Sex pheromones and olfactory proteins in Antheraea moths: A. pernyi and A. polyphemus (Lepidoptera: Saturniidae)

Olfaction is essential for regulating the physiological and behavioral actions of insects with specific recognition of various odors. Antheraea moths (Lepidoptera: Saturniidae) possess relatively large bodies and antennae so that they are good subjects for exploring molecular aspects of insect olfaction. Current knowledge of the molecular aspects of Antheraea olfaction is focused on the Chinese tussah silkmoth A. pernyi Guérin-Méneville and another species A. polyphemus (Cramer) in their pheromones, odorant-binding proteins (OBPs), odorant receptors (ORs), odorant receptor coreceptors (ORCOs), sensory neuron membrane proteins (SNMPs), and odorant-degrading enzymes (ODEs). The first insect OBP, SNMP, and ODE were identified from A. polyphemus. This review summarizes the principal findings associated with the olfactory physiology and its molecular components in the two Antheraea species. Three types of olfactory neurons may have specific ORs for three respective sex-pheromone components, with the functional sensitivity and specificity mediated by three respective OBPs. SNMPs and ODEs are likely to play important roles in sex-pheromone detection, inactivation, and degradation. Identification and functional analysis of the olfactory molecules remain to be further performed in A. pernyi, A. polyphemus, and other Antheraea species.     

Functional morphology of the ommatidia in the compound eye of the moth,Antheraea polyphemus (Insecta,Saturniidae)

Summary The fine structure of the superposition eye of the Saturniid moth Antheraea polyphemus Cramer was investigated by electron microscopy. Each of the approximately 10000 ommatidia consists of the same structural components, but regarding the arrangement of the ommatidia and the rhabdom structure therein, two regions of the eye have to be distinguished. In a small dorsal rim area, the ommatidia are characterized by rectangularly shaped rhabdoms containing parallel microvilli arranged in groups that are oriented perpendicular to each other. In all other ommatidia, the proximal parts of the rhabdoms show radially arranged microvilli, whereas the distal parts may reveal different patterns, frequently with microvilli in two directions or sometimes even in one direction. Moreover, the microvilli of all distal cells are arranged in parallel to meridians of the eyes. By virtue of these structural features the eyes should enable this moth not only discrimination of the plane of polarized light but also skylight-orientation via the polarization pattern, depending on moon position. The receptor cells exhibit only small alterations during daylight within the natural diurnal cycle. However, under illumination with different monochromatic lights of physiological intensity, receptor cells can be unbalanced: Changes in ultrastructure of the rhabdomeres and the cytoplasm of such cells are evident. The effects are different in the daytime and at night. These findings are discussed in relation to the breakdown and regeneration of microvilli and the influence of the diurnal cycle. They are compared with results on photoreceptor membrane turnover in eyes of other arthropod species.     

Sex pheromone of the moth,Antheraea polyphemus

The sex pheromone system of Antheraea polyphemus was characterized from female abdominal tips by classical and electroantennogram techniques as rans-6,cis-11-hexadecadienyl acetate and trans-6,cis-11-hexadecadienal. A 90 : 10 mixture of acetate and aldehyde was highly attractive to wild males in the field. The synthetic pheromone and A. polyphemus females were not attractive to released Antheraea pernyi males.     

Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi.

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K. , Krieger, J. & Breer, H. (1989) FEBS Lett. 256, 2215-2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta 1088, 277-284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6, 11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.     

