Oral Communications and Posters

A31: VIII National Postharvest Technology Conference, Chiang Mai (Thailand), 1-3 September 2010, plenary lecture Learning insect olfaction to build novel highly sensitive olfactory-based biosensors Angeli S. Faculty of Science and Technology, Free University of Bolzano, Italy. Piazza Università 5, 39100 Bolzano, Italy E-mail address: sergio.angeli@unibz.it Coming soon ...   A30: VIII National Postharvest Technology Conference, Chiang Mai (Thailand), 1-3 September 2010 Antennal responses of rice weevils to volatile organic compounds released by jasmine paddy rice 1Thakeow P., 2Angeli S. 1 Department of Product Development Technology, Faculty of Agro-Industry, Chiang Mai University, 50100 Chiang Mai, Thailand 2 Faculty of Science and Technology, Free University of Bolzano, 39100 Bolzano, Italy. The most famous and favoured variety of Thai rice is the aromatic jasmine rice, Khao Dok Mali 105 (KDML 105). It possesses unique flavour, good appearance, and good texture. In each year there has been loss of rice during storage due to the rice pest Sitophilus oryzae (Coleoptera: Curculionidae). Therefore, there have been several attempts aiming to prevent and control the rice weevil infestations. For example, stored rice is often treated with low toxic organophosphorous insecticides and fumigant treatments. Nowadays, for insect control, semiochemicals often plays an important role in modem agriculture. Insects use volatile organic compounds (VOCs) for orientation in mating, host finding, and food source orientation. These chemicals are generally not dangerous to human health, and are used as a valid alternative to reduce the economic losses of pest insects. Therefore, in this research, we examined volatiles released from KDML105 paddy rice using four sampling techniques, namely, (i) solid phase micro extraction, (ii) close loop stripping analysis, (iii) thermal desorption system, and (iv) direct headspace analysis. The collected volatiles were identified using gas chromatography-mass spectrometry. The VOCs detected from headspace of paddy rice belongs to different chemical groups, but most of them are aldehydes. Afterwards, the VOCs were tested with S. oryzae dissected antennae using a gas chromatograph-mass spectrometer coupled with electroantennographic detector (GC-MS/EAD). It was found that insect antennae responded consistently to hexanal. This finding could lead to an alternative and environmental friendly pest control technique for the rice weevil S. oryzae. Keywords: jasmine rice, rice weevil, VOCs   A29: XXIII IUFRO World Congress, "Forests for the Future: Sustaining Society and the Environment, Seoul (Republic of Korea), 23-28 August 2010 Olfactory cues of female Hypsipyla robusta (Moore) (Lepidoptera: Pyralidae) to mahoganies of the genera, Entandrophragma and Khaya 1Abraham J., 2Angeli S., 3Schütz S., 3Weissbecker B. and 1Opuni-Frimpong E. 1 Forest Research Institute of Ghana, Kumasi, Ghana 2 Faculty of Science and Technology, Free University of Bolzano, 39100 Bolzano, Italy 3 Institute of Forest Zoology and Forest Conservation, Georg-August-University Göttingen, D-37077 Göttingen, Germany The mahogany shoot borer, Hypsipyla robusta (Moore) (Lepidoptera: Pyralidae) is a serious pest insect attacking and damaging indigenous mahogany plantations. It is believed that H. robusta relies on olfaction and gravid females are attracted by volatile compounds of mahogany trees. This research was conducted to determine which volatile compounds influence the attack of mahogany trees in the genera, Entandrophragma and Khaya. Volatile samples were collected from shoots of Entandrophragma angolense, Entandrophragma utile, Khaya anthotheca and Khaya ivorensis in field and laboratory sampling in Ghana by Closed Loop Stripping Analysis (CLSA). Gas-chromatography mass-spectrometry electroantennographic detection and electroantennogram experiments were performed with the volatile samples. The results revealed significant responses of the female moth antennae to 1-hexanol, 2-ethyl-1-hexanol, beta-caryophyllene, cis-ocimene, cis-3-hexenyl acetate, cis-3-hexenyl butyrate, cis-3-hexenyl hexanoate, decanal, germacrene D, naphthalene and nonanal. Dose-response curves of electroantennogram responses for these compounds showed that female H. robusta are able to perceive 1-hexanol and nonanal at very low concentrations. We therefore propose that these compounds may serve as long-range attractants for female H. robusta to localized mahogany host trees and may be used as trap baits.   A28: XXII Italian National Congress of Entomology, Ancona (Italy), 15-18 June 2009 Studio delle interazioni nella codifica di composti volatili in Cydia pomonella (L.) mediante la superposizione di stimoli olfattivi 1Vitagliano S., 2Angeli S., 2Thakeow P., 1De Cristofaro A. 1 Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via De Sanctis, 86100, Campobasso, Italy 2 Institute of Forest Zoology and Forest Conservation, Georg-August-University Göttingen, D-37077 Göttingen, Germany Cydia pomonella (L.) (Lepidoptera: Tortricidae) è fitofago di primaria importanza per melo, pero e noce. (E, Z) - 2,4-etil-decadienoato, un estere estratto da pere mature, esercita un’elevata attività attrattiva sul fitofago. La possibilità di utilizzo di questo composto volatile per il monitoraggio degli adulti di entrambi i sessi, o come mezzo ausiliario nel controllo delle larve, potrebbe giocare un ruolo determinante nella gestione delle infestazioni di C. pomonella. Studi recenti hanno focalizzato l’attenzione sull’effetto combinato del principale componente del feromone sessuale di C. pomonella, il (E,E) - 8,10-dodecadien-1-olo o codlemone, ed il (E,Z) - 2,4-etil-decadienoato, nella prospettiva di migliorare l’efficienza delle trappole per il monitoraggio. Nel presente lavoro è stato utilizzato un elettroantennografo portatile, che applica la tecnica della superposizione, per lo studio delle interazioni esistenti fra la percezione del codlemone, del 2,4-etil-decadienoato e di altri composti volatili nelle antenne di C. pomonella. Il codlemone è stato utilizzato come odore di fondo, mentre altri composti organici volatili quali l’esenil-acetato, l’1-octen-3-olo, l’octanale, l’esanolo, il