How is Pheromone Specificity Encoded in Proteins?

Pheromone specificity in the Lepidoptera is encoded in proteincomponents of the antennal sensillum lymph and dendritic membrane.In this paper, we highlight recent work on the molecular determinantsof pheromone binding affinity of pheromone binding proteins(PBPS) of three genera. First, we describe new cDNA sequencesfor Lymantria dispar (Lymantriidae) and Agrotis segetum (Noctuidae).These data enrich the conclusions derived from our functionalstudies. Secondly, we indicate how preparation of multimilligramquantities of the recombinant PBP ‘Apol-3’ (originallyfrom Antheraea polyphemus) has provided a platform (i) to determinethe ligand binding sites using photoaffinity labeling, (ii)to conduct structural analysis by CD and NMR, and (iii) to measurebinding affinities using a new binding assay. Thirdly, we describethe use of expression-cassette PCR technology to prepare tworelated PBPS from Antheraea perneyi to test binding affinitiesof naturally-occurring homologous PBPs. Our results supporta model in which ligand specificity for chain length, doublebond position, and terminal functionality is partially encodedin the PBPS. We propose that the final decoding is accomplishedwhen the PBP-pheromone complex activates a G-protein coupledseven-transmembrane domain receptor that contains recognitionsites for both the presented pheromone and the presenting PBP.Chem. Senses 20: 461–469, 1995.     

Are Odorant-binding Proteins Involved in Odorant Discrimination?

Pheromone-sensitive sensilla trichodea of nine moth speciesbelonging to six families and three superfamilies of Lepidopterawere immunolabelled with an antiserum against the pheromone-bindingprotein of Antheraea polyphemus. Strong immunolabelling of thesensillum lymph was observed in all long sensilla trichodeaof A. polyphemus, A. pernyi (Saturniidae), Bombyx mori (Bombycidae)and Manduca sexta (Sphingidae). Very weak labelling was foundwith all sensilla trichodea of Dendrolimus kikuchii (Lasiocampidae)and Lymantria dispar (Lymantriidae). In three noctuid species,some long sensilla trichodea were labelled strongly, some onlyweakly and some were not labelled at all. The fraction of longsensilla trichodea that were strongly labelled was large inHelicoverpa armigera, but small in Spodoptera littoralis andAutographa gamma. The observed cross-reactivity was not correlatedwith taxonomic relatedness of the species but rather with chemicalrelatedness of the pheromones used by these species, as a highlabelling density was consistently observed in sensilla tunedto pheromones with an alcyl chain of 16 carbon atoms. The highlydivergent specificity of pheromone-receptor cells in Noctuidaeappears to be mirrored by a similar diversity of the pheromone-bindingproteins in the sensilla trichodea. These data support the notionthat pheromone-binding proteins participate in odorant discrimination.Chem. Senses 21: 719–727, 1996.     

INHIBITION OF OLFACTION IN THE MOTH HELIOTHIS VIRESCENS BY THE SULFHYDRYL REAGENT FLUORESCEIN MERCURIC ACETATE

A combined electrophysiological, behavioral, and biochemicalstudy was initiated to determine the effects of the sulfhydryl-specificreagent fluorescein mercuric acetate (FMA) on olfaction in thetobacco budworm moth Heliothis virescens. The electroantennogram(EAG) response to the standard odorant n-pentyl acetate showedboth a time and concentration dependent inhibition by FMA. Treatmentof insect antennae with 2.52 x 10^–5 M FMA for 2 min reducedthe EAG by 50%, while treatment for 17 min reduced the EAG by80%. Incubation of antennae for 7 min with 2.52 x 10^–6M FMA resulted in 30% inhibition, while incubation with 2.52x 10^–6 M FMA for 7 min resulted in 65% inhibition. Antennalgrooming behavior was inhibited by FMA in a similar time andconcentration dependent manner as the EAG. Regeneration of previouslyinhibited behavioral and EAG responses has been observed withina 24-hr period. The interaction of protein, obtained by sonicatingintact antennae in phosphate buffer, with FMA was monitoredfluorometrically. Successive additions of antennal sonicateto FMA resulted in stepwise decreases in fluorescence. Partialrecovery of fluorescence was obtained by addition of cysteineto the FMA-antennal sonicate solution. The polarization of theFMA-antennal sonicate fluorescence decreased upon addition ofcysteine. These data indicate that FMA is reacting with a relativelylarge antennal protein (s) by mercaptide linkage and blockingthe olfactory transduction process.     

