Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth<Emphasis Type="Italic"> Antheraea polyphemus</Emphasis>

SNMP-1 (sensory neuron membrane protein 1) is an olfactory-specific membrane-bound protein which is homologous with the CD36 receptor family. Previous light level immunocytochemical studies suggested that SNMP-1 was localized in the dendrites and distal cell body of sex-pheromone-specific olfactory receptor neurons (ORN); these studies further suggested SNMP-1 was expressed in only one of two to three neurons in male-specific pheromone-sensitive trichoid sensilla. To better understand the expression and localization of SNMP-1, an immunocytochemical study was performed using electron microscopy to visualize the distribution of SNMP-1 among the neurons of several classes of olfactory sensilla of both male and female antennae of the silkmoth Antheraea polyphemus. SNMP-1 antigenicity was primarily restricted to the receptive dendritic membranes of ORNs of all sensilla types examined and was observed in cytosolic granules, but not plasma membranes, of the cell soma. Mean labeling densities ranged from 1 to 16 gold particles per micrometer of dendrite circumference; dendrites of trichoid and intermediate sensilla showed significantly higher labeling densities than those of basiconic sensilla. Larger dendrites of trichoid sensilla showed significantly higher mean labeling densities (13-16/micron) than smaller diameter dendrites (3-7/micron). Immunofluorescence studies using baculovirus expressed SNMP-1 and multiphoton photon laser scanning microscopy (MPLSM) indicated that rSNMP-1, which was post-translationally processed to the in vivo molecular weight, was inserted into the plasma membrane in a topography presenting extracellular epitopes. These studies suggest SNMP-1 is a common feature of the ORNs, is asymmetrically expressed among functionally distinct neurons, and possesses a topography which permits interaction with components of the extracellular sensillum lymph.     

Molecular elements of pheromone detection in the female moth,Heliothis virescens

Pheromones play pivotal roles in the reproductive behavior of moths, most prominently for the mate finding of male moths. Accordingly, the molecular basis for the detection of female‐released pheromones by male moths has been studied in great detail. In contrast, little is known about how females can detect pheromone components released by themselves or by conspecifics. In this study, we assessed the antenna of female Heliothis virescens for elements of pheromone detection. In accordance with previous findings that female antennae respond to the sex pheromone component (Z)‐9‐tetradecenal, we identified olfactory sensory neurons that express its cognate receptor, the receptor type HR6. All HR6 cells coexpressed the “sensory neuron membrane protein 1” (SNMP1) and were associated with supporting cells expressing the pheromone‐binding proteins PBP1 and PBP2. These features are reminiscent to male antennae and point to congruent mechanisms for pheromone detection in the two sexes. Further analysis of the SNMP1‐expressing cells revealed a higher number in females compared to males. Moreover, in females, the SNMP1 neurons were arranged in clusters, which project their dendrites into a common sensillum, whereas in males there were only solitary SNMP1‐neurons and only 1 per sensillum. Not all SNMP1 positive cells in female antennae expressed HR6 but instead the putative pheromone receptors HR11 and HR18, respectively. Neurons expressing 1 of the 3 receptor types were assigned to different sensilla. Together the data indicate that on the antenna of females, sensory neurons in a subset of sensilla trichodea are equipped with molecular elements, which render them responsive to pheromones.     

EXPRESSION OF SNMP1 AND SNMP2 GENES IN ANTENNAL SENSILLA OF Spodoptera exigua (HÜBNER)

Sensory neuron membrane proteins (SNMPs) are olfactory‐specific, two‐transmembrane proteins. Previous publications reported that SNMP1 is expressed on the dendrite membrane of pheromone‐sensitive neurons in Heliothis virescens and is an essential cofactor for pheromone detection in Drosophila. In this study, we cloned two SNMP genes (GenBank accession nos. JX469106 and JX469107) from the antenna of the beet armyworm moth Spodoptera exigua (Lepidoptera: Hübner). These SNMP genes are classified into two highly conserved subclades, indicating their importance in physiological activity of lepidopteran insects. SexiSNMP1 is antenna‐specific in male and female adults, while SexiSNMP2 is antenna‐abundant but also expressed in other chemosensory tissues, particularly proboscises and maxillary palps of adults both sexes. In situ hybridization revealed that both SNMPs are broadly expressed in long and short trichoid and basiconic sensilla. We infer that SNMP1 and SNMP2 act in the detection of the sex pheromone and general odorants.     