cariofillene, il linalolo, il farnesene e il (Z)-3-esen-1-olo, venigano saggiati in superposizione a diverse concentrazioni in paraffina. Comparando la risposta delle antenne di adulti di C. pomonella al solo odore di fondo e ai soli composti volatili con la risposta agli odori sovrapposti è stato possibile verificare l’esistenza di un’interazione nella percezione di alcuni di questi composti. Infatti, in alcuni casi si è osservato che le risposte in superposizione hanno ampiezza minore della somma delle risposte ai singoli stimoli. Ciò è imputabile alla presenza di recettori olfattivi comuni per le due sostanze, confermando quanto riportato in precedenti lavori dove è stato ipotizzato che la codifica olfattiva del codlemone e del 2,4-etil-decadienoato sia mediata, almeno in parte, da neuroni olfattivi che esprimono gli stessi recettori olfattivi. In futuro un utilizzo più esteso della tecnica per superposizione potrà contribuire a quantificare in maniera dettagliata le interazioni esistenti nella codifica olfattiva di diversi composti volatili della pianta ospite e dei componenti feromonici di C. pomonella. Parole chiave: Carpocapsa, EAG, elecroantennografia, interazioni pianta-insetto, E8,E10-12:OH, VOCs.   A27: Entomologentagung DGaaE (=German Society for General and Applied Entomology), Göttingen (Germany), 16-19 March 2009 Attractive defence compounds and repellent host odours: unexpected behaviour of Bolitophagus reticulatus (Coleoptera: Tenebrionidae) Holighaus G., Blome R. and Angeli S. Institute of Forest Zoology and Forest Conservation, Georg-August-University Göttingen, Germany Büsgenweg 3, D-37077 Göttingen, Germany The black tinder fungus beetle Bolitophagus reticulatus is a model organism to study landscape fragmentation effects on deadwood insects. This beetle feeds all life stages exclusively on fruiting bodies of the bracket fungus, Fomes fomentarius. Although B. reticulatus is a long-living organism and a good flyer, genetic isolation occurs in fragmented landscapes. Here, we investigated the chemoecology of the beetle to elucidate the function of some volatile organic compounds (VOCs) in terms of insect behaviour, feeding and reproduction. VOCs of F. fomentarius fruiting bodies at different degree of colonisation as well as the ones of abdominal defensive glands were analysed by gas chromatography (GC-MS). Selected VOCs were tested in a dual choice walking bioassay. 1-Octen-3-ol showed high level in young fruiting bodies of F. fomentarius, contrary to old, colonised fruiting bodies. Abdominal glands contain defensive compounds as methyl- and ethyl-benzoquinone. Moreover, we found m-cresol, which was never described in Tenebrionidae. The choice tests showed a strong repellence of 1-octen-3-ol, whereas a significant attraction to m-cresol was recorded. 1-Octen-3-ol has been previously characterised as an antifeedant against slugs. B. reticulatus avoids young fruiting bodies where 1-octen-3-ol level is comparably high. Therefore, we suggest that here 1-octen-3-ol plays a similar role as in the case of slugs. In a previous research, considerable aggregations of beetles within single fruiting bodies of F. fomentarius were found. The m-cresol of the abdominal glands may work as an aggregation pheromone, either for feeding or overwintering strategies. Further experimental research will be required to determin whether 1-octen-3-ol has a direct toxicity or characterises the fruiting body suitability. Moreover, field experiments could demonstrate the efficacy of m-cresol as aggregation pheromone.   A26: Entomologentagung DGaaE (=German Society for General and Applied Entomology), Göttingen (Germany), 16-19 March 2009 Response of the rice weevil Sitophilus oryzae L. to volatile compounds of jasmine rice KDML105 1Thakeow P., 23Vearasilp S., 2Schütz S. 1 Department of Product Development Technology, Faculty of Agro-Industry, Chiangmai University, Thailand 2 Department of Forest Zoology and Forest Conservation, Büsgen Institute, Faculty of Forestry, Georg-August-University Göttingen, Germany 3 Department of Seed Technology, Faculty of Agriculture, Chiangmai University, Thailand The rice weevil Sitophilus oryzae L. (Coleoptera: Curculionidae) is one of the most destructive pest insects in stored products. Rice weevil infests the seeds of rice, maize, sorghum, wheat, barley, and other cereals. In Thailand this insect is a severe pest species on rice, a very important agricultural product of the country. Therefore, we have started a study on the semiochemical communication of the rice weevil which could lead to develop an alternative approach of integrated pest management of this species. In this experiment, the jasmine rice KaoDawkMali105 (shortly called KDML105) was used. This rice variety is the most famous and most exported rice of Thailand. The volatile organic compounds (VOCs) of brown KDML105 were sampled using solid phase microextraction technique (SPME). Afterwards, the VOCs were further characterized using gas chromatography-mass spectrometry (GC-MS). The odor perception of the rice weevil was recorded using gas chromatography-mass spectrometry/electroantennographic detection (GC-MS/EAD) and electroantennography (EAG). Broad chemical classes of VOCs were detected in brown KDML105 rice: acetone, hexanal, 3-carene, 2-pentyl furan, limonene, octan-3-one, o-cymene, 1-octen-3-ol, 2-ethyl hexan-1-ol, camphor, N-ethylbenzenamine, and naphthalene. The results of GC-MS/EAD and EAG will be further described.   