Sexual dimorphism in neuronal projections from the antennae of silk moths (Bombyx mori,Antheraea polyphemus) and the gypsy moth (Lymantria dispar)

Summary The antennal lobe of both sexes of the silk moth Bombyx mori contains 55–60 ventrally located antennal glomeruli; in addition, that of the male contains a dorsal macroglomerular complex (MGC). A group of identifiable glomeruli consisting of two lateral large glomeruli (LLG) and four medial small glomeruli (MSG) is present in both sexes, but the LLG are greatly enlarged in the female. A MGC is also present in the male gypsy moth Lymantria dispar and male giant silk moth Antheraea polyphemus. The MGC in all of these species is organized into 3–4 distinct levels of glomeruli. Antennal sensory fibers were stained by cobalt backfills in B. mori, A. polyphemus, and L. dispar. Most fibers stained from cut long hairs (sensilla trichodea) projected to MGC in males and LLG in both sexes of B. mori. The distribution of fibers in the MGC of B. mori was topographically biased in that a majority of fibers from anterior branches projected medially in MGC while most fibers from posterior branches projected laterally or anteriorly. Terminal arborizations of single fibers were each restricted to a single glomerular level of the MGC. Fibers projecting to the posterior antennal center were frequently stained in cut-hair and control preparations, apparently by uptake of cobalt through intact sensilla on flagellar branches.     

Adaptation of male moth antennal neurons in a pheromone plume is associated with cessation of pheromone-mediated flight

Recordings of the firing rates of single antennal neurons whenAgrotis segetum antennac were placed 70 cm downwind of a pheromonesource revealed that cells sensitive to the most volatile componentadapted rapidly in a plume from a high–concentration sourceknown from previous studies to cause in-flight arrestment ofprogress towards the source. No adaptation was found in responseto lower-concentration plumes known to promote high levels ofsustained flight to the source with little premature arrestment.Adaptation was not observed in antennal neurons of a secondspecies, Heliothis virescens, when they were placed in plumesof this species' sex pheromone blend, regardless of the concentration.In flight-tunnel tests these same pheromone sources evoked highlevels of source location with little arrestment. These resultsindicate that adaptation or attenuation of antennal neuronalburst frequencies in response to rapidly arriving pheromonefilaments in a plume may be important peripheral determinantsof whether or not prolonged upwind flight and successful pheromonesource location occurs.     

Heterogeneity of Odorant-binding Proteins in the Antennae of Bombyx mori

Different odorant-binding proteins (OBPs) were isolated fromtotal antennal homogenates of male and female Bombyx mori. Proteinswere separated according to their isoelectric point by usingpreparative fast-flow isoelectrofocusing. Odorant-binding proteinswere identified in immunoblots by antisera raised against thepheromone-binding protein (anti-PBP) and the general odorant-bindingprotein (anti-GOBP2) of Antheraea polyphemus. Four proteinscross-reacting with anti-PBP were detected in males and twoin females, while three proteins cross-reacting with anti-GOBP2were found in males and five in females. Both anti-PBP and anti-GOBP2cross-reacting proteins had an apparent molecular weight of15–16 kDa. In parallel, the same two antisera were usedin immunocytochemical studies in order to determine the distributionof these proteins within the various subtypes of olfactory sensilla.The presence of multiple odorant-binding proteins within onemoth species as well as their complex distribution pattern supportthe suggestion that soluble OBPs might have a function in odorantdiscrimination. Chem. Senses 22: 503–515, 1997.     

