrovalve, is described. The device is fully automatic and allows insertion of constant cyclic air puffs of the test compounds to the antenna at predetermined intervals.     

旋花蛾性信息素成分的鉴定

提取和鉴定了杂草田旋花Convolvulus arvensis的天敌旋花蛾Tyta luctuosa的性信息素主要成分,并应用风洞实验对雄蛾进行了测试。结果表明,腺体提取物和雌蛾求偶时所释放的主要性信息素成分均为2种化合物：顺-9-十四碳烯醛（Z9-14∶Ald）和顺-11-十六碳烯醛（Z11-16∶Ald）。腺体提取物中2种化合物的总量在个体间差异很大（22～167 ng）,但二者的比率相对稳定（Z9-14∶Ald/Z11-16∶Ald=0.3±0.15）。雌蛾所释放的性信息素量及比率在个体间也存在差异。每只雌蛾求偶时平均每小时释放94 ng Z9-14∶Ald和45 ng Z11-16∶Ald,平均比率为2.2。风洞实验结果显示,48%的雄蛾可被合成的性信息素混合物（0.4 μg Z9-14∶Ald, 2 μg Z11-16∶Ald）引诱完成逆风飞行并最终触及刺激源,而2种对照组（以求偶期雌蛾或性信息素腺体提取物为刺激源）的引诱率则分别为62%和44%。     

Gas chromatography column as an ambient‐temperature volatile trap

Open‐tube volatile traps have largely been shunned in favor of solid adsorbent containing traps for the collection of volatile pheromones and attractants. Solid adsorbents require large solvent rinses and glass capillaries can be difficult to maneuver for the collection of volatiles from small or hard‐to‐reach odor sources. A gas chromatograph (GC) column (DB‐1), an open‐tube glass capillary, and a SuperQ^®‐containing capillary were compared for their collection efficiencies from rubber septa and live calling insects. All three traps captured similar ratios of test compounds from septa at airflows >10 ml per min. Eluting analytes from a packed adsorbent, SuperQ, required at least 30× more solvent than was required to collect all the pheromone from the open‐tube glass capillaries, and the GC column enjoyed an additional three‐fold reduced solvent volume compared to the glass capillary. Thus, analytes could be eluted from the GC‐column trap and directly analyzed on GC without solvent evaporation. We placed glass wool ‘plugs’ in both GC columns and glass capillaries and found no volatiles in these plugs, indicating that breakthrough did not occur during 1‐h collections at 25 ml per min. We demonstrate here that at ambient laboratory temperatures, a DB‐1 GC column effectively collects Oriental fruit moth sex pheromone volatiles from a rubber septum and live pheromone‐releasing moths. Release ratios of pheromone from rubber septa are consistent with earlier reports from static air systems, whereas the release ratio of the (Z)‐8‐dodecenyl alcohol (Z8‐12:OH) from female Grapholita molesta Busck (Lepidoptera: Tortricidae) differed from published results and is likely due to different collection methods or moth‐strain origin.     

Hydrolysis of sex pheromone by antennal esterases of the cabbage looper, Trichoplusia ni

Esterases were isolated from chemosensory sensilla on the antennaeof Trichoplusia ni (Hübner). The disc gel electrophoreticpatterns of these esterases from males and females were similar;however, more bands were observed in the antennae than in 8other tissues examined. Most of the esterases detected in the100,000 g supernatant of the antennal preparation could be dissociatedfrom the 100,000 g membrane pellet. Esterases from both maleand female antennae hydrolyzed the sex attractant, (Z)-7-dodecen-l-olacetate, more rapidly than did the legs, fat body or Malpighiantubules. The enzymes primarily responsible for pheromone catabolismwere less sensitive to paraoxon, eserine and p-(hydroxymercuri)benzoatethan those hydrolyzing 1-napthyl acetate. This suggested thata major portion of the observed pheromone-hydrolytic activitywas due to acetylesterases. Measurement of pheromone hyrolysisin sections of disc gels that contained separated antennal orabdominal body wall esterases revealed 2 peaks of activity withboth tissues; however, the rate of pheromone hydrolysis by theabdominal esterases was slower than that of the antennae. Thesignificance of these findings is discussed in relation to thepossibility of antennal esterases having a functional role inthe olfactory process of males of T. ni.     