A25: XXII International Socity of Chemical Ecology Annual Meeting (XXII ISCE), Barcelona (Spain), 15-19 July 2006 Epicuticular hydrocarbons of Leptinotarsa decemlineata (Say): a sexual dimorphism Angeli S., Thakeow P., Weissbecker B. and Schütz S. Institute of Forest Zoology and Forest Conservation, Georg-August-University Göttingen, Germany Büsgenweg 3, D-37077 Göttingen, Germany E-mail address: angeli@sssup.it Insect epicuticular hydrocarbons play a key role as odorants in several species. During last years they have been found to be involved in chemoreception processes such as nest recognition of Apis mellifera, sexual pheromones in several Diptera, and the aggregation phenomenon of the desert locust Schistocerca gregaria. In the present study we characterise the epicuticular composition of the Colorado Potato Beetle, Leptinotarsa decemlineata. Despite this insect is considered as one of the most dangerous pest species in agriculture, so far no sexual pheromone has been reported. In a previous work we identified a new volatile amino-aromatic compound from the elytra of males and females. Here we focus our attention on the pattern of hydrocarbon extracted from the cuticle of male and female elytra in order to characterize a possible sexual communication. In a previous research a sex-dependent composition of cuticular hydrocarbons of L. decemlineata has been already described but the methodologies we adopted and the results differ substantially from the reported ones. We extracted cuticular hydrocarbons from the elytra of single animals in different life stage, e.g. freshly emerged, mating and ovipositing females. We analyzed the heptane extracts by the mean of GC-MS using a specific temperature program reaching 310°C. The chromatograms were interpreted with NIST Standard Reference Database, and in comparison with standards chemicals. The results confirmed that there is no n-alkanes in the epicuticular hydrocarbon mixture of L. decemlineata, but -contrary of what previously reported- females did not show a higher quantity of hydrocarbons with more than 34 carbons atoms. In our experiments males showed a simpler mixture of hydrocarbons, where hydrocarbons with higher molecular weight are actually predominant in comparison with females. Moreover, three hydrocarbons are exclusively or highly more abundant in females. These three compounds appeared in the first half of the chromatogram, therefore with a relatively low molecular weight. The same data were also analyzed by using principal component analysis (PCA) Pirouette® software (Infometrix). A PCA scores plot showed a clear separation between male and female hydrocarbons. A24: XVII Congress European Chemoreception Research Organization (XVII ECRO), Granada (Spain), 4-8 September 2006 Volatile organic compounds of Thai aroma rice Thakeow P. (1,3), Angeli S. (1), Ripon S. (2), Simapaisan P. (3), Thanapornpoonpong S. (2) and Schütz S. (1) 1 Institute of Forest Zoology and Forest Conservation. Georg-August-University Göttingen. Germany. 2 Postharvest Technology Institute. Chiang Mai University. Thailand. 3 Dept. Product Development. Faculty of Agro-Industry. Chiang Mai University. Thailand. Contact: pthakeo@gwdg.de Aroma rice, e.g. Basmati and Jasmine rice, is very popular among rice consumers in South and South-East Asia and is becoming famous in USA and Europe. Thailand is one of the major rice exporters; rice produced from Thailand possesses peculiar properties as aroma, appearance, and texture. One outstanding property of Thai rice, known as “Jasmine rice”, is its fragrance. The characteristic flavour of this rice is principally contributed by 2-acetyl-1-pyrroline. There are many varieties of aroma rice produce in Thailand, among which Khao Dawk Mali 105 (KDLM105) variety is the most famous one. In this experiment, we investigated volatile organic compounds (VOC) of Thai rice (Oryza sativa) by using five varieties: KDLM105, RD15, PTN1, HSP and CNT1. All of them were planted in the experimental field in 2005. The first four varieties are aroma rice, the last one is a rice variety of high yield and good grain. The Jasmine rice bought from the retail store was also used. VOC released to the head space from dry stored and moistured-heated rice were sampled using solid phase microextraction (SPME). In order to optimise the device suitability, three different fibers, Carboxen/PDMS, Carbowax/DVB and PDMS/DVB, were chosen. The resulting compounds were analysed using GC-MS. In parallel, sensory evaluation of rice samples was carried out focussing on odour perception of the panel considering the parameters “variety” and “ageing”. The majority of VOC identified as highly abundant were aldhydes. Hexanal, considered as an off odour, was detected even in recently harvested rice. P. Thakeow holds PhD fellowship by the Thai government and Chiang Mai University, Thailand. A23: XVII Congress European Chemoreception Research Organization (XVII ECRO), Granada (Spain), 4-8 September 2006 Assessment of fungivorous insect antennae as biosensors for detecting wood rotting fungi Thakeow P., Angeli S., Weissbecker B. and Schütz S. Institute for Forst Zoology and Forest Conservation, Göttingen University, Büsgenweg 3, D-37077 Göttingen Trametes versicolor, Gloeophyllum trabeum and Poria placenta are known as wood destructive fungi, causing high economic losses in construction wood and wood composite products. To evaluate the quality of wood as raw-material for the production of high engineering property products, a reliable and sensitive detection of wood rotting fungi is needed. A rapid and non-destructive method for the evaluation of fungal infestation can focus on volatile organic compounds (VOC) released by fungi. Beech wood (Fagus sylvatica) was inoculated either with G. trabeum, P. placenta or T. versicolor and incubated at 22 °C for one week prior to sampling of VOC. Volatiles released to the head space were collected using solid phase microextraction (SPME), 85 µm Carboxen/PDMS StableFlex fiber type, and were analysed by GC-MS. Each fungus growing on beech released a specific pattern of VOC. The detected VOC can be categorised into two broad groups, aliphatic C5-C8 compounds and isoprenoids. Typical fungal odour compounds like 1-octen-3-ol and 3-octanone were produced by all three fungi. Two monoterpenes, alpha-pinene and 3-carene were found. However, dominant components with respect to abundance were sesquiterpenes. Sesquiterpene patterns were characteristic for each species. Moreover, 6-protoilludene was specifically produced by G. trabeum in high amounts. Alpha- and beta-barbatene were typical and dominant VOC of T. versicolor. Cis boleti, an insect typically feeding on fruiting bodies of wood rotting fungi like T. versicolor was used for electroantennographic recordings of fungal volatiles. On the basis of these measurements the feasibility of a biosensor for the detection of wood rotting fungi was assessed. P. Thakeow holds PhD fellowship