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.     

Pheromone-evoked potentials and oscillations in the antennal lobes of the sphinx moth Manduca sexta

Using intra- and extracellular recording methods, we studied the activity of pheromone-responsive projection neurons in the antennal lobe of the moth Manduca sexta. Intracellularly recorded responses of neurons to antennal stimulation with the pheromone blend characteristically included both inhibitory and excitatory stages of various strengths. To observe the activity of larger groups of neurons, we recorded responses extracellularly in the macroglomerular complex of the antennal lobe. The macroglomerular complex is part of a specialized olfactory subsystem and the site of first-order central processing of sex-pheromonal information. Odors such as the pheromone blend and host-plant (tobacco) volatiles gave rise to evoked potentials that were reproducible upon repeated antennal stimulation. Evoked potentials showed overriding high-frequency oscillations when the antenna was stimulated with the pheromone blend or with either one of the two key pheromone components. The frequency of the oscillations was in the range of 30–50 Hz. Amplitude and frequency of the oscillations varied during the response to pheromonal stimulation. Recording intracellular and extracellular activity simultaneously revealed phase-locking of action potentials to potential oscillations. The results suggest that the activity of neurons of the macroglomerular complex was temporally synchronized, potentially to strengthen the pheromone signal and to improve olfactory perception. Accepted: 19 December 1997     

Swimming in formation in krill (Euphausiacea), a hypothesis: dynamics of the flow field, properties of antennular sensor systems and a sensory-motor link

The act of swimming in formation by species such as Euphausiasuperba, Antarctic krill, is assumed to be regulated by a sensitivityto the characteristic and spatially elaborate flow field producedby this species of shrimp. We used a related species, Meganyctiphanes,North Atlantic krill, to visualize the flow field produced bytethered shrimps in an aquarium. In this situation, the propulsionjet flow some centimeters behind the shrimp is surrounded bya vortex ring of recoiling water motion from which, if the vortexis also produced by unrestrained swimming shrimp, a followingshrimp hypothetically can draw forces of lift and propulsionto decrease energy expense in long-distance migration. Two antennularsensitivities to water vibration in frequency ranges 5–40and 40–150 Hz were calibrated, and the activity of connectedinterneurons was traced into the abdominal pleopod-carryingsegments. Water oscillation of 3–10 Hz frequency, appliedto the antennules, was shown to entrain a closely synchronouspleopod beat in the stimulated specimens.     

Discrimination of mixtures of female sex pheromone components by male Trichoplusia ni (Hubner)

Pheromone mixture discrimination by male cabbage loopers, Trichoplusiani (Hübner), was assessed in a wind tunnel by responsedifferences to paired stimuli. They discriminate differencesbetween mixtures that contain three and all six of the pheromonecomponents that are emitted by virgin females. The ability todiscriminate between these two mixtures is perplexing becauseonly three antennal pheromone specialist neurons respond atnatural stimulus intensities. The cabbage looper also discriminatedbetween mixtures whose component ratios differed slightly fromthat of the female. There was some interaction between the stimulusintensity, the distance between the stimulus sources, and possiblythe extent of the perceptual difference. The mixtures of pheromonecomponents failed to enhance the amount of upwind flight responseover that of Z7–12:Ac alone. Other evidence argues thatthe instantaneous release of pheromone may exceed time-averagedmeasures.     