Immunolocalization of odorant-binding proteins in noctuid moths (Insecta, Lepidoptera)

Odorant-binding proteins were studied in the noctuid moths Agrotis segetum, Autographa gamma, Helicoverpa armigera, Heliothis virescens and Spodoptera littoralis using antisera raised against the pheromone-binding protein (PBP) and general odorant-binding protein 2 (GOBP2) of Antheraea polyphemus (Saturniidae). Proteins immunoreacting with these antisera were only found on the antennae and PBP and GOBP2 could be identified on western blots of males and females of all five species. PBPs were predominantly localized in sensilla trichodea and GOBP2 in sensilla basiconica, in good correlation with the stimulus specificity of the receptor cells in these sensilla. In H. armigera and H. virescens the majority of the s. trichodea immunoreacted with the antiserum against PBP of A. polyphemus; in A. segetum, A. gamma and S. littoralis, on the other hand, a high percentage of s. trichodea remained unlabelled. Probably, the PBP expressed in these sensilla is so different that it does not immunoreact with the antiserum used. Such a protein was found by native PAGE of antennal extracts of A. segetum and S. littoralis. These data correlate with the fact that the two heliothine species use pheromones with the same alkyl chain length as A. polyphemus, while the other three species use pheromones with shorter chains. In H. armigera, H. virescens, A. gamma and S. littoralis female antennae were also immunolabelled and a large number of PBP-expressing s. trichodea was consistently found. In S.littoralis this fits with the electrophysiologically recorded high pheromone sensitivity of female s. trichodea, whereas in females of H. armigera and H. virescens no or only weak responses to pheromone stimulation have been reported. Therefore, PBP expression in a sensillum does not necessarily imply pheromone sensitivity of its receptor cells.     

Antennal SNMPs (sensory neuron membrane proteins) of Lepidoptera define a unique family of invertebrate CD36-like proteins

SNMP1-Apol is an antennal-specific protein of the wild silk moth Antheraea polyphemus; the protein is abundantly expressed and localized to the receptor membranes of sex-pheromone specific olfactory sensory neurons (OSNs). SNMP1-Apol is thought to function in odor detection based on its olfactory-specific expression, localization within OSNs, developmental time of expression, and apparent homology to the CD36 family of membrane-bound receptor proteins. In the current study, SNMP1-Apol homologues were identified from the moths Bombyx mori, Heliothis virescens, and Manduca sexta. These species posses antennal mRNAs encoding proteins with amino acid sequence identities ranging from 75-80%; these proteins are collectively designated SNMP1. A second M. sexta SNMP homologue, previously identified and partially sequenced [Robertson et al.: Insect Mol Biol 8:501-518, 1999] was fully sequenced and characterized. The encoded protein shares only 26-27% sequence identity with the SNMP1 proteins, and is thus designated SNMP2-Msex. The SNMP sequences were used to identify 14 and four possible homologues in Drosophila melanogaster and Caenorhabditis elegans genome databases, respectively; thus, greatly expanding CD36 family membership among the invertebrate lineages. Despite their sequence difference, SNMP1-Msex and SNMP2-Msex expression is localized to OSNs and occurs simultaneously with the onset of olfactory function. These findings suggest that SNMPs play a central role in odor detection in insects, and that the CD36 gene family is widely represented among animal phyla. The SNMPs are the only identified neuronal members of the CD36 family, and as such expand the activities of this gene family into roles influencing brain function and behavioral action.     

Immunolocalization of general odorant-binding protein in antennal sensilla of moth caterpillars

Antennae of Bombyx mori and Helicoverpa armigera larvae were immunolabelled with antisera raised against the pheromone-binding protein or the general odorant-binding protein 2 of Antheraea polyphemus to assign the expression of these proteins to individual sensilla and to compare the localization pattern with that in sensilla of adult moths. Specific labelling of antennal sensilla was only obtained with the antiserum against general odorant-binding protein 2. Among the few sensilla present on the antenna the three large sensilla basiconica, which are suspected to be olfactory in function, were labelled. These sensilla are compound sensilla consisting of several sensillum units which form a common sensory hair. The hair is single-walled and pierced by many pores. Labelling of sensillum compartments was the same as in sensilla of adults. Prominent labelling of the sensillum lymph is accompanied by labelling of secretory organelles in the two outermost auxiliary cells and of endocytotic pathways in all sensillum cells. The results suggest that general odorant-binding protein is expressed in single-walled multiporous sensilla of presumed olfactory function on the antenna of moth larvae. The overall identity of the localization pattern for general odorant-binding protein between larval and adult sensilla implies a similar role of these proteins in olfactory stimulus transduction.     