by the Thai government and Chiang Mai University, Thailand. A22: Proc. of the IX European Symposium on Insect Taste and Olfaction (IX ESITO), Villasimius (CA, Italy), 24-30 September 2005 Purification and first characterisation of soluble proteins involved in odorant recognition of Colorado potato beetle Angeli S. and Schütz S. Institute for Forest Zoology and Forest Conservation, Büsgenweg 3, D-37077 Göttingen E-mail: angeli@sssup.it Odorant Binding Proteins (OBP) and Chemo-Sensory Proteins (CSP) are two classes of soluble globular proteins secreted in a very high concentration in the neuron-bathing fluid of insect chemosensory sensilla. In the last ten years, members of both classes were discovered and fully characterized in several insects mainly belonging to the Orders of Lepidoptera, Hymenoptera and Diptera. Despite some members of both classes are recently fully characterized in terms of three-dimensional  structure, binding activity and histological localization, a full explanation of their specific role in the sensillar lymph is still missing. However, several evidence addresses to an important function in the early coding process of odorant and taste compounds during the olfactory perception of insects. These proteins may also be a useful tool in developing artificial biomimetic chemosensors, since all members characterized so far have a high stability, no post-translational modification and can be easily expressed as recombinant proteins in a full functional form. Therefore, we decided to investigate the chemosensory perception of Colorado potato beetle (Leptionotarsa decemlineata (Say)), where this type of protein had not been discovered. Extracts of body parts were obtained from males and females of our lab population and analyzed by SDS and NATIVE-PAGE. Protein profile of chemosensory organs, like mouthpart, antennae and tarsi, did not show any specific band in SDS condition, but a peculiar weak band specific for these extract became visible when the extracts were run in their native state.  Therefore, a mass-rearing of males and females were performed in order to obtain about 2000 antennae for each sex and to reach a full purification of the target proteins.  A combination of gel-filtration and ion-exchange chromatography allowed the purification of a protein of an apparent molecular weight of 12 kDa and a low isoelectric point. A Western blot experiment with the polyclonal antibody raised against the CSP-Sg4 was performed in order to check if this protein belongs to the CSP or OBP family. No reaction of the polyclonal antibody was observed against our purified protein.  A further purification was achieved by HPLC, and the isolated peak was subjected to Edman N-terminal degradation. The aminoacid sequence will be used to design a specific degenerated primer in order to clone the full sequence with RT-PCR and later to characterize the structure and the possible function of this new protein. A21: XX Congresso Nazionale Italiano di Entomologia, Assisi (Italy), 12-18 June 2005 Caratterizzazione chimica, fisiologica e comportamentale di un nuovo feromone della dorifora della patata, Leptinotarsa decemlineata (Say) Angeli S. and Schütz S. Institute for Forst Zoology and Forest Conservation, Göttingen University, Büsgenweg 3, D-37077 Göttingen La dorifora della patata rappresenta l’insetto più dannoso nella coltura della patata e di altre Solanacee. Recentemente questa specie ha raggiunto una resistenza ad oltre 25 insetticidi comunemente impiegati per la protezione delle colture. Lo sviluppo di metodi di lotta alternativa si rende perciò necessario, in particolare in seguito al diffondersi sempre più marcato dell’agricoltura biologica dove questo insetto ritorna ad essere una vera calamità. Promettente potrebbe essere l’utilizzo di composti volatili ad azione feromonica. Diverse ricerche sono attualmente in corso per l’identificazione di questi composti, in particolare nel tentativo di determinare la struttura chimica del feromone sessuale che, avendo una potente azione attrattiva, permetterebbe di monitorare in maniera molto precisa la dinamica delle popolazioni in pieno campo. Nel 2002 è stato isolato nella dorifora della patata il feromone di aggregazione, (S)-3,7-dimethyl-2-oxo-6-octene-1,3-diol, inaspettatamente emesso dai maschi (Dickens et al., 2002). Ricerche precedenti avevano inoltre messo in evidenza una diversa composizione di idrocarburi cuticolari fra maschi e femmine (Dubis et al., 1987). Ogni ricerca sulla natura chimica di un feromone sessuale di discreta volatilità non ha, fin’ora, portato a nessun risultato, sebbene prove comportamentali abbiano dimostrato che i maschi sono attratti da volatili emessi dalle femmine (Edwards and Seabrook, 1997). In questa comunicazione gli autori riportano l’individuazione di un nuovo composto volatile, il 2-aminoacetofenone, identificato attraverso tecniche di GC/MS dagli estratti delle elitre di individui adulti sessualmente attivi di Leptinotarsa decemlineata. La secrezione di questo composto aromatico e di natura amminica risulta sorprendente, sebbene in altri coleotteri siano stati ritrovati altri feromoni di natura aromatica, ad esempio negli ultimi anni è stato identificato il 1,4-benzoquinone in Melolontha melolontha e il japonilure Popillia japonica. Il composto isolato da noi, 2-aminoacetofenone, potrebbe essere un buon candidato come feromone sessuale e per questo motivo sono state successivamente eseguite prove di elettroantennografia (EAG) su antenne di maschi e femmine e di orientazione in olfattometro. I risultati di EAG e di GC-MS-EAD confermano come questo composto venga percepito selettivamente dalle antenne in entrambi i sessi, a differenza dei composti simili 3- e 4-aminoacetofenone. Prove comportamentali in un olfattometro a 4 vie sono in corso e i risultati dell’analisi statistica verranno presentati in questa comunicazione. Parole chiave: Coleoptera, Crisomelidae, semiochimici, comunicazione chimica, gas-cromatografia. A20: Entomologentagung Dresden 2005 DGaaE (=German Soc. for General and Applied Entomology), Dresden(Germany) 21-24 March 2005 Pheromones of Leptinotarsa decemlineata (Say): an integrated approach of electorophysiology, behaviour and biochemical study