Mechanoreception in marine copepods: electrophysiological studies on the first antennae

Neural activity was recorded extracellularly at the base ofthe first antenna in 15 marine copepods. Controlled mechanicalstimuli were delivered with a vibrator driven by a waveformgenerator. Many species exhibited responses characterized bya large number of small spikes, while others were characterizedby the presence of a small number of large units. Two bay species,Labidocera madurae and Acartia fossae, exhibited large unitsthat could be easily distinguished from the background activityof smaller units. In these species, the antennal receptors firedshort latency (>5 ms) trains of one to several impulses inresponse to a brief mechanical stimulus and sustained trainsto a prolonged sinusoidal stimulus. They were extremely sensitiveto small displacements and sensitivity increased with stimulusfrequency. The receptors responded to stimuli between 40 and1000 Hz and receptors required displacement velocities of 20µm s^–1 or more to fire. Displacements as small as10 nm were capable of triggering spikes. With an increase inthe amplitude of the displacement, a decrease in the latencyand an increase in the number of units recruited and/or firingfrequency was recorded. Phase-locking to oscillatory stimuliwas observed over a frequency range of 80–500 Hz. Neuralactivity increased in response to bending of individual setae.Setae appear innervated and structurally constrained to movementsin specific directions. These experiments suggest that (i) somecopepod setal receptors may be more nearly velocity detectorsthan purely displacement sensors, (ii) they may be capable ofsensing closely spaced stimuli, (iii) the patterns of responsemay code for intensity and duration of the stimulus, and (iv)receptors may be capable of supplying directional information.     

Some Low-temperature Effects on Sieve Tube Translocation in Salix viminalis

The data presented on translocation of ¹⁴C-labelled compoundsin Salix viminalis show that above a temperature close to –4°C translocation occurs, whilst below –4 °C inmost cases it does not. Stoppages were irreversible when the temperature of the cooledstem was raised again to normal (approximately 20 °C) andappeared to involve some disruption of the phloem. The temperaturesat which the bark was found to freeze were found to be closelysimilar, with respect to level and variability, to the stoppingtemperature. Respiration measurements on isolated strips of bark at –2°C showed that oxygen uptake fell to approximately 5 percent of its value at 25 °C. The decreased level of radioactivity in the cooled regions ofthe stems is considered to be evidence that the exchange oflabelled compounds between the sieve tubes and the surroundingcells was slowed up at low temperatures.     

The Osmotic Pressure of Concentrated Solutions of Polyethylene Glycol 6000, and its Variation with Temperature

The vapour pressures of aqueous solutions of polyethylene glycol6000 have been measured (by equilibration with sucrose solutions)up to the saturation point at 25 °C (1.45 g g^–1 water).The reduced-osmotic-pressure (/c), when plotted versus concentration(c), rapidly and linearly increased up to a concentration ofabout 0.8 g g^–1 (crossing the similar plot for sucrose).Above this concentration, the reduced-osmotic-pressure rosemore slowly, but still more rapidly than sucrose. The maximumosmotic pressure achieved at saturation was nearly 18 MPa. Usingthe virial equation: /c= RT/M + RTA₂c, the calculated secondvirial coefficient (A₂) for the linear part is 4.5 x 10^–3mol g^–1, a value slightly greater than most literaturevalues at 25 °C. Data are cited showing that A₂ varies linearlyfrom 5–6 x 10^x3 at 0 °C, to zero at 80–90 °C     

The energetics of Calanus euxinus: locomotion, filtration of food and specific dynamic action

The effect of locomotor activity on respiration rate was studiedin the food-deprived copepod Calanus euxinus tethered to a forcesensor. The power generated by mouth appendages during cruisinglocomotion, with a frequency of 40 Hz, accounted for 0.026 and0.0031 W for metabolic and mechanical processes, respectively.To overcome total hydrodynamic drag during foraging with a meanswimming speed of 3.2 cm s^–1, the copepods need 0.4 x10^–3 W, equating to 1.3% of total metabolism. The lossesof mechanical energy for body propulsion amounted to 1.3 x 10^–3W, whilst the cost of feeding current generation run up to 1.8x 10^–3 W, or 58% of the total. Changing of locomotor activityand respiration rate during feeding was examined separatelyin tethered and free-swimming copepods. At algal concentrationof 300 µg C L^–1, the magnitude of specific dynamicaction (SDA) averaged 1.2 ± 0.44 nL O₂ µg C^–1h^–1 in copepodites V and females, with similar movingactivity before and during feeding. The contribution of SDAinto total metabolism varied from 23 to 85% in C. euxinus withlow activity level and constituted only 10% in active animals.In starved copepods, with low locomotor activity, feeding eventsstimulated the increase in frequency and total duration of locomotionwhich resulted in elevated energy expenditure enhancing the‘apparent SDA’.     

