Differentiation of insect sensory neurons in the absence of their normal synaptic targets.

Sensory neurons in the antenna of the moth, Manduca sexta, arise and differentiate during the 18 days of metamorphosis from pupa to adult, sending axons to the brain. To assess the trophic dependence of developing antennal neurons on their targets, we studied antennae from surgically debrained animals. If the brain is removed 1 to 45 hr after pupal ecdysis (before and during the period when antennal neurons arise by cell divisions), adult development can be triggered by injection of β-ecdysone; if the brain is removed 50 to 60 hr after pupal ecdysis (before antennal axons contact the brain), metamorphosis proceeds spontaneously. Neurons proliferate normally and differentiate extensively in the antennae of debrained animals. They acquire a characteristic size and shape, elaborate axons and dendrites, migrate to appropriate positions in the sensilla, accumulate components of a neurotransmitter system (acetylcholine, choline acetyltransferase, and acetylcholinesterase), and generate electrical responses to olfactory and mechanical stimuli. Antennal sensory neurons thus differ from a variety of vertebrate neurons, which fail to mature when deprived of their synaptic targets.     

Acetylcholine and its metabolic enzymes in developing antennae of the moth, Manduca sexta.

Antennae of the moth, Manduca sexta, are thickly populated with sensory neurons, which send axons through antennal nerves to the brain. These neurons arise by cell divisions and differentiate synchronously during the 18 days of metamorphosis from pupa to adult. Biochemical studies support the hypothesis that antennal neurons use acetylcholine (ACh) as a neurotransmitter: (1) Antennae incubated with [¹⁴C]choline synthesize and store [¹⁴C]ACh; several other transmitter candidates do not accumulate detectably when appropriate radioactive precursors are supplied; (2) antennae and antennal nerves contain endogenous ACh; and (3) extracts of mature antennae contain choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) with properties similar to those reported for the enzymes from other arthropods. Levels of ACh, ChAc, and AChE begin to increase in antennae soon after the sensory neurons are “born.” Levels rise exponentially for over a week as the neurons differentiate and then reach a plateau, at about the time the neurons reach morphological maturity, that is maintained into adulthood. In contrast, levels of carnitine acetyltransferase, cholinesterase, and soluble protein, presumably not confined to nervous tissue, change little during metamorphosis. Levels of ACh, ChAc, and AChE rise in an intracranial segment of antennal nerve at about the same time as in the antenna, indicating that axons can transport neurotransmitter machinery at an early stage in their development.     

Behavioural and Electrophysiological Responses of Mosquito Vectors <Emphasis Type="Italic">Aedes aegypti,Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> to an Ethyl Ester: Ethyl 2-aminobenzoate

Mosquito control using different methods remains an integral component of intervention programmes which aim to protect humans from various mosquito-borne diseases. The host seeking behaviour of mosquitoes is essentially guided by odorant receptor neurons housed in the antenna, maxillary palps and proboscis. The odorant receptor neurons are responsible for detecting chemical cues from hosts and also useful for developing sustainable mosquito-control strategies that exploit host-seeking behaviours. The present investigation evaluates host seeking behavioural responses of a novel, non-toxic and environment friendly repellent, ethyl 2-aminobenzoate against three known vector species of mosquitoes viz. Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus maintained in laboratory. The flight orientation of the test mosquitoes was studied using Y-tube olfactometer, whereas the antennae of adult female mosquitoes were used to investigate the effect of ethyl 2-aminobenzoate on the peripheral olfactory system using electroantennogram (EAG). The findings demonstrate that ethyl 2-aminobenzoate exhibited significant response in Y-tube olfactometer against all the three known vector species of mosquitoes. However, only Anopheles stephensi significantly elicited responses in EAG experiments, while the responses obtained for Aedes aegypti and Culex quinquefasciatus were not statistically significant. The results conclude that currently evaluated chemical ethyl 2-aminobenzoate has potential against some well established mosquito vector species and could be exploited to develop new and comparatively more effective anti-mosquito formulations.     