Reconstruction and morphometry of silkmoth olfactory hairs: A comparative study of sensilla trichodea on the antennae of male Antheraea polyphemus and Antheraea pernyi (Insecta,Lepidoptera)

Summary Olfactory trichoid hairs on the antennae of male Antheraea silkmoths were reconstructed with respect to the following parameters: number, shape, course, and dimensions of outer dendritic segments as well as the numbers of their microtubules; inner and outer dimensions of the cuticular hair shafts; and number and distribution of pores and pore tubules in the hair walls. The smallest distances between dendritic membranes and inner hair surfaces were determined with respect to the possibility of pore tubule contacts. It was shown that most hairs contain one thick and one, or frequently two, thin dendrites. The number of microtubules in the dendrites is correlated with dendrite diameter, which decreases towards the hair tip. The dendrites form numerous swellings and constrictions: this beading occurs especially along the thin dendrites. The dendrites do not run straight, but rather follow a sinuous course in the hairs. The density of wall pores is lowest in the basal region of the hairs. Only in relatively few places do the dendritic membranes get near enough the hair walls to come into the probable range of the pore tubules. In the sensilla trichodea of A. polyphemus, the hairs as well as the dendrites have markedly smaller diameters than in A. pernyi.     

Foliage of oaks grown under elevated CO2 reduces performance of Antheraea polyphemus (Lepidoptera: Saturniidae)

To understand how the increase in atmospheric CO2 from human activity may affect leaf damage by forest insects, we examined host plant preference and larval performance of a generalist herbivore, Antheraea polyphemus Cram., that consumed foliage developed under ambient or elevated CO2. Larvae were fed leaves from Quercus alba L. and Quercus velutina Lam. grown under ambient or plus 200 microl/liter CO2 using free air carbon dioxide enrichment (FACE). Lower digestibility of foliage, greater protein precipitation capacity in frass, and lower nitrogen concentration of larvae indicate that growth under elevated CO2 reduced the food quality of oak leaves for caterpillars. Consuming leaves of either oak species grown under elevated CO2 slowed the rate of development of A. polyphemus larvae. When given a choice, A. polyphemus larvae preferred Q. velutina leaves grown under ambient CO2; feeding on foliage of this species grown under elevated CO2 led to reduced consumption, slower growth, and greater mortality. Larvae compensated for the lower digestibility of Q. alba leaves grown under elevated CO2 by increasing the efficiency of conversion of ingested food into larval mass. Despite equivalent consumption rates, larvae grew larger when they consumed Q. alba leaves grown under elevated compared with ambient CO2. Reduced consumption, slower growth rates, and increased mortality of insect larvae may explain lower total leaf damage observed previously in plots in this forest exposed to elevated CO2. By subtly altering aspects of leaf chemistry, the ever-increasing concentration of CO2 in the atmosphere will change the trophic dynamics in forest ecosystems.     

Characterization of lipophorin from Antheraea mylitta drury (Lepidoptera: Saturniidae)

The high molecular weight (680 KDa) glycolipoprotein from the haemolymph of male larvae of Antheraea mylitta Drury (Lepidoptera: Saturniidae) was identified as lipophorin by gradient KBr ultracentrifugation and SDS-PAGE. This lipophorin is composed of two subunits: apolipoprotein 1 (234 KDa) and apoprotein II (80 KDa). The density of the native molecule is 1.1941 g/ml. By weight, it contains 53.7% protein, 3.7% carbohydrate, and 42.6% lipid. Neutral lipids and phospholipids are 66.2 and 33.8% of the total lipids, respectively. Mannose and N-acetylglucosamine are the only sugars detected by gas liquid chromatography. The amino acid composition of both the native molecule and its two subunits was determined, and yielded similar amino acid compositions. © 1996 Wiley-Liss, Inc.     

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.     

Atomic-force microscopy on the olfactory dendrites of the silkmoths Antheraea polyphemus and A. pernyi

Olfactory transduction is thought to occur in the outer dendritic membrane of insect olfactory receptor neurons. Electrophysiological studies have indicated that the outer dendritic membrane has non-specific cation channels and inositol-triphosphate-dependent Ca²⁺ channels. The presence of such channels is further supported by the observation that pheromone-stimulated dendrites take up cobalt. However, to date, there is no structural evidence for these channels. Therefore, in order to search for putative ion channels, we have imaged the membrane of the olfactory dendrites in the scanning electron microscope (SEM) and the atomic-force microscope (AFM), after extruding the dendrites out of the olfactory hairs and fixing them on plastic coverslips. With the aid of the SEM, we could see the beaded structure of the dendrite but no fine structural details, as the membrane was sputtered with gold. With the use of the contact mode of the AFM, we could see “pores” that were deeper than 3 nm and with a diameter of about 15 nm. The density of the “pores” was approximately 20/µm² or 10?000 pores per thick dendrite. We believe these to be putative ion channels based on indirect evidence.     