Size-specific swimming behavior of Daphnia pulex

Body length affects several aspects of the behavior of quietlyswimming Daphnia pulex Swimming and sinking rates were measuredat 0.033 s intervals during the ‘hops’ characteristicof Daphnia swimming behavior Larger animals swim faster, covermore distance, and produce more powerful swimming strokes. LargerDaphnia also sink faster, but the sinking rate scales as lengthto the 0 58 power, far lower than the power of 2 00 predictedby Stokes Law considerations. The number of hops s^–1 wasindependent of body size, although a theoretical analysis predictshopping rate (antennal beat frequency) should increase as bodylength squared. Turning behavior, measured as the ratio of displacementto total distance, during 5 s, is also independent of body sizeIndependence of several parameters of body motion and body sizeimplies that factors other than simple mechanics affect Daphniaswimming behavior     

Glomerular interactions in olfactory processing channels of the antennal lobes

An open question in olfactory coding is the extent of interglomerular connectivity: do olfactory glomeruli and their neurons regulate the odorant responses of neurons innervating other glomeruli? In the olfactory system of the moth Manduca sexta, the response properties of different types of antennal olfactory receptor cells are known. Likewise, a subset of antennal lobe glomeruli has been functionally characterized and the olfactory tuning of their innervating neurons identified. This provides a unique opportunity to determine functional interactions between glomeruli of known input, specifically, (1) glomeruli processing plant odors and (2) glomeruli activated by antennal stimulation with pheromone components of conspecific females. Several studies describe reciprocal inhibitory effects between different types of pheromone-responsive projection neurons suggesting lateral inhibitory interactions between pheromone component-selective glomerular neural circuits. Furthermore, antennal lobe projection neurons that respond to host plant volatiles and innervate single, ordinary glomeruli are inhibited during antennal stimulation with the female’s sex pheromone. The studies demonstrate the existence of lateral inhibitory effects in response to behaviorally significant odorant stimuli and irrespective of glomerular location in the antennal lobe. Inhibitory interactions are present within and between olfactory subsystems (pheromonal and non-pheromonal subsystems), potentially to enhance contrast and strengthen odorant discrimination.     

Discrimination of female sex pheromone by male Trichoplusia ni (Hubner)

The olfactory discrimination process of male cabbage loopermoths, Trichoplusa ni (Hübner), was assessed by measuringtheir response to one of two emission sources within a windtunnel. The males discriminated between (1) Z7–12:Ac concentrations;(2) Z7–12:Ac alone and the volatile emission from excisedfemale sex pheromone glands; and (3) Z7–12:Ac and theemission from a mixture of six synthetic pheromone componentsthat mimics the volatile emissions of a female gland. Althoughmales could discriminate between a freshly excised female sexpheromone gland and 7.4x 10^–11 M Z7–12:Ac, theycould not discriminate between a gland and 78.5x10^–11M Z7–12:Ac. Males also could not discriminate betweenthe mixture of six volatile compounds and 28.7x10^–11 Mof Z7–12:Ac. The data show that male cabbage looper mothshave difficulty discriminating between Z7–12:Ac aloneand in mixtures with other female-emitted volatile compounds.     