Perception of potential sex pheromones and host-associated volatiles in the cotton plant bug,Adelphocoris fasciaticollis (Hemiptera: Miridae): morphology and electrophysiology

Highly developed chemoreception allows insects to detect foods, find mates, and escape natural enemies. We described the structures and distributions of antennal chemosensilla in Adelphocoris fasciaticollis Reuter by scanning electron microscopy and transmission electron microscopy. Seven major types of antennal sensilla were identified in adults of both sexes. Types 1 and 2 are sensilla chaetica and have thick cuticular walls with conspicuous grooves at their surfaces. Types 3 and 4 are multiporous sensilla trichodea with 1–3 dendrites located at the sensillum lymph, indicating a putative olfactory function. Types 5 and 6 are typical sensilla basiconica but share different characteristics in both external morphology and internal ultrastructure, and may be involved in the perception of host-associated odorants. The last sensilla were Böhm bristles. In addition to the morphological characterization, electrophysiological responses of antennal chemosensilla to 51 semiochemicals were investigated based on electroantennogram (EAG) recordings. Results revealed that different chemical stimuli elicited significantly different dose-dependent EAG responses, in which potential sex pheromone components and green leaf volatiles showed relatively higher EAG responses, but neither monoterpenes nor sesquiterpenes can elicit favorable EAG values. The results provided direct morphological and electrophysiological evidence that the adult antennae of A. fasciaticollis could function in searching for mates and host plants.     

Electroantennogram responses of the male moth, Argyrotaenia velutinana to mixtures of sex pheromone components of the female

Electroantennogram (EAG) responses were recorded from male redbanded leafroller moth, Argyrotaenia velutinana, antennae using mixtures of the three female-produced sex pheromone components: cis-11-tetradecenyl acetate, trans-11-tetradecenyl acetate, and dodecyl acetate (12:Ac). Binary mixtures containing 3%, 8%, and 15% trans in cis elicited significantly higher amplitude responses than other isomeric mixtures as well as pure cis and trans alone. The higher responses to such mixtures were less than additive at high dosages and additive at lower dosages. Receptor adaptation studies using the two isomers support a previous single unit study demonstrating the presence of at least two functionally different receptor sites on male A. velutinana antennae; adaptation to an airstream containing one isomer did not eliminate response to the opposite isomer presented concurrently. An airstream containing the third component, 12:Ac, caused a significant slowing of the recovery rate during the entire recovery period of EAG responses to cis but not trans, suggesting that a possible temporal modulation of neuronal response by 12:Ac may be a means of coding for this component by antennal sensory neurons.     

Olfactory attraction to aggregation pheromone is mediated by disti-flagellum of antennal segments in Riptortus pedestris

Riptortus pedestris (Hemiptera: Alydidae) is a serious pest of soybean and sweet persimmon and uses male produced aggregation pheromone, (E)-2-hexenyl (Z)-3-hexenoate, (E)-2-hexenyl (E)-2-hexenoate, and tetradecyl isobutyrate to facilitate food location and recognition by conspecifics. Using electroantennogram (EAG) and greenhouse bioassay, we determined which antennal segment is involved in the detection of their aggregation pheromone. In the first EAG test using individual antennal segments, significant EAG responses to 1:1:1 mixture of the aggregation pheromone were observed only from the disti-flagellum segments of both male and female antennae at both pheromone doses tested (1 µg and 100 µg). In the following EAG tests using gradually removed antennal segment(s), EAG response was still maintained when the distal half of a disti-flagellum was surgically removed, while EAG response was lost when whole segment of disti-flagellum or other whole segments were gradually removed from intact antenna of both sexes. In greenhouse experiment, removing one or both segment(s) of disti-flagellum from male or female antennae resulted in significant reduction in their attraction to the aggregation pheromone. Together, these findings support that the disti-flagellum of R. pedestris houses olfactory neurons associated with attraction to their aggregation pheromone.     

Function and central projections of gustatory receptor neurons on the antenna of the noctuid moth Spodoptera littoralis

Chemosensory information is crucial for most insects to feed and reproduce. Olfactory signals are mainly used at a distance, whereas gustatory stimuli play an important role when insects directly contact chemical substrates. In noctuid moths, although the antennae are the main olfactory organ, they also bear taste sensilla. These taste sensilla detect sugars and hence are involved in appetitive learning but could also play an important role in food evaluation by detecting salts and bitter substances. To investigate this, we measured the responses of individual taste sensilla on the antennae of Spodoptera littoralis to sugars and salts using tip recordings. We also traced the projections of their neuronal axons into the brain. In each sensillum, we found one or two neurons responding to sugars: one NaCl-responsive and one water-sensitive neuron. Responses of these neurons were dose-dependent and similar across different locations on the antenna. Responses were dependent on the sex for sucrose and on both sex and location for glucose and fructose. We did not observe a spatial map for the projections from specific regions of the antennae to the deutocerebrum or the tritocerebrum/suboesophageal ganglion complex. In accordance with physiological recordings, back-fills from individual sensilla revealed up to four axons, in most cases targeting different projection zones.     