Immunolocalization of a candidate pheromone receptor in the antenna of the male moth, Heliothis virescens

Pheromone recognition in insects is thought to involve distinct receptor proteins in the dendritic membrane of antennal sensory neurons. We have generated antibodies directed against a peptide derived from the sequence of the candidate pheromone receptor HR13 from Heliothis virescens. The antibodies specifically labelled the cell bodies of a distinct neuron population housed in male-specific pheromone-sensitive sensilla. Combining antibody staining with in situ hybridization the reactive cells were found to express the HR13 gene. In addition, dendrites projecting into sensilla hairs as well as the axonal processes of immunoreactive cells were labelled. Labelling of axons has allowed visualization of their fasciculation within antennal segments and permits tracking of axons as they merge into the antennal nerve. The HR13 protein was first detected 1 day before eclosion. Thus, the distribution of HR13 protein in the antennal neurons of the male moth strongly suggests a role of the HR13 receptor in recognition of pheromones.     

Identification and localization of two sensory neuron membrane proteins from Spodoptera litura (Lepidoptera: Noctuidae)

Sensory neuron membrane proteins (SNMPs), which are located on the dendritic membrane of olfactory sensory neurons (OSNs), are proposed to be associated with odor reception in insects. Recent studies have demonstrated that SNMP1 is essential for electrophysiological responses of OSNs to the sex pheromone, cis‐vaccenyl acetate (cVA) in Drosophila melanogaster. To investigate the function of Lepidoptera SNMPs, we cloned two SNMP genes, SlituSNMP1 and SltiuSNMP2, from Spodoptera litura (Lepidoptera: Noctuidae). Sequence alignment and phylogenetic analysis showed that both genes bear the general characteristics of SNMPs, including six conserved cysteine residues and two transmembrane domains. Further expression profile experiments showed that SlituSNMP1 is mainly expressed in the antenna, while SlituSNMP2 is broadly expressed in various tissues. By in situ hybridization experiments, it was found that SlituSNMP1 expressing cells are surrounded by the SlituSNMP2 expressing cells in the pheromone sensitive sensilla, suggesting different functions of the two SNMPs in insect olfaction.     

Cloning,expression and immunocytochemical localization of a general odorant-binding protein gene from Helicoverpa armigera (Hübner)

A cDNA clone coding for general odorant-binding protein2 was isolated from the antenna of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. Results of sequencing and structural analyses showed that the full-length of GOBP2Harm was 636 bp, possessing 162 amino acid residues and a signal peptide of 21 amino acids. Its predicted molecular weight and isoelectric point were 18.2 kDa and 5.21, respectively. This deduced amino acid sequence shared some common structural features with odorant-binding proteins from several moth species, including the six conserved cysteine motif, typical of insect's OBPs. Northern blot showed that GOBP2Harm is specifically expressed in the antenna of Helicoverpa armigera at similar levels in both sexes. In order to obtain sufficient GOBP2 for further determining its biochemical and physiological properties, a bacterical expression vector of GOBP2 was constructed and successfully expressed. The protein was obtained mainly as insoluble inclusion bodies, that, however, could be solubilized and refolded. The rGOBP2 was purified by affinity chromatography and gel filtration. The rGOBP2 was shown to cross-react with an anti-GOBP antiserum from Antheraea polyphemus. Finally, polyclonal antibodies against GOBP2Harm were used to mark the distribution of the protein in olfactory sensilla and were tested by immuno-electron microscopy. In the male, GOBP2Harm is mainly expressed in sensilla basiconica, while in the female, it is equally expressed in sensilla basiconica and in sensilla trichodea.     