Angeli S., Weissbecker B. and Schütz S. Institute for Forst Zoology and Forest Conservation, Göttingen University, Büsgenweg 3, D-37077 Göttingen It has been documented that males of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) respond selectively to the wing extract of fertile females, suggesting that a sexual pheromone may be involved. In the past, several attempts have been made to elucidate the chemical structure of this compound. However, till now only an aggregation pheromone emitted by the males has been identified, while there is a complete lack regarding the female sexual pheromone. In the present communication, the authors would like to show a set of data regarding chemical communication of Colorado potato beetle using different approaches, like GC-MS analyses, antennal electrophysiology (EAG), behavioural assays and protein biochemistry. By the means of GC-MS we were able to identify a new volatile amino-aromatic compound from the forewing of adults, with females showing a higher concentration. We hypothesise that this compound may be a pheromone, in agreement with a previous research where only the methanol extract of female elytra stimulate male mating behaviour. By means of EAG we demonstrated that this odorant is clearly perceived by the antennae of both sexes and behavioural assays are in progress. We also planned to find out chemosensory proteins (CSP) and odorant binding proteins (OBP), which are involved in the first steps of insect olfactory transduction. An antennal specific protein has been recently extracted from both sexes and partially characterised. Similar proteins may occur also in pheromone emitting glands, since their ability of reversible bind specific odorants. Therefore, proteins were extracted from male and female forewings of L. decemlineata and partially characterised. A19: 97. Jahresversammlung der Deutschen Zoologischen Gesellschaft, Rostock (Germany) 31 May-4 June 2004 Characterization of a cuticle protein of the sphinx moth Manduca sexta Angeli S. and Treckzek T. Institute of Zoology, Justus-Liebig University of Giessen, D-35390 Giessen, Germany Cuticular proteins of insects play an important role in defining mechanical and biological functions of their integument. These proteins may constitute more than 50% of the total cuticular weight, comprising several members such as arthropodin, sclerotin, and resilin. The different arrangement of the embedded chitin fibers, in association with the local protein secretion and the disposition of the layers, can change widely the mechanical properties of the integument in a way that confer optimal local plasticity and elasticity to the integument. In the present study, a protein of the larval endocuticular layer of Manduca sexta, is recognised by a monoclonal antibody (MS#77), previously raised against Manduca sexta haemocyte. In an effort to understand the specific property of this protein, a combined histological and biochemical study has been made. Firstly, immunohistological investigation of larval semi-thin transversal sections were performed with the original MS#77 hybridoma and a series of lectin labelling assays. An overlapping binding pattern to of the cuticular layers was found with the Vicia villosa lectin, which recognise preferentially alpha- or beta-linked terminal N-acetylgalactosamine, linked to serine or threonine in a glycopeptide. Western blot analyses were made after extraction of cuticular proteins with different buffers. A strong band of approximately 48 kDa appeared only when cuticle is extracted with high concentration of SDS and the yield increased when EDTA was added. Moreover, biotinylated V.v. lectin bind to the same protein, showing that the MS#77 antigen is likely to be a glycoprotein. Therefore, a large preparation of cuticular proteins were made under optimal buffer condition and the extracted proteins were subjected to gelfiltration, mono-Q and preparative electrophoresis purification, allowing us to fully purify the antigen. Finally, enzymatic and chemical deglycosylation techniques were performed. Results will be shown in the present communication. This research was supported by the EU network Insect Chemical Ecology (INCHECO) - Contract n. HPRN-CT-1999-00054 A18: Innate Immunity Workshop: bridging the gap between ecology and molecules, 6-9 May 2004 How to characterize the immune competence of an insect? Treckzek T., Schmidtberg H. and Angeli S. Institute of Zoology, Justus-Liebig University of Giessen, D-35390 Giessen, Germany Research in insect immunity has developed an overwhelming amount of data sets and knowledge about antimicrobial peptides, diverse humoral factors, melanization responses, hemocyte types, phagocytosis, nodule formation and encapsulation of a variety of insect species. However, in most cases the description of an immune reaction of a certain insect is restricted to one or two measurable phenomena. Since a successful immune system depends on the interaction of all its parameters in a network as well as on the life circumstances (selection pressure) related to its ecological niche, it is important to analyze several components of the immune system within one insect species to come close to an understanding of the real competence of the system. As examples, different aspects of the immune systems of the white cabbage butterfly, Pieris brassicae, of the Colorado potato beetle, Leptinotarsa decemlineata and of sawflies, Thentredinidae will be described. The work was supported by the SNF, the BMBF and EU-network INCHECO A17: IX International Congress of International Society for Developmental and Comparative Immunology, St Andrews (Scotland), 29 June- 4 July 2003 Hemocytes of Calliphora vomitoria (L.): morphological, histological and functional characterization 1Wang J.X., 2Trenczek T., and 2Angeli S. 1 School of Life Sciences, Shandong University, Jinan 250100, China 2 Institute of Zoology, Justus-Liebig University of Giessen, D-35390 Giessen, Germany The most common hemocyte types of insects are prohemocytes, plasmatocytes and also granular cells. In addition, spherulocytes and oenocytoids have been described in Lepidoptera, cristal cells in Drosophila melanogaster and throbocytoids in some other Diptera. Here