Electroantennogram responses of aphid nymphs to plant volatiles

Abstract. Electroantennogram (EAG) responses of immature aphids were investigated for 30 plant volatile compounds in third‐ and fourth‐stadium nymphs of the black bean aphid, Aphis fabae. The nymphs were destined to develop into adult alate (winged) virginoparae. The EAG response profiles were similar to those previously reported in the adults. Among the compounds tested, hexanonitrile elicited the largest EAG responses in both nymphal stadia, corresponding to previously reported results with adults. Six‐carbon aliphatic compounds showed relatively higher EAG activities in the nymphs but, in contrast, (E)‐2‐hexenal, benzaldehyde, α‐pinene and β‐pinene, and citronellal elicited relatively smaller EAG responses in nymphs than adults. Although overall EAG response profiles were similar between the third and the fourth stadia for the majority of the volatiles, four aldehyde compounds, hexanal (E)‐2‐heptenal, 2‐hydroxybenzaldehyde and citronellal, showed relatively higher EAG activities in the third than in the fourth stadium. The present study indicates that aphid nymphs possess a functional olfactory receptor system before the antennae are fully developed morphologically and physiologically.     

Ultrastructure and potential role of integumentary glandular cells in adult male and female Callosobruchus subinnotatus (Pic) and C. maculatus (Fabricius) (Coleoptera : Bruchidae)

Transmission and scanning electron microscopy methods were used to study the ultrastructure of integumentary glandular cells that may be involved in the production of sex pheromones and other semiochemicals in Callosobruchus subinnotatus and C. maculatus (Coleoptera : Bruchidae). Additionally, we measured electroantennogram (EAG) responses of male and female antennae to solvent extracts and glassadsorbed volatiles from both sexes of C. maculatus in order to localize the source of the putative sex pheromone. Both species have numerous cuticular pores on the abdomen and thorax. These pores open via an epicuticular duct into a single type 3 secretory cell. Solvent extracts of the pygidium from the female elicit the highest EAG responses from male antennae, suggesting this area of the abdomen to be the source of the putative sex pheromone in C. maculatus.     

Antennal responses of Cydia pomonella (L.) exposed to surfaces treated with methoxyfenozide

Electroantennogram (EAG) measurements were recorded from the antennae of male and female codling moth, Cydia pomonella L., to determine whether adult moths exposed to surfaces treated with the ecdysteroid agonist methoxyfenozide experience a decline in their antennal reception and thus olfactory sensitivity. Such a phenomenon would offer a possible mechanism for the previously reported decreased responsiveness from moths treated with methoxyfenozide to pheromone‐ and plant volatile‐based monitoring lures. Mean EAG data revealed that the antennae from methoxyfenozide‐treated male moths appear to be just as sensitive to various doses of synthetic codlemone as the antennae from the control and surfactant‐treated moths, but they appeared to be less sensitive to the pheromone component 12OH (collected from female effluvia) than the control male antennae. Mean male EAG responses to the pheromone components E8,E10‐12Al and codlemone collected from methoxyfenozide‐treated females were significantly less than the responses towards those two pheromone components collected from the control and surfactant‐treated females. Female moth exposure to methoxyfenozide did not negatively impact the sensitivity of female antennae to the plant volatile pear ester, but it did towards the apple volatile butyl hexanoate. Data from this study show that adult C. pomonella exposure to methoxyfenozide‐treated surfaces does appear to negatively impact, in a minor way, the (i) olfactory sensitivity (or detection) of male antennae towards some components of the female sex pheromone, (ii) the female antennal sensitivity towards a key apple volatile and (iii) the attractiveness of female pheromone effluvia.     

Electroantennogram (EAG) responses of Microplitis croceipes and Cotesia marginiventris and their lepidopteran hosts to a wide array of odor stimuli: Correlation between EAG response and degree of host specificity?