苹毛丽金龟触角嗅感器超微结构

利用扫描电镜和透射电镜对苹毛丽金龟Proagopertha lucidula (Faldermann)雌雄成虫触角嗅感器超微结构特征进行研究。结果表明:嗅感器集中于触角鳃片上,分布在触角鳃片表皮内陷形成的凹腔里,有5种感器,以板状感器为主,以外还有锥状感器、腔状感器、腔锥状感器和毛状感器。板状感器根据盘体形状的不同可分为4种类型,锥状感器根据锥体形状的差异可分为2种类型,腔锥状感器根据形状分为2种类型。嗅感器表皮有微孔和孔道微管。嗅感器内神经元的数目并不一致,1～2个不等。雄虫鳃片嗅感器总数显著多于雌性,是雌虫嗅感器总数1.8倍。其中雄性锥状感器的数目是雌性2倍,雌虫腔锥状感器数量是雄虫的4倍,雄虫板状感器数量是雌虫的2倍,雌雄腔状感器的数目无显著性差异。     

Receptors and neurons for fly odors in Drosophila

Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila[1]. We systematically examined the trichoid sensilla, one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, by electrophysiological analysis. We find that none respond strongly to food odors but that all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone transferred from males to females during mating [2-4]. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin-female extract as well. Thus, males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. We then systematically tested all members of the Odor receptor (Or) gene family [5-7] that are expressed in trichoid sensilla [8] by using an in vivo expression system [9]. Four receptors respond to fly odors in this system: Two respond to extracts of both males and virgin females, and two respond to cVA. We propose a model describing how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic.     

Putative circadian pacemaker cells in the antenna of the hawkmoth Manduca sexta

Antennal sensory neurons in the fruit fly Drosophila melanogaster express circadian rhythms in the clock gene PERIOD (PER) and appear to be sufficient and necessary for circadian rhythms in olfactory responses. Given recent evidence for daily rhythms of pheromone responses in the antenna of the hawkmoth Manduca sexta, we examined whether a peripheral PER-based circadian clock might be present in this species. Several different cell types in the moth antenna were recognized by monoclonal antibodies against Manduca sexta PER. In addition to PER-like staining of pheromone-sensitive olfactory receptor neurons and supporting cells, immunoreactivity was detected in beaded branches contacting the pheromone-sensitive sensilla. The nuclei of apparently all sensory receptor neurons, of sensilla supporting cells, of epithelial cells, and of antennal nerve glial cells were PER-immunoreactive. Expression of per mRNA in antennae was confirmed by the polymerase chain reaction, which showed stronger expression at Zeitgeber-time 15 compared with Zeitgeber-time 3. This evidence for the expression of per gene products suggests that the antenna of the hawkmoth contains endogenous circadian clocks.     

桑天牛头部附器感器的扫描电镜观察

利用扫描电镜观测桑天牛Apriona germari(Hope)成虫的触角、下颚须和下唇须上感器的分布及超微结构。结果表明,雌、雄桑天牛触角上共存在6种感器,即毛形感器、锥形感器、刺形感器、芽形感器、鳞形感器和棒形感器,其中锥形感器分5种亚型,刺形感器分2种亚型,且部分感器在雌雄成虫触角上的分布模式及数量存在差异,如棒形感器及角锥形感器丛模式仅在雄虫触角上发现,而雌虫触角上的细锥形感器和耳锥形感器多于雄虫,芽形感器少于雄虫;下颚须和下唇须存在5种感器,即毛形感器、刺形感器、末梢锥形感器、钟形感器和隙缝感器,各种感器在雌、雄天牛下颚须和下唇须上的分布和数量无明显的区别。     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. I. The leaf-shaped antenna of the pupa from diapause to apolysis

The antenna of the male silkmoth Antheraea polyphemus is a featherlike structure consisting of a central stem and ca. 120 side branches, which altogether carry about 70,000 olfactory sensilla. We investigate the development during the pupal phase. At the end of diapause, the antennal rudiment consists of a leaf-shaped, one-layered epidermal sac. It is supplied with oxygen via a central main trachea, which gives off numerous thin side branches. These are segmentally arranged into bundles which run to the periphery of the antennal blade. When the epidermis retracts from the pupal cuticle (apolysis; stage 1), it consists of cells which are morphologically uniform. The epidermal cells form a network of long, irregular basal protrusions (epidermal feet), which crisscross the antennal lumen. During the first day post-apolysis (stage 2), the antennal epidermis differentiates into alternating thick 'sensillogenic' and thin 'non-sensillogenic' areas arranged in stripes which run in parallel to the tracheal bundles. Numerous dark, elongated cells, which might be the sensillar stem cells, are scattered in the sensillogenic epithelium. A number of very early sensilla has been found at the distal edges of the sensillogenic stripes in positions which later will be occupied by sensilla chaetica. The whole antennal blade is enveloped by the transparent ecdysial membrane, consisting of the innermost layers of the pupal cuticle which are detached during apolysis.     

