Identification of cytochrome P450 and glutathione-S-transferase genes preferentially expressed in chemosensory organs of the swallowtail butterfly, Papilio xuthus L

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. We undertook an expressed sequence tag (EST) analysis to identify the chemosensory-related genes that are expressed in chemosensilla on the tarsus of P. xuthus. Several genes that showed similarity with biotransformation enzymes were identified from the ESTs. Among them, a cytochrome P450 and a glutathione-S-transferase (GST) were preferentially expressed in the chemosensory organs. We have determined the structure of both cDNA and genomic sequences encoding these enzymes and designated the P450 as CYP341A2, a novel member of CYP341A subfamily, and the GST as GST-pxcs1, respectively. We observed a localized expression of CYP341A2 at the base of tarsal chemosensilla by in situ hybridization. These results suggest that these degrading enzymes play a role in the chemosensory reception for host plant recognition.     

Tyramine from the leaves of wild parsnip: a stimulant and synergist for oviposition by the black swallowtail butterfly

Abstract .Oviposition stimulants from the foliage of wild parsnip, Pastinaca sativa (Apiaceae), were isolated by column chromatography and HPLC and tested in bioassay experiments with hand-held female black swallowtail butterflies, Papilio polyxenes (Lepidoptera: Papilionidae). Two of the stimulants were identified as tyramine and trans -chlorogenic acid. A combination of tyramine, trans -chlorogenic acid and an active neutral fraction was needed to elicit a significant oviposition response. These results are discussed in the context of previous research on the oviposition stimulants of swallowtail butterflies and on the significance of tyramine as a neuromodulator of physiological processes in invertebrates.     

Taste input from tarsal sensilla is related to egg‐laying behavior in Papilio hospiton

In herbivorous insects, host selection involves various sensory modalities (sight, smell, taste), but the contact chemoreceptors capable of detecting stimuli both from host and non‐host plants play an important role in the final steps of oviposition behavior. Female butterflies scratch and drum the leaf surface and taste the compounds present in plant saps with their tarsal chemosensilla. We assumed that tarsal taste sensitivity may be related to the breadth of host selection in ovipositing females of Papilio hospitonGéné (Lepidoptera: Papilionidae). The spike activity of tarsal taste basiconic sensilla was recorded in response to stimulation with NaCl, bitter compounds, and carbohydrates, with the aim of characterizing the gustatory receptor neurons (GRNs) and of comparing the response patterns in the light of differences in acceptability of host plants. Then we studied the sensitivity of GRNs to saps of the host plants Ferula communis L., Peucedanum paniculatumLoisel, Pastinaca latifolia (Duby) DC. (all Apiaceae), and Ruta lamarmorae Bacch., Brullo et Giusso (Rutaceae), and evaluated the relationship between taste sensitivity and oviposition preference. The results indicate that (1) each sensillum houses sugar‐, bitter‐, and salt‐sensitive cells; (2) the spike activity of the gustatory neurons in response to plant saps produces a different response pattern across all active GRNs; and (3) the number of eggs laid on each plant is highest on F. communis and lowest on R. lamarmorae. These results suggest that the varying activity of the tarsal GRNs may affect host plant acceptability and that ovipositing females of P. hospiton seem to be able to discriminate between host plants.     

Identification and genomic structure of chemosensory proteins (CSP) and odorant binding proteins (OBP) genes expressed in foreleg tarsi of the swallowtail butterfly Papilio xuthus

Chemoreception is a key feature for selection of host plants by phytophagous insects. Female swallowtail butterflies recognize their host plants using chemosensilla present on foreleg tarsi. We constructed a cDNA library of female tarsi and a genome library of Papilio xuthus. We identified 11 chemosensory protein (CSP) genes and three odorant binding proteins (OBP) genes from the cDNA library and eight additional CSP genes from the genome library using the ESTs as probes. A sequence similarity tree of insect CSPs showed that lepidopteran CSPs constructed big branches of the order. Small numbers of CSPs have been identified from the whole genomes of several insect orders which belong to branches separated from those of Lepidoptera. The CSP gene family of Lepidoptera may have diverged in at least two steps, the first on a small scale and the second on a large scale before and after the diversification of insect orders, respectively. Seventeen of 19 CSP genes of P. xuthus clustered in a specific region of the genome, suggesting that they were diversified by gene duplication from a common ancestral gene.     