In order to test whether the electroantennogram (EAG) response spectrum of an insect correlates to its degree of host specificity, we recorded EAG responses of two parasitoid species with different degrees of host specificity, Microplitis croceipes (specialist) and Cotesia marginiventris (generalist), to a wide array of odor stimuli including compounds representing green leaf volatiles (GLVs), herbivore-induced plant volatiles (HIPV), ecologically irrelevant (not used by the parasitoid species and their hosts for host location) plant volatiles, and host-specific odor stimuli (host sex pheromones, and extracts of host caterpillar body and frass). We also tested the EAG responses of female moths of the caterpillar hosts of the parasitoids, Heliothis virescens and Spodoptera exigua, to some of the odor stimuli. We hypothesized that the specialist parasitoid will have a narrower EAG response spectrum than the generalist, and that the two lepidopteran species, which are similar in their host plant use, will show similar EAG response spectra to plant volatiles. As predicted, the specialist parasitoid showed greater EAG responses than the generalist to host-specific odor and one HIPV (cis-3-hexenyl butyrate), whereas the generalist showed relatively greater EAG responses to the GLVs and unrelated plant volatiles. We detected no differences in the EAG responses of H. virescens and S. exigua to any of the tested odor.     

Antijuvenile influence of the precocene on the development of adult antennae in the large fruit-tree tortrix Archips podana Scop. (Lepidoptera: Tortricidae)

The influence of precocene II, an antijuvenile agent, on the development of adult antennae in the large fruit-tree tortrix A. podana Scop. was demonstrated. Treatment of the fifth instar larvae and prepupae with different doses of precocene proved to cause different sensitivity of the specimens to the juvenile hormone deficit. Treatment with 450 and 600 ??g precocene per specimen during the first days after ecdysis to the fifth instar caused the death of larvae. Treatment with 300, 450, and 600 ??g per specimen on the third day of the fifth instar larvae and prepupae caused a delay in the development of adult antennae. The results are discussed with respect to the role of the juvenile hormone in the development of imaginal structures during metamorphosis.     

Structure and development of antennae in a moth,Manduca sexta

The antenna of the moth, Manduca sexta, comprises two small basal segments and a long (2 cm) flagellum, which is divided into nearly 80 annuli. The annuli bear cuticular scales and small sensory organs, sensilla. A trachea, a blood vessel, and two nerve trunks run through the lumen of the antenna and into the head. Sensilla are arranged in an orderly pattern that is repeated on each flagellar annulus. Each flagellum bears about 10⁵ sensilla, which contain about 2.5 × 10⁵ primary sensory neurons. Clumps of undifferentiated cells (imaginal disks), present in the larva, form pupal antennae during the larval-pupal molt. During the subsequent metamorphic development of the adult, cell divisions, changes in cell shape, and cellular differentiation transform pupal into adult antennae. Sensilla and scales arise and differentiate in the antenna during metamorphosis; regions in which sensilla and scales will arise can be recognized before overt differentiation occurs. All of the flagellar annuli develop synchronously. The dense innervation and neuronal simplicity of antennal flagella, as well as their synchronous development at a late and accessible stage in the animal's life cycle, suit them for studies of neuronal differentiation.     

Electrophysiological responses of Batocera horsfieldi (Hope) adults to plant volatiles

The olfactory stimuli from the maturation feeding‐plant, Rosa multiflora Thunb., and larval‐host plant, Populus deltoides, of the longhorned beetle, Batocera horsfieldi (Hope), were investigated by using TCT‐GC/MS (Thermal desorption and cold trap, and GC/MS) and electroantennogram recordings (EAG). A total of 20 plant compounds were identified from plant headspace volatile, including aliphatic, aromatic and terpenoid compounds. Five compounds were common to both plants. Eight compounds eliciting strong EAG response were E‐3‐hexenyl acetate, 3‐carene, 1‐penten‐3‐ol, 3‐pentanol, Z‐2‐penten‐1‐ol, hexanal and E‐2‐hexenal. Female and male B. horsfieldi exhibited broad overlap in their EAG responses to individual plant odour, and there was no clear pattern of difference between responses of female and male antennae to different compounds. Mating status had little effect on the EAG responses of females.     