Analysis of immunocytochemical staining patterns in the antennal system of Drosophila melanogaster

By immunizing mice with homogenized brains, heads, or a mixture of heads and antennae of D. melanogaster, we obtained six monoclonal antibodies (mabs) that bind to the olfactory system of Drosophila with various degrees of specificity. They can be divided into three groups with respect to their staining pattern: (1) The antibodies ca51/2, na21/2, and nb230 label both in the third (olfactory) antennal segment and in the visual ganglia. All of them bind to antennal structures that can be correlated with basiconic sensilla. The antibody ca51/2 labels sensory neurons of these sensilla. In the antenna of the lozenge ³ mutant, which lacks basiconic sensilla, no labeling is present. In Western blots ca51/2 recognizes in the antenna an antigen of 43.5 kDa, which is expressed in the antenna only in the presence of basiconic sensilla. The antibody na21/2 binds to basiconic and coeloconic sensilla, most likely to the apical part of sheath cells. In immunoblots it recognizes in the antenna two antigens of 42.2 kDa and 46.7 kDa. The latter appears to be correlated in the antenna with the presence of basiconic sensilla. (2) The staining pattern of antibody nc10 is associated with the sheath cells of basiconic and coeloconic sensilla. Moreover, nc10 binds to a subset of glomeruli in the antennal lobe. (3) The staining pattern of the antibodies VG2 and I24B5 is restricted to the antenna. I24B5 recognizes coeloconic sensilla and VG2 recognizes both coeloconic and basiconic sensilla. Staining patterns in both cases include sheath cells.     

Analysis of immunocytochemical staining patterns in the antennal system of Drosophila melanogaster

By immunizing mice with homogenized brains, heads, or a mixture of heads and antennae of D. melanogaster, we obtained six monoclonal antibodies (mabs) that bind to the olfactory system of Drosophila with various degrees of specificity. They can be divided into three groups with respect to their staining pattern: (1) The antibodies ca51/2, na21/2, and nb230 label both in the third (olfactory) antennal segment and in the visual ganglia. All of them bind to antennal structures that can be correlated with basiconic sensilla. The antibody ca51/2 labels sensory neurons of these sensilla. In the antenna of the lozenge ³ mutant, which lacks basiconic sensilla, no labeling is present. In Western blots ca51/2 recognizes in the antenna an antigen of 43.5 kDa, which is expressed in the antenna only in the presence of basiconic sensilla. The antibody na21/2 binds to basiconic and coeloconic sensilla, most likely to the apical part of sheath cells. In immunoblots it recognizes in the antenna two antigens of 42.2 kDa and 46.7 kDa. The latter appears to be correlated in the antenna with the presence of basiconic sensilla. (2) The staining pattern of antibody nc10 is associated with the sheath cells of basiconic and coeloconic sensilla. Moreover, nc10 binds to a subset of glomeruli in the antennal lobe. (3) The staining pattern of the antibodies VG2 and I24B5 is restricted to the antenna. I24B5 recognizes coeloconic sensilla and VG2 recognizes both coeloconic and basiconic sensilla. Staining patterns in both cases include sheath cells.     

Pheromone-binding proteins contribute to the activation of olfactory receptor neurons in the silkmoths antheraea polyphemus and Bombyx mori

The sensilla trichodea of the silkmoth Antheraea polyphemus are innervated by three types of receptor neurons each responding specifically to one of three pheromone components. The sensillum lymph of these sensilla surrounding the sensory dendrites contains three different types of pheromone-binding proteins (PBPs) in high concentrations. The sensilla trichodea of the silkmoth Bombyx mori are supplied by two receptor neurons each tuned specifically to one of the two pheromone components bombykol and bombykal, but only one type of PBP has been found so far in these sensilla. Recombinant PBPs of both silkmoth species in various combinations with pheromone components were applied to the receptor neurons via tip-opened sensilla during electrophysiological recordings. Over a fairly broad range of pheromone concentrations the responses of the receptor neurons depended on both, the pheromone component and the type of the PBP. Therefore, the PBPs appear to contribute to the excitation of the receptor neurons. Furthermore, bombykal in combination with the expressed PBP of B. mori failed to activate the corresponding receptor neuron of B. mori, but did so if combined with one of the PBPs of A. polyphemus. Therefore, a still unknown binding protein involved in bombykal transport might be present in B. mori.     