we report about hemocytes investigation of Calliphora vomitoria larvae and pupae by phase contract microscopy, fluorescence microscopy using antibodies and lectins as well as in vitro and in vivo functional assays. Four kinds of cells have been distinguished by phase contrast microscopy and were named: prohemocytes, plasmatocytes, thrombocytoids and oenocytoids. About 20 monoclonal antibodies raised against Manduca sexta hemocytes and one developed against Hyalophora cecropia hemocytes have been screened and five of them showed to bind specific epitopes of C. vomitoria hemocytes. Preliminary SDS-PAGE followed by Western blot analysis were made in the efford to characterise their specific antigens. Lectin labeling of C. vomitoria hemocytes revealed several differences in their carbohydrate composition. In particular, the lectin from Arachis hypogea [ß-Gal,(1-3),GalNAc] did not bind to throbocytoids whereas the lectin of Vicia villosa B4 [GalNAc] did, reflecting a difference in the GalNAc-composition of these cells. The phagocytic activity of C. vomitoria hemocytes was investigated by using bacteria (Escherichia coli, Micrococcus luteus, and Bacillus megaterium) as well as yeast (Saccharomyces cerevisae), all labeled with fluorescein-isothiocynate (FITC). The results showed that yeast was efficiently phagocytosed, mainly in vitro by plasmatocytes and in vivo by thrombocytoids. Finally, we compared the data of C. vomitoria hemocytes with those obtained with studies on hemocytes from Musca domestica and Drosophila melanogaster. A16: IX International Congress of International Society for Developmental and Comparative Immunology, St Andrews (Scotland) 29 June- 4 July 2003 A comparative study of immune system and easy bleeding phenomenon of sawflies (Hymenoptera; Tenthredinidae) Institute of Zoology, Justus-Liebig University of Giessen, D-35390 Giessen, Germany The immune system has been well studied in respect both to humoral and cellular components in several insects. Hemocytes are characterised according to their morphological features, specific protein expressions and cellular activities, although there has been controversy over the number of hemocytes types and on the nomenclature adopted in different species. Till now in sawflies no results have been shown about their hemocyte and only one investigation is published on their hemolymph antibiotic activity. A very interesting phenomenon, called “easy bleeding”, has recently been discovered in several sawfly species. It is linked to an easy cuticular disruption with the formation of hemolymph drops along their cuticle surface, when larvae are bothered from other animals. This phenomenon is interpreted as a defence mechanism against predators. We hypothesised that “easy bleeding” sawflies might have stronger immune mechanism than “non-bleeding” in order to survive after periodically hemolymph losses. In this work we show various investigations on the immune system of eight different sawflies species. We have characterized sawfly hemocytes with phase contrast microscopy, immunohistochemistry and lectin labelling assays, in particular, of Athalia rosae and Gipinia hercyniae. In immunohistological analyses some monoclonal antibodies, specific for Manduca sexta hemocytes epitops, bind to cuticular layers in sawflies. In addiction several antibiotic tests on infected and non-infected larvae were made in the effort to verify the efficiency of their humoral immune response. As a first result we can clearly demonstrate that easy bleeders express a very sensitive humoral response, showing a high antibiotic activity after bacterial infection. This research was supported by the EU network Insect Chemical Ecology (INCHECO) - Contract n. HPRN-CT-1999-00054 A15: 96. Jahresversammlung der Deutschen Zoologischen Gesellschaft und Deutschen Gesellschaft für Parasitologie, Berlin (Germany) 9-13 June 2003 Morphological and immunohistological investigations of “easy bleeding” and “non-easy bleeding” Tenthredinidae Angeli S. and Treckzek T. Institute of Zoology, Justus-Liebig University of Giessen, D-35390 Giessen, Germany An interesting defence strategy, called “easy bleeding”, has recently been discovered to be specific of several sawfly species (Hymenoptera: Tenthredinidae). When larvae of easy bleeding species are bothered from other animals, they quickly form drops of hemolymph along the cuticle, which is highly deterrent in feeding bioassays. This phenomenon has therefore considered as a defence strategy mainly against insect predators. In this work we hypothesise that easy bleeding sawflies might have stronger immune mechanisms than non-bleeding ones, in order to survive after hemolymph exposure and a possible microorganismal challenge. Investigations were made with phase contrast microscopy, immunohistochemistry and lectin labelling assays. An easy bleeder, Athalia rosae, has very well defined plasmatocytes, granular cells, a few big globular oenocytoids and a forth cell type similar to the spherulocytes of Manduca sexta. A non-easy bleeder, Gilpinia hercyniae (belonging to the Diprionidae family), has a lower hemocyte concentration and only plasmatocytes, granular cells and oenocytoids. Moreover, semi-thin sections of 15 sawfly species were made in order to perform histological investigations. Several monoclonal antibodies specific for M. sexta hemocytes and fluorescent lectins were then tested. Surprisingly, the results show that two of them (MS39 and MS77) bind specifically to cuticular layers, similar as the binding pattern of selected lectins. These results can now provide a powerful tool in order to explain the easy bleeding phenomenon at molecular level. This research was supported by the EU network Insect Chemical Ecology (INCHECO) - Contract n. HPRN-CT-1999-00054 A14: XIX Congresso Nazionale Italiano di Entomologia, Catania (Italy) 10-15 June 2002 Stimoli chimici nelle interazioni tra individui di Ceratomegilla undecimnotata (Schneider) (Coleoptera, Coccinellidae) in fase di aggregazione 1Boddi G., 2Rosi M.C., 1Angeli S., 1Petacchi R. and 2Sacchetti P. 1 Scuola Superiore Sant’Anna di Studi Universitari e Perfezionamento, Pisa, Italia 2 Dipartimento di Biotecnologie Agrarie, Università di Firenze, Italia