Interrelationships between the Nature of the Light Source, Ambient Air Temperature, and the Vegetative Growth of Different Species within Growth Cabinets

The interacting effects within growth cabinets of the natureof the light source and the temperature of the ambient air on14 parameters of vegetative growth have been examined for Gotsypiumhirsutum, Helianthus annuus, Phaseolus vulgaris, and Zea mays.The comparisons were between fluorescent lighting and mixtureswith tungsten lamps (17 and 26 per cent of the total wattage),adjusted to give 32 400 lx and covering six equally spaced andconstant air temperatures from 10 to 35 °C. The relationships are highly complex. Species differ in theirresponses to the three light sources. Moreover, for some parameters,but only for some species, there are highly significant interactionsbetween the nature of the light source and the temperature level.Part of this complexity is due to the varying growth potentialof species at low and high temperatures but important contributingfactors are the differences between the temperatures of theambient air and the plant parts. Such disparities may be positiveor negative and are greater for leaf as against root temperature.The divergencies are larger during the day and largest for leaftemperature. With the higher proportion of tungsten lamps theleaf day temperature ranges from 7.1 °C above an ambienttemperature of 10 °C to 2.5 °C above 35 °C ambient.For fluorescent lighting the leaves are 2.8 °C above ambientat 10 °C and 2.5 °C below at 35 °C. When variations in the response are examined against the meaninternal day temperature of the plant, for some parameters,but not others, differences between light sources are no longerevident. Here again the reactions of species differ. It is concluded firstly that if only ambient air temperaturesare recorded errors will arise in the interpretation of theinduced biological responses, and secondly that departures fromthe ambient air temperatures will be dependent on the designof the cabinet and the lighting system.     

小地老虎雄蛾对雌蛾性信息的EAG反应

利用触角电位记录（EAG）技术,测定了小地老虎Agrotis ypsilon (Rottemberg) 雄蛾对性信息素标准化合物Z7-12：Ac（A）、Z9-14：Ac（B）、Z11-16：Ac（C）、Z5-10：Ac（D）和Z8-12：Ac（E）的EAG反应。结果表明：这些标准化合物均能引起EAG反应,其中组分A（Z7-12：Ac）引起的反应最强,为5.65 mV,组分B（Z9-14：Ac）和C（Z11-16：Ac）居中; 组分D（Z5-10：Ac）的EAG反应值最小,为2.50 mV。二元混合物、三元混合物、四元混合物和全组分的EAG反应较高,其EAG反应值均显著高于单组分的反应值。三元混合物ABC的反应值最高,与5头雌蛾腺体的正己烷浸提液的EAG值相当。在使用剂量为0.01 ng～100 μg反应内,小地老虎雄蛾触角对性信息素各组分及其混合物的剂量反应曲线大致呈“S”形。从不同日龄雄蛾对标准化合物的反应中,发现在羽化后第3天达到最高值,之后则随日龄增加EAG反应降低。     

Localized adaptation processes in olfactory sensilla of Saturniid moths

Receptor potentials and nerve impulses were recorded extracellularlyfrom the two olfactory cells innervating most sensilla trichodeaon the antenna of male Antheraea polyphemus and Antheraea pemyimoths that respond to two key compounds, the sex pheromone components(E)-6, (Z)-11-hexadecadienyl acetate and (E)-6, (Z)-11-hexadecadienal.Stimulation with the key compound of one receptor cell auto-adaptsthis cell and also cross-adapts the other cell in the same sensillumbut cross-adaptation is weaker than auto-adaptation. Local stimulationexperiments demonstrate that sections of the olfactory receptorcell can be selectively adapted as monitored by the receptorpotential response. The mechanism of impulse generation canadapt separately from the mechanism generating the receptorpotential as indicated by an altered relationship between impulseresponse and receptor potential. These results demonstrate multipleand distributed adaptation processes in an olfactory bipolarneuron as studied in a time domain of seconds. Cross-adaptationmay indicate extracellular alterations caused by excitationof one cell but could also be caused by direct inhibitory actionof the stimulus compound.     