Feeding of Sagitta enflata and vertical distribution of chaetognaths in relation to low oxygen concentrations

Zooplankton samples were collected in Mejillones Bay, northernChile (23°00'15'S, 70°26'43'W ). Sampling was conductedat 4 h intervals, for 24 h during three seasons, austral spring(October 2000), summer ( January 2001) and winter (August 2001)at three different strata (0–25, 25–50 and 50–100m). Five species of chaetognaths were collected. Sagitta enflatawas the most abundant species, representing up to 65% of allchaetognaths in total numbers, followed by Sagitta bierii, makingup 34% of the total abundance of chaetognaths. S. enflata wasdistributed mainly above the Oxygen Minimum Zone, while S. bieriiremained below this zone. Feeding rates were relatively constantwithin the upper layer (0–25 m depth), for each samplingdate, averaging 1.2 prey S. enflata day^–1, and decreasingwith depth. Gut content analyses demonstrated that predationwas principally focused on small copepods (<1500 µm),with greatest feeding activity occurring at night. The dailypredation impact on the total standing stock of small copepodsvaried seasonally between 6% in spring and 0.4% in winter. Thispercentage may represent a negligible impact on the entire copepodcommunity, but it is relevant at the species or genus level,since S. enflata removed more than 20% of the standing stockof Centropages brachiatus and Corycaeus sp. Thus, during someperiods of the year, chaetognaths may strongly influence theabundance and size distribution of copepods in coastal upwellingecosystems.     

The Effect of Sucrose Supply on the Energization of Amino Acid Uptake by Cotyledons of Euphorbia lathyris L

The cotyledons of Euphorbia lathyris L. take up sucrose andamino acids from the endosperm. The interaction between theuptake of sucrose and that of amino acids by cotyledons of intactseedlings was investigated. Sucrose (100 mol m^–3) reducedvaline uptake to 75% of the control rate; the active uptakecomponent of valine uptake was reduced from 45 to 25 % of thetotal uptake rate. In a reverse experiment, 100 mol m^–3valine inhibited sucrose uptake by 25%. At 500 mol m^–3sucrose, valine uptake was completely restored to the controlrate, whereas high valine concentrations failed to restore sucroseuptake. The stimulation of valine uptake by sucrose is linkedto the role of sucrose as a primary respiratory substrate. Whenthe cotyledons were bathed in sucrose concentrations rangingfrom 0 to 100 mol m^–3 (these concentrations are non-saturatingwith respect to sucrose uptake), a constant 1.8% of the sucrosetaken up was respired. The K_m of the concentration-dependentsucrose oxidation (44±6 mol m^–3) agreed reasonablywell with that for sucrose uptake (29±6 mol m^–3).When the external sucrose concentration was increased from 100to 600 mol m^–3, the sucrose uptake increased by 30% again,while sucrose oxidation was increased by 300%. This increasewas not due to an increased engagement of the alternative (cyanide-resistant)pathway for respiration. Alternative pathway, Euphorbia lathyris L., fermentation, seedling, sucrose uptake, valine uptake     

Purification and Characterization of a Novel Sex Pheromone that Induces the Release of Another Sex Pheromone during Sexual Reproduction of the Heterothallic Closterium peracerosum-strigosum-littorale Complex

A sex pheromone, PR-IP Inducer, which is released from mating-typeminus cells of Closterium, was purified by monitoring its biologicaleffect on the induction of the release of protoplast-release-inducingprotein (PR-IP) from mating-type plus cells. The purified PR-IPInducer had an apparent molecular mass of 22 kDa and of 18.7kDa as determined by SDS-PAGE and mass-spectrometric analysis,respectively. Staining with periodic acid-Schiff reagent indicatedthat PR-IP Inducer included a glycan chain. From the analysisof a dose-response curve, it seemed that PR-IP Inducer was ableto exert its activity over quite wide range of concentrations(1 x 10^–10–3 x 10^–7 M). It appears that PR-IPInducer is a novel glycoproteinaceous pheromone, as is PR-IP,and that it exerts its effect at the earliest stages of thesexual reproduction of the Closterium peracerosum-strigosum-littoralecomplex. ¹Recipient of a Fellowship for Japanese Junior Scientists fromthe Japan Society for the Promotion of Science.