Chemical stimuli involved in interactions among individuals of Ceratomegilla undecimnotata (Schneider) (Coleoptera Coccinellidae) in aggregations. - Among Coccinellids, Ceratomegilla (=Semiadalia) undecimnotata is one of the most studied species because it migrates in large number towards overwintering sites (located both in hill or mountain) before diapausing. In this paper the results of bioassays carried out both in two-way olfactometer and arena are reported, in order to show the possible role of volatile and non volatile chemicals on such species. In all the experiments aggregating individuals of C. undecimnotata, collected from overwintering sites located either in France or Italy, have been used. In the olfactometer bioassays we evaluated the amount of time spent in each of the two arms; one arm was injected with pure air, while the other was injected with pure air passing through an aggregation of males or females. In arena three set of experiments have been performed. In the first two an heptane extract of both sexes C. undecimnotata elytra was applied either on glass and paper. In the third experiment the elytra were used as a dummy after having been washed with solvents characterised by different polarity. In all bioassays the behaviour of single individuals was observed for the described treatments, evaluating the time spent in the treated arena and the number of individuals that showed arrestment on the same arena. Data analysis showed that in olfactometer females spent a significantly lower time in the arm with aggregated males. In the arena bioassays males spent a longer time over the glass slide treated with male elytra extract, while in the paper experiments a lower number of females showed arrestment over the female extract treated areas. In the dummy experiments, a higher number of females were found over elytra treated with alcohol compared with those washed with chloroform and with those unwashed. Although these results are preliminary and will need more research, it is possible to show, for the first time in this species, how chemical stimuli can play a role in the interaction among aggregating individuals. Key words: Semiadalia, behaviour, olfactometer, arena. A13: XIX Congresso Nazionale Italiano di Entomologia, Catania (Italy) 10-15 June 2002 L’aggregazione in Ceratomegilla undecimnotata Schn. (Coleoptera: Coccinellidae): indagini preliminari sulla caratterizzazione chimica degli idrocarburi cuticolari e dei composti volatili presenti 1Angeli S., 1Boddi G., 2Dani F., 3Rosi C., 3Sacchetti P. and 1Petacchi R. 1 Scuola Superiore Sant’Anna di Studi Universitari e Perfezionamento, Pisa, Italia 2 Dipartimento di Biologia Animale e Genetica, Università di Firenze, Italia 3 Dipartimento di Biotecnologie Agrarie, Università di Firenze, Italia Aggregation in Ceratomegilla undecimnotata Schn. (Coleoptera: Coccinellidae): preliminary analyses on chemical characterisation of cuticular hydrocarbons and volatile compounds. - Some species of ladybirds aggregate during summery and winter period. This phenomenon has been object of numerous studies in order both to comprise its ethological meaning, and -potentially- to improve the employment of these predators in organic farming techniques. Induction to summer migration seems correlated to variation in photoperiod, temperature and hormonal pattern while orientation towards the tops of mountains appears more related to visual stimuli. In this work, authors report some results in the attempt to identify contact and volatile compounds involved in the aggregation phenomena of Ceratomegilla undecimnotata. Analyses were made in GC-MS both of heptane extracts and volatiles collected using SPME (solid phase micro-extraction) technique. These experiments allowed us to identify hydrocarbon profiles of aggregate individuals. In particular approximately 28 compounds have been described, the most abundant of which are saturated and monounsaturated hydrocarbons with long chains from 23 to 33 carbons. No significant differences in hydrocarbons composition were found between males and females and also between an Italian and French population. Analyses of head spaces in SPME have shown low concentrations of volatile compounds, among which the presence of palmitic aldehyde is interesting. These data, although preliminary, are the first GC-MS investigation on potential pheromones in aggregates of C. undecimnotata and will be used in order to devise behavioural bioassays and to establish which is the function of identified compounds. Keywords: pheromones, hydrocarbons, behaviour, SPME, GC-MS. A12: Green-Tech® 2002 and 5th European Symposium of Industrial Crops and Products, The Netherlands, 24-26 April 2002 Effect of environmental on crop production and indigo yield in Polygonum tinctorium in Central Italy 1Angelini L., 1Tozzi S., 1Angeli S., and 2John P. 1 Dipartimento di Agronomia e Gestione dell’Agroecosistema, The University of Pisa, San Michele degli Scalzi 2, 56100 Pisa, Italy 2 Department of Agricultural Botany, School of Plant Sciences, Plant Science Laboratories, The University of Reading, Reading RG6 6AS, UK In order to introduce introduce indigo-producing crops into European agriculture, research have been undertaken within a EU-funded research project (SPINDIGO). The current European market for indigo is supplied entirely by the synthetic product and the aim of this project is that a significant proportion of the European market will be met by natural indigo generating a useful income to the rural sector. Although natural dyes are rarely used in modern dyeing except by specialist fashion companies (e.g. to dye denim jeans), an increasing trend in textile industries is toward replacing synthetic products by natural ones and it has prompted research toward natural plant pigments. Natural indigo is indistinguishable from its synthetic counterpart: by extracting indigo from plants, blue dyes can be produced from a totally renewable resources. Natural indigo can be obtained from a wide range of tropical, sub-tropical and temperate plants from many different species, genera and families (Cardon and Chatenet, 1990; Stoker, 1997; Balfour-Paul, 1998). In tropical and sub-tropical areas, the plants most widely