Ultrastructural Changes of the Egg Apparatus Associated with Fertilization and Proembryo Development of Soybean, Glycine max (Fabaceae)

As part of a study involving pod retention in soybean, Glycinemax (L.) Merr., we investigated changes occurring in the eggapparatus of non-abscised flowers from the time immediatelypreceding fertilization through early embryogeny. Prior to theentry of the pollen tube into the embryo sac, one of the synergidsbegins to degenerate as evidenced by increased electron densityand a loss of volume. This cell serves as the site of entryfor the pollen tube. The cytoplasm of the second, or persistentsynergid, remains unaltered until after fertilization. Bothsynergids contain, in addition to a filiform apparatus, a singleunidentified inclusion of flocculent material located in thechalazal portion of each cell. The zygote can be distinguishedfrom the egg by its consistently narrow wall; and it dividesto form a proembryo, a mass of cells not yet differentiatedinto embryo proper and suspensor. The basal cells of the proembryoare more vacuolate than the apical ones, characteristic of thebasal vacuolation of both egg and zygote. Cells of the proembryoare connected to one another via plasmodesmata, and with theexception of the basal-most cell, are isolated symplasticallyfrom the surrounding endosperm. Wall ingrowths frequently occurin certain cells of the proembryo, notably those cells in contactwith the degenerate synergid and embryo sac wall. At a laterstage of ontogeny, by which time the globular embryo properhas become distinct from the suspensor, the wall ingrowths areconcentrated in the suspensor. Glycine max, soybean, embryogeny, synergids     

枫杨枝把在田间对棉铃虫的引诱作用

观察了枫杨枝把和性诱剂在田间对棉铃虫Helicoverpa armigera (Hübner) 的引诱作用。结果表明,枫杨枝把在田间对棉铃虫成虫有较强的引诱作用,所诱雌蛾略多于雄蛾。枫杨枝把上所诱雄蛾量与上把的雌蛾量呈正相关。棉铃虫性诱剂对枫杨枝把的诱蛾作用没有显著的影响。讨论了枫杨枝把在田间引诱棉铃虫的作用。     

Attraction between social crab spiders: silk pheromones in Diaea socialis

The snare web is used as a medium for communication betweenindividuals within colonies of social spiders and has thereforebeen suggested as necessary for the evolution of sociality inthe Araneae. The social spider Diaea socialis (Thomisidae) isan exception because it does not build a snare web. Experimentsdemonstrate that silk attracts all spiders and that a chemicaldeposited onto the silk attracts adult female spiders, suggestingthat the group living of this species is mediated by a pheromone.The pheromone attracts spiders differentially: females are notattracted to juvenile silk, and it repels gravid females. Thepheromone appears to be stable but volatile, is ether-soluble,and retains its viability after dissolution. Molecular-ionicmasses for 7-8 different compounds were found in the range 220–281atomic units; the pheromone may be one or a combination of severalof these.     

A receptor and binding protein interplay in the detection of a distinct pheromone component in the silkmoth Antheraea polyphemus

Male moths respond to conspecific female-released pheromones with remarkable sensitivity and specificity, due to highly specialized chemosensory neurons in their antennae. In Antheraea silkmoths, three types of sensory neurons have been described, each responsive to one of three pheromone components. Since also three different pheromone binding proteins (PBPs) have been identified, the antenna of Antheraea seems to provide a unique model system for detailed analyzes of the interplay between the various elements underlying pheromone reception. Efforts to identify pheromone receptors of Antheraea polyphemus have led to the identification of a candidate pheromone receptor (ApolOR1). This receptor was found predominantly expressed in male antennae, specifically in neurons located beneath pheromone-sensitive sensilla trichodea. The ApolOR1-expressing cells were found to be surrounded by supporting cells co-expressing all three ApolPBPs. The response spectrum of ApolOR1 was assessed by means of calcium imaging using HEK293-cells stably expressing the receptor. It was found that at nanomolar concentrations ApolOR1-cells responded to all three pheromones when the compounds were solubilized by DMSO and also when DMSO was substituted by one of the three PBPs. However, at picomolar concentrations, cells responded only in the presence of the subtype ApolPBP2 and the pheromone (E,Z)-6,11-hexadecadienal. These results are indicative of a specific interplay of a distinct pheromone component with an appropriate binding protein and its related receptor subtype, which may be considered as basis for the remarkable sensitivity and specificity of the pheromone detection system.