Contact chemoreception of oviposition-stimulating glucosinolates and an oviposition-deterrent cardenolide in two subspecies of Pieris napi

Abstract. Electrophysiological responses to ten glucosinolates, oviposition stimulants isolated from their cruciferous host plants, were recorded from tarsal taste neurones of two subspecies of Pieris napi: P. napi napi L. and P. napi olerucea (Harris) (Lepidoptera: Pieridae). In the cluster of fourteen medial B-sensilla on the fifth tarsomere, strong responses to glucosinolates were recorded. Three receptors cells, identified on the basis of spike amplitude and shape, were typically active in these responses, with the cell producing the smallest spike predominating. The response intensity of the latter cell to a particular glucosinolate at 1 mm concentration depended both on the chemical structure of the stimulus and on the subspecies of insect. In P. napi napi , aromatic glucosinolates were just as effective as aliphatic glucosinolates, whereas in P. napi oleracea the long-chain aliphatic glucosinolates gluconapin and progoitrin were stronger stimuli. At 10 and 100 pg/ml concentrations, significant differences between the subspecies were found in the absolute response intensity to seven compounds. A second cell in P. napi olerucea , producing a medium-sized spike, was excited most by sinalbin, whereas in Pnupi nupi three aliphatic glucosinolates were equally effective stimulants for a similar cell. In both subspecies the cell producing the medium-sized spikes in response to glucosinolates was also highly sensitive to erychroside, an oviposition deterring cardenolide occurring in a host of P. napi olerncea. The evolutionary divergence between the two geographically separated subspecies is reflected by differences in chemosensory recognition mechanisms.     

Neighbourhood of host plants influences oviposition decisions of a stem-boring weevil

The spatial arrangement of suitable host plants in the field may significantly constrain insects to find optimal hosts. Plant neighbours around a focal host plant can either lead to lower (associational resistance) or higher (associational susceptibility) herbivore loads. We tested whether the spatial arrangement of hosts of different suitability for the larval development of the shoot-base boring weevil Apion onopordi affects oviposition decisions in the field. Host plants in our study were healthy creeping thistles (Cirsium arvense; suboptimal hosts) and thistles infected by a rust pathogen (Puccinia punctiformis; optimal hosts). For analysis, we used nearest neighbour methods that disentangle the spatial distribution of organisms that are dependent on the position of other species (e.g. phytophagous insects and their host plants). Although theory predicts that the small-scale spatial infestation pattern can have major consequences for the population dynamics in insect–plant systems, field studies quantifying spatial pattern of phytophagous insects are rare.

The spatial arrangement of host plants clearly influenced oviposition pattern in A. onopordi. In contrast to previous studies, we demonstrated that not the rust infection itself determined if a plant was infested by weevils, but rather the density of rusted shoots within a certain neighbourhood. We found strong indications for associational susceptibility of healthy thistle shoots to weevil oviposition when growing in the neighbourhood of rusted thistles. Weevil-infested plants were spatially aggregated, indicating that A. onopordi is limited in its dispersal ability within patches. Other stem-boring insects on creeping thistle were affected in their oviposition decisions by other factors than A. onopordi. Thus, it may be difficult to find general rules for oviposition choice in phytophagous insects.

Our study showed that the spatial arrangement of host plants in the field critically determines oviposition choice and should thus be included as constraint in theories of optimal host selection.     

Molecular response of Drosophila melanogaster tyramine receptor cascade to plant essential oils

This paper reports the role of the tyramine (TA) receptor cascade in the insecticidal activity of plant essential oils. A TA receptor cDNA encoding a putative seven transmembrane domain G-protein coupled receptor was amplified from Drosophila melanogaster head cDNA phage library. The encoded protein contains 601 amino acids and has a sequence similar to other biogenic amine receptors. This protein was expressed in Drosophila S2 cells for radioligand binding studies with the ligand 3H-TA. Competitive binding studies comparing biogenic amines that could potentially function as endogenous ligands have demonstrated that this receptor had the highest affinity for TA (Ki=1.27 microM) followed by DL-octopamine, dopamine, serotonin and histamine. TA decreased the forskolin-increased cAMP levels (IC50=5.802 microM) and increased [Ca2+]i through the receptor expressed in S2 cells. The toxicity rank order of the tested plant essential oils against wild type D. melanogaster fly demonstrated a pattern similar to their effect on receptor binding activity and changes in cAMP level and [Ca2+]i. The toxicity of two of these chemicals was eliminated when tested against the TA receptor mutant (TyrRneo30) Drosophila strain. Therefore, the data indicates a correlation between cellular changes and insecticidal activity of tested plant essential oils, and suggests that the toxicity of at least two of these chemicals is mediated through the TA receptor.     