Offset response of the olfactory projection neurons in the moth antennal lobe

We investigated a population activity of central olfactory neurons after the termination of odor input. Olfactory response of projection neurons in the moth primary olfactory center was characterized using in vivo intracellular recording and staining techniques. The population activity changed rapidly to the different states after the stimulus offset. The response after stimulus offset represents information regarding odor identity. We analyzed the spatial distribution of offset-activated glomeruli in a virtual neuronal population that was reconstructed using accumulated individual recordings obtained from different specimens. The offset-activated glomeruli tended to be widely distributed, whereas the onset-activated glomeruli were relatively clustered. These results suggest the importance of lateral interaction in shaping the offset olfactory response.     

Analysis of the electrical responses of antennal thermo- and hygroreceptors of Antheraea (Saturniidae,Lepidoptera) to thermal,mechanical, and electrical stimuli

Summary Antennal styloconic thermo-hygro sensilla of Antheraea were studied with DC-coupled transepithelial recordings. — The transepithelial voltage changed by about 2 mV · °C^–1. The spike frequency of the cold cell reached 300 Hz at the onset of negative temperature steps, but only 30 Hz at static temperatures (as with metal electrodes). The cold cell spikes showed a brief afterhyperpolarization that increased with temperature. The spikes of the cold- and warm-stimulated cells facilitated each other at low temperature. Mechanical stimuli (push against the sensillum, hydrostatic pressure of < ± 50 kPa, ultrasonic vibrations 120 kHz) modified the responses of the cold- and the warm-stimulated cells. Latency of cold cell responses to ultrasonic stimulation was occasionally less than 3 ms. — The impulse frequencies of the warm and the cold cells depend on the temperature and the magnitude of temperature change. When the firing rate is high enough by either or both of these parameters, it can be forced still higher by application of clamp current (outside positive). The higher the firing rate prior to clamping, the greater the effect of the current. — By analogy with sensilla for other modalities, this relationship between frequency and clamp current strongly suggests that stimulus-dependent changes in the conductance of dendritic membranes control the excitation of the warm and cold cells.Abbreviations DC direct voltage - TER transepithelial slope resistance between recording electrode and reference electrode in the hemolymph - NTC thermistor with negative temperature coefficient - TEV transepithelial voltage between electrodes - THS thermo-hygro sensillum     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. VI. Experimental disturbance of antennal branch formation

In the male silkmoth Antheraea polyphemus, the formation of the side branches of the quadripectinate antennal flagellum was disturbed by an experimental manipulation. Normally the side branches develop in the pupa via deep incisions which proceed from the periphery towards the centerline of the leaf-shaped antennal anlage. Local removal of the uppermost, pigmented cuticular layers of the pupal antennal pocket ('cuticular window') led to a local standstill of branch formation in the manipulated region of the pocket, most probably caused by increased evaporation of water through the remaining layers of meso- and endocuticle. These parts of the antenna retained an unbranched, plate-like shape. This early morphogenetic stage was conserved by the secretion of antennal cuticle. Besides cuticle formation, development of sensilla is not impeded by the manipulation. In the plate-shaped regions, the initial pattern formed by the sensilla in the antennal epidermis is preserved, because they maturate at their birthplaces. In the individual segments, the pattern of sensilla shows a mirror-like symmetry with respect to the segmental midline. From the edge to the midline, we found large s. trichodea, followed by small s. trichodea, s. basiconica, and s. coeloconica on the dorsal side whereas on the ventral side, there are only large s. trichodea and s. campaniformia. We conclude that the development of the featherlike antennal shape on the one hand and the development of sensilla and cuticle on the other hand are independent processes.     