Functional specialisation and polyphenism in aphid olfactory sensilla

Electroantennogram (EAG) responses to the aphid sex pheromone components, (1R,4aS,7S,7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone, and a plant volatile, (E)-2-hexenal, were investigated at three different positions (5/6th, 4/5th and 3/4th inter-segmental regions) along the antennae of four different morphs in two host-alternating aphid species, Aphis fabae Scopoli and Rhopalosiphum padi (L.). Position-dependent and morph-specific EAG responses were elicited in both species. The nepetalactol and nepetalactone isomers elicited large EAG responses at all three recording positions in males of both species, such that primary rhinaria as well as secondary rhinaria appeared to respond. Asexual female morphs showed relatively smaller EAG responses to these compounds. The secondary rhinaria, which have been reported as sex pheromone receptors in males, were not very different in their number and distribution between gynoparae and alate virginoparae, but the gynoparae showed significantly larger EAG responses to nepetalactol and nepetalactone. The alate virginoparae showed EAG responses that were similar to those of apterous virginoparae, which lack secondary rhinaria. Taking the EAG response profiles together with the distribution of the secondary rhinaria, it is suggested that the function of secondary rhinaria differs between the morphs. Secondary rhinaria appear to detect sex pheromone components in males and gynoparae but not in the alate virginoparae. If they are functional in the latter morph, they are likely to play a role in detecting specific, but as yet unknown, volatile compounds. Some 30 plant volatiles were tested but none evoked an EAG response that could be allocated to the secondary rhinaria. In contrast to the very different EAG response profiles to the pheromone compounds between morphs, EAG responses to (E)-2-hexenal were similar in all forms and both species. These findings suggest that this plant volatile was detected only by the two primary rhinaria, which are common to all morphs. The present study showed that EAG responses were not a simple summation of receptor potentials between recording and reference electrodes in aphids. The localised distribution pattern of olfactory receptor neurones around the recording electrode was also likely to contribute to the EAG responses.     

The circadian rhythm of flight activity of Spodoptera exigua males in response to sex pheromone

In many moths, male attraction to the blend of synthetic sex pheromone releasing continuously in the field shows an apparent circadian rhythm similar to that of locomotion activity. In this study, the daily rhythms of electroantennography (EAG) and behavioral responses to sex pheromone, and the daily rhythms of locomotion activity were measured in male beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). The peaks of males trapped by light and sex pheromone were all during the latter part of the night in the field. However, there was no significant variation among time intervals in the EAG responses of male antennae to sex pheromone stimuli. The principal period of locomotion activity under L15:D9 (LD) continued to occur during the scotophase and the subjective scotophase in the day of constant darkness (DD1) and the second of two consecutive days of constant darkness (DD2). The majority of males contacted the sex pheromone source in a wind tunnel during the latter part of the scotophase under LD and the subjective scotophase for DD1 and DD2. There were significant associations between the rhythm of the behavioral response to sex pheromone and locomotion activity. These results suggested that the male's behavioral response to sex pheromone in the beet armyworm could be observed only until locomotion activity of the male occurred at the end of the dark period, despite sex pheromone being released continuously from synthetic pheromone‐baited traps in the field.     

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.     

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.     

Electroantennogram and behavioural responses of the gypsy moth to enantiomers of disparlure and its trans analogues

The attractive power of both enantiomers of disparlure and its trans analog was tested electrophysiologically (electroantennogram = EAG) and behaviourally (whole organism response). EAG responses correlated very well with the behaviourally determined effectiveness sequence: cis(+)-disparlure was the most effective substance; the authentic racemic disparlure came second; cis(?)-disparlure inhibited the activity of cis(+)-disparlure; and trans enantiomers were not significantly different from the control. These results allow us to conclude that cis(+)-disparlure is the natural sex pheromone of the gypsy moth, and that the male antennae have a chiral receptor system in the antenna for the reception of the sex pheromone.     

Electroantennogram responses to the sex pheromone and other odours in the American cockroach

Electroantennogram responses of adult American cockroaches were recorded for the sex pheromone and 30 kinds of monoterpenoids. Strong responses to the sex pheromone were obtained from the antennae of male adults, but not from females. The compounds possessing a hydroxyl or carbonyl group produced significant EAG responses in the antennae. Bornyl acetate, however, was an exception and gave strong responses in both male and female antennae. There was basically no difference between the antennal responsiveness to monoterpenoids tested in both sexes.