Morphological and electrophysiological mapping of tarsal chemoreceptors of oviposition-deterring pheromone in Rhagoletis pomonella flies

Light microscopy and S.E.M. observations revealed that Rhagoletis pomonella female tarsi, bearing the principal receptors of oviposition-deterring fruit marking pheromone (ODP), have 3 types of chemosensilla: B, C and D. Using electrophysiological hair tip-recording techniques, we found that D chemosensilla located in pairs on distal ventrolateral portions of the 2nd, 3rd and 4th tarsomeres of each leg were highly sensitive to stimulation by the pheromone. D chemosensilla located in pairs on the 5th tarsomere of each leg were moderately sensitive to the pheromone, while the B and C chemosensilla of all tarsi were nearly or completely insensitive to it. For reasons discussed, the D chemosensilla on the prothoracic tarsi may be the most important in providing sensory input eliciting oviposition deterrence.     

Antioxidant effects of dopamine and related compounds.

The antioxidant and free radical scavenging effects of dopamine, noradrenaline, tyramine, and tyrosine were investigated and compared with alpha-tocopherol. The antioxidant effect of dopamine and its related compounds on peroxidation of linoleic acid were in the order of dopamine > alpha-tocopherol = tyramine > tyrosine > noradrenaline as measured by the thiocyanate method. These amine compounds had reducing power, and a scavenging effect on reactive oxygen species, i.e., superoxide anion and hydroxyl radical. The results for reducing power and scavenging effect of these amine compounds had a similar trend as their inhibition of linoleic acid peroxidation. The antioxidant activity of these amine compounds in soybean oil was also evaluated by the Rancimat method. The induction time to reach 100 meq/kg peroxide value (POV) of soybean oil for dopamine, alpha-tocopherol, tyramine, tyrosine, noradrenaline, and control were 9.0, 8.2, 8.0, 6.4, 4.6, and 4.3 h, respectively. The antioxidant efficacy of amine compounds seems to be correlated with the numbers of hydroxy groups and their position on the phenolic ring.     

Eleven new putative aminergic G-protein coupled receptors from Amphioxus (<Emphasis Type="Italic">Branchiostoma floridae</Emphasis>): identification,sequence analysis and phylogenetic relationship

We have identified eleven novel aminergic-like G-protein coupled receptor (GPCRs) sequences (named AmphiAmR1-11) by searching the genomic trace sequence database for the amphioxus species, Branchiostoma floridae. They share many of the structural motifs that have been used to characterize vertebrate and invertebrate aminergic GPCRs. A preliminary classification of these receptors has been carried out using both BLAST and Hidden Markov Model analyses. The amphioxus genome appears to express a number of D1-like dopamine receptor sequences, including one related to insect dopamine receptors. It also expresses a number of receptors that resemble invertebrate octopamine/tyramine receptors and others that resemble vertebrate alpha-adrenergic receptors. Amphioxus also expresses receptors that resemble vertebrate histamine receptors. Several of the novel receptor sequences have been identified in amphioxus cDNA libraries from a number of tissues.     

A new family of insect tyramine receptors

The Drosophila Genome Project database contains a gene, CG7431, annotated to be an "unclassifiable biogenic amine receptor." We have cloned this gene and expressed it in Chinese hamster ovary cells. After testing various ligands for G protein-coupled receptors, we found that the receptor was specifically activated by tyramine (EC(50), 5x10(-7)M) and that it showed no cross-reactivity with beta-phenylethylamine, octopamine, dopa, dopamine, adrenaline, noradrenaline, tryptamine, serotonin, histamine, and a library of 20 Drosophila neuropeptides (all tested in concentrations up to 10(-5) or 10(-4)M). The receptor was also expressed in Xenopus oocytes, where it was, again, specifically activated by tyramine with an EC(50) of 3x10(-7)M. Northern blots showed that the receptor is already expressed in 8-hour-old embryos and that it continues to be expressed in all subsequent developmental stages. Adult flies express the receptor both in the head and body (thorax/abdomen) parts. In addition to the Drosophila tyramine receptor gene, CG7431, we found another closely related Drosophila gene, CG16766, that probably also codes for a tyramine receptor. Furthermore, we annotated similar tyramine-like receptor genes in the genomic databases from the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera. These four tyramine or tyramine-like receptors constitute a new receptor family that is phylogenetically distinct from the previously identified insect octopamine/tyramine receptors. The Drosophila tyramine receptor is, to our knowledge, the first cloned insect G protein-coupled receptor that appears to be fully specific for tyramine.     

Characterization of chemoreceptive protein binding to an oviposition stimulant using a fluorescent micro-binding assay in a butterfly

A native female-specific chemoreceptive protein of a swallowtail butterfly [oviposition stimulant binding protein (OSBP)] was shown to specifically bind to aristolochic acid, a main stimulant for oviposition from its host plant. Oviposition stimulants are recognized by chemoreceptive organs of insects. OSBP isolated previously from the chemoreceptive organs was assumed to bind to an oviposition stimulant. Using a highly sensitive fluorescent micro-binding assay, we clarified OSBP bound to aristolochic acid. Three-dimensional molecular modeling revealed the structure of the OSBP-aristolochic acid complex. This is the first report of a native chemoreceptive protein binding to an oviposition stimulant as a ligand in insects.     