Morphogenesis of the antenna of the male silkmoth. Antheraea polyphemus, III. Development of olfactory sensilla and the properties of hair-forming cells

During adult development of the male silkmoth Antheraea polyphemus, the anlagen of olfactory sensilla arise within the first 2 days post-apolysis in the antennal epidermis (stage 1-3). Approximately on the second day, the primary dendrites as well as the axons grow out from the sensory neurons (stage 4). The trichogen cells start to grow apical processes approximately on the third day, and these hair-forming 'sprouts' reach their definite length around the ninth day (stages 5-6). Then the secretion of cuticle begins, the cuticulin layer having formed on day 10 (stage 7a). The primary dendrites are shed, the inner dendritic segments as well as the thecogen cells retract from the prospective hair bases, and the inner tormogen cells degenerate around days 10/11 (stage 7b). The hair shafts of the basiconic sensilla are completed around days 12/13 (stage 7c), and those of the trichoid sensilla around days 14/15 (stage 7d). The trichogen sprouts retract from the hairs after having finished cuticle formation, and the outer dendritic segments grow out into the hairs: in the basiconic sensilla directly through, and in the trichoid sensilla alongside, the sprouts. The trichogen sprouts contain numerous parallel-running microtubules. Besides their cytoskeletal function, these are most probably involved in the transport of membrane vesicles. During the phase of cuticle deposition, large numbers of vesicles are transported anterogradely from the cell bodies into the sprouts, where they fuse with the apical cell membrane and release their electron-dense contents (most probably cuticle precursors) to the outside. As the cuticle grows in thickness, the surface area of the sprouts is reduced by endocytosis of coated vesicles. When finally the sprouts retract from the completed hairs, the number of endocytotic vesicles is further increased and numerous membrane cisterns seem to be transported retrogradely along the microtubules to the cell bodies. Here the membrane material will most probably be used again in the formation of the sensillum lymph cavities. Thus, the trichogen cells are characterized by an intensive membrane recycling. The sensillum lymph cavities develop between days 16-20 (stage 8), mainly via apical invaginations of the trichogen cells. The imago emerges on day 21.     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. II. Differential mitoses of 'dark' precursor cells create the Anlagen of sensilla

The antenna of the male silkmoth Antheraea polyphemus develops from a one-layered, flattened epidermal sac during the pupal phase. Within the first day post-apolysis (developmental stages 1 and 2), this epithelium differentiates into 'sensillogenic' and 'nonsensillogenic' regions, while numerous slender 'dark cells' interpreted as the precursor cells of sensilla arise in the former. Approximately between the first and second day post-apolysis (developmental stage 3), the dark cells retract to the apical pole of the epidermis, assume a round shape, and undergo a series of differential mitoses with spindles usually oriented parallel to the epidermal surface. These mitoses finally yield the Anlagen of the olfactory sensilla trichodea, each consisting of mostly 6-7 dark cells arranged side by side. In most of the Anlagen, 3-4 of these cells are situated more basally, each giving off a slender apical process which together are arranged in a fascicle. These are the prospective 2-3 sensory neurons plus the thecogen cell, which most probably is a sister cell of the former. Three additional cells are arranged more apically and partly enclose the fascicle of presumed sensory and thecogen cell processes. These are interpreted as the trichogen plus 2 tormogen cells, one of the latter degenerating later during development. In the basal region of the sensillogenic epidermis, massive signs of cell degeneration have been found. At stage 3, the basal epidermal feet in the non-sensillogenic regions have assumed a more uniform orientation as compared with the preceding stages.     

棉铃虫感觉神经元膜蛋白基因克隆和表达

从棉铃虫Helicoverpa armigera触角中克隆了一条全长1 690 bp的cDNA序列,该序列阅读框全长1 572 bp,编码523个氨基酸残基,序列中有2个跨膜区,具有昆虫感觉神经元膜蛋白（sensory neuron membrane protein, SNMP）的典型特征。SNMP与已报道的其他昆虫的感觉神经元蛋白的氨基酸序列有很高的同源性。半定量RT-PCR研究结果显示,SNMP在棉铃虫中不仅在触角中表达,也在去掉触角的头、足中表达。但是在触角中的表达量最高,在雌雄触角中的表达量差异不显著。在喙、下颚须和下唇须中也有表达。SNMP在卵、蛹和成虫体内也都有表达,但在卵中表达量相对较低。将SNMP编码区克隆到表达载体pET21b中,成功地进行了原核表达,表达出带有6个组氨酸标签的重组蛋白。