Microsatellite markers for the Indian golden silkmoth, Antheraea assama (Saturniidae: Lepidoptera)

Antheraea assama, an economically important and scientifically unexplored Indian wild silkmoth, is unique among saturniid moths. For this species, a total of 87 microsatellite markers was derived from 35 000 expressed sequence tags and a microsatellite‐enriched sub‐genomic library. Forty individuals collected from Tura and West Garo Hills region of Northeast India were screened for each of these loci. Ten loci from expressed sequence tags and one from genomic library were found to be polymorphic. These microsatellite markers will be useful resources for population genetic studies of A. assama and other closely related species of saturniids. This is the first report on development of microsatellite markers for any saturniid species.     

Age-related changes in the dendrites of olfactory receptor neurons in the male silkmoth Antheraea peryni (Insecta, Lepidoptera: Saturniidae).

A study was conducted to evaluate the changes that occur with aging at the dendrite level of the olfactory receptor neuron in the male silkmoth Antheraea pernyi. Using calcein AM/ethidium homodimer-1 solutions, we found increased numbers of dendrites with damaged membrane with aging. Correspondingly there was an overall decrease in the electrophysiological activity as evidenced by the decreased number of cells discharging nerve impulse in response to female pheromone. It was also seen that the number of membrane swellings increased with age. In young animals aged 1-4 days, swellings showed intact membrane, and in older animals aged 5-15 days, they showed damaged membranes. With TUNEL assay that detects fragmented DNA in dying cells, an increased number of dendrites showing cytoplasmic labelling with age was found. The presence of fragmented DNA within aged dendrites was also confirmed in polyacrylamide gels after DNA extraction and PCR amplification. When tested for reversal of phosphatidylserine from the inner leaflet to the outer leaflet of plasma membrane no reactivity was seen. It appears that changes that occur during aging of dendrites may reflect some of the recognized symptoms of both apoptosis and necrosis.     

Olfactory dendrites of the silkmoth antheraea polyphemus cramer (Lepidoptera : Saturniidae) : immunofluorescent staining of cytoskeletal proteins and transmission electron microscopy

In this paper we show the presence of major cytoskeletal proteins : tubulin, globular actin, and intermediate filament-like protein, 2 motor proteins : kinesin and unconventional myosin, and fodrin, in the olfactory dendrites of the silkmoth Antheraea polyphemus (Cramer) (Lepidoptera : Saturniidae) by means of immunofluorescence microscopy. We also show cross-bridges between the membrane and the microtubules and between the microtubules themselves, as well as particle-like structures close to the microtubules, by means of transmission electron microscopy. Within the long and thin dendrites, the cytoskeletal proteins provide structural support to the membrane, and, together with the motor proteins, are most probably involved in transport, motility and olfactory transduction.     

A chromosome‐scale genome assembly of Antheraea pernyi (Saturniidae,Lepidoptera)

Antheraea pernyi is a semi‐domesticated lepidopteran insect species valuable to the silk industry, human health, and ecological tourism. Owing to its economic influence and developmental properties, it serves as an ideal model for investigating divergence of the Bombycoidea super family. However, studies on the karyotype evolution and functional genomics of A. pernyi are limited by scarce genomic resource. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first high‐quality A. pernyi genome from a single male individual. The genome is 720.67 Mb long with 49 chromosomes and a 13.77‐Mb scaffold N50. Approximately 441.75 Mb, accounting for 60.74% of the genome, was identified as repeats. The genome comprises 21,431 protein‐coding genes, 85.22% of which were functionally annotated. Comparative genomics analysis suggested that A. pernyi diverged from its common ancestor with A. yamamai ~30.3 million years ago, and that chromosome fission contributed to the increased chromosome number. The genome assembled in this work will not only facilitate future research on A. pernyi and related species but also help to progress comparative genomics analyses in Lepidoptera.