Biogenic amines and the control of neuromuscular signaling in schistosomes

Biogenic amines are small cationic monoamines that function broadly as neurotransmitters and/or neuromodulators in every animal phylum. They include such ubiquitous substances as serotonin, dopamine and invertebrate-specific phenolamines (tyramine, octopamine), among others. Biogenic amines are important neuroactive agents in all the flatworms, including blood flukes of the genus Schistosoma, the etiological agents of human schistosomiasis. A large body of evidence spanning nearly five decades identifies biogenic amines as major modulators of neuromuscular function in schistosomes, controlling movement, attachment to the host and other fundamental behaviors. Recent advances in schistosome genomics have made it possible to dissect the molecular mechanisms responsible for these effects and to identify the proteins involved. These efforts have already provided important new information about the mode of action of amine transmitters in the parasite. Moreover, these advances are continuing, as the field moves into a post-genomics era, and new molecular tools for gene and protein analysis are becoming available. Here, we review the current status of this research and discuss future prospects. In particular, we focus our attention on the receptors that mediate biogenic amine activity, their structural characteristics, functional properties and "druggability" potential. One of the themes that will emerge from this discussion is that schistosomes have a rich diversity of aminergic receptors, many of which share little sequence homology with those of the human host, making them ideally suited for selective drug targeting. Strategies for the characterization of these important parasite proteins will be discussed.     

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.     

Comparison of the egg-laying behaviour and electrophysiological responses of Delia radicum and Delia floralis to cabbage leaf compounds

Abstract.  The behaviour and the sensitivity of adult cabbage root fly, Delia radicum and turnip root fly, Delia floralis are compared with host-plant extracts and isolated crucifer compounds previously identified as oviposition stimulants for D. radicum . The oviposition behaviour of both species is similar; 7–10-day-old females are stimulated to lay eggs by the methanol extract of cauliflower leaves that contains thia-triaza-fluorenes (CIF) as well as glucosinolates. The glucosinolate fraction is mainly composed of glucobrassicin, which alone stimulate both fly species to lay eggs. The C₅ and D_3,4 sensilla on the prothoracic tarsae of newly-emerged D. radicum contain neurones sensitive to the glucosinolate fractions tested and to glucobrassicin, whereas the CIF specifically stimulate a neurone in the C₅ sensillum. By contrast, newly-emerged D. floralis respond less to glucosinolates, especially to glucobrassicin, and have sensitive neurones to CIF in other sensilla than D. radicum . Recordings are also made from the longest sensilla present on the labellum because they are apparently sensitive to glucosinolates. By contrast to earlier investigations, no remarkable phasic-tonic responses of these neurones are seen. The two species are difficult to discriminate visually, have the same host plants, show identical host-selection behaviour, apparently respond to the same physical and chemical properties of their host-plants, but have a clearly different distribution of receptor neurones in the tarsal sensilla.     

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.     

Contact chemoreception related to host selection and oviposition behaviour in the monarch butterfly, Danaus plexippus

Abstract. Behavioural events during host selection by ovipositing monarch butterflies (Danaus plexippus (L.), Danainae, Nymphalidae) include tapping the leaf surface with fore-tarsi and touching this surface with mid-tarsi (‘drumming’) and antennae. Flavonoids identified from host plant extracts are known to stimulate oviposition. Scanning electron microscopy revealed the presence of contact-chemoreceptor sensilla on all appendages that contact the leaf surface. This electrophysiological study was conducted to identify the contact chemoreceptors that are sensitive to the known oviposition stimuli and are therefore probably involved in host recognition. Receptor cells of conspicuous sensilla grouped in clusters on fore-tarsi of females were sensitive to the behaviourally active butanol fraction of host plant (Asclepias curassavica) extract. However, these receptors generally had low sensitivity to three oviposition-stimulating flavonoids identified from this fraction, but they were also sensitive to the butanol fraction of a non-host (Brassica oleracea). Chemoreceptors in sensilla of the tarsomers 2–4 of the mid-legs also responded to the behaviourally active fraction of host plant extract and showed some sensitivity to two of the flavonoids that stimulate oviposition. Similar results were obtained from receptor cells in sensilla on the tip of the antennae. Most of these sensilla had cells responding to the butanol fraction of A. curassavica extract but only 25% of them were also sensitive to one of the behaviourally active flavonoids. These electrophysiological results, in combination with behavioural observations, suggest that host selection in monarch butterflies relies on a complex pattern of peripheral sensory information from several types of tarsal and antennal contact chemoreceptors.     

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.