used for indigo production were Indigofera spp. (Martin-Leake, 1975). In temperate climates, the most commonly used species was Isatis tinctoria or dyers woad. Polygonum tinctorum (Ai), known as “Chinese indigo”, had been used in Japan, China and Russia for large-scale indigo production (Perkin and Everest, 1918; Hill, 1992). Polygonum tinctorum is a subtropical annual herbaceous plant, belonging to the family Polygonaceae with large dark bluish-green and it was introduced in Europe at the end of the eighteenth century. It has some glycosides as secondary metabolites, the major one is a colourless glucoside called indican (indoxyl b-D-glucoside). When the plant leaves are put in water, indican is extracted and it is degraded to indoxyl and glucose. A dimerization of this indoxyl by air oxidation follows and indigo is formed: this is the compound that is commonly used as a blue dye since ancient time (Minami, 2000). Although indigo can be obtained from plants in large amounts, the decrease of the natural indigo use had been related to the fact that the process is not well understood. The precursor pathway may also be relied to the intensity of sunlight, which has been found to play a vital role in the production of indigo yield (Stoker et al., 1998). Polygonum tinctorum is a relatively new species for Italy and research is needed to improve cultivation methods in order to produce plant material with the highest quality and the lowest production costs. Research has been carried out within Spindigo project, closely cooperating with other European scientific institutions, in order to better understand the effect of environment on crop production and indigo yield. Three Polygonum tinctorum populations and three sowing times were tested under field conditions in Central Italy in a split plot experimental scheme with four replications. To evaluate maximum crop yield, plants were maintained under optimum nutrient and water supply conditions. The changes in air temperature, rainfall, global radiation and photosynthetically active radiation (PAR ) were recorded along the growing season. The productive determinations (fresh and dry leaves, stems and total plant yield in t ha-1; leaves:stems ratio and Harvest Index) were performed on a minimal area of 2 m2 per plot on the middle rows. Plants had been sampled prior of each harvest and fresh leaves were extracted in an adequate water volume. Indigo content was determined spectrophotometrically at 600 nm. Indican content was assessed by HPLC coupled with an ELS (Evaporating Light Scattering) detector and determined against a calibration curve obtained with an indican standard solution. Changes in PAR had been recorded and had been plotted against variation in leaves and indigo yield. A11: Proc. of  the VII  European Symposium on Insect Taste and Olfaction, Villasimius (CA, Italy), 22-28 September 2001 Purification of Chemosensory Proteins (CSP) in Locusta migratoria 1Ban L., 2Brandazza A., 3Angeli S., 1Zhang L., 1Yan Y. H. and 2Pelosi P. 1 Department of Entomology, China, Agricultural University, Beijing 100094, China 2 Dip. Chimica e Biotecnologie Agrarie, via S. Michele, 4, 56124 Pisa, Italy 3 Scuola Superiore S. Anna; 56124 Pisa, Italy Locusta migratoria represents the major pest in Asia,producing enourmous damage to crops. Based on our previous information on the related species Schistocerca gregaria, we have started investigating the biochemical mechanisms of chemical communication in this insect, focusing our attention on soluble proteins of chemosensory organs. Electrophoretic analysis both in native and denaturing conditions of crude extracts of antennae, tarsi and palpi, revealed the presence of acidic proteins of low molecular weight. Some of these bands were present only in antennal extracts, while others were present also in tarsi and palpi. These proteins were purified by gel filtration chromatography, followed by preparative electrophoresis in native conditions. N-terminal sequences have indicated such proteins to be members of the family of Chemosensory Proteins (CSPs). Amplification of the relative genes by PCR with degenerate primers, cloning and sequencing allowed the establishment of nucleotide sequences encoding several members of this group. Their derived amino acid sequences share about 90% of their residues with CSPs of S. gregaria and between 50 and 60% with those described in L. migratoria. Polyclonal antibodies have been raised in mice agains the protein purified from the antennae and are currently used in immunocytochemistry experiments to label its sites of production and accumulation within the chemosensory organs.  Western blot experiments have been used to monitor the expression of these proteins during the developmental stages of the insect.  As in the case of S. gregaria, a similar protein was purified from the wings, where it is associated with organic compounds of low volatility, whose structures are currently being investigated. A10: Proc. of the VII European Symposium on Insect Taste and Olfaction, Villasimius (CA, Italy), 22-28 September 2001 Structure and function of Chemosensory Proteins (CSP) in insects 1Picone D., 2Crescenzi O., 3Scaloni A., 4Angeli S. and 5Pelosi P. 1 Dipartimento di Chimica, University Federico II, 80100 Napoli, Italy 2 Dipartimento di Chimica Organica e Biochimica, University Federico II, 80100 Napoli, Italy 3 I.A.B.B.A.M. - Centro Internazionale Servizi di Spettrometria di Massa, CNR, via Argine 1085, 80147 Napoli, Italy 4 Scuola Superiore S. Anna, 56124 Pisa, Italy 5 Dip. Chimica e Biotecnologie Agrarie, via S. Michele 4, 56124 Pisa, Italy Chemosensory proteins (CSPs) is the name given to a class of small soluble proteins found in chemosensory organs of insects. Some charcteristics suggest that they represent a second class of binding proteins for chemical stimuli. These proteins are slighly smaller than OBPs, generally, but not always acidic and very soluble. Like OBPs, their concentration in chemosensory structures is extremely high. The first protein of this class was identified in Drosophila, later members were described in most insect Orders, from the very primitive Phasmids to Lepidoptera and Hymenoptera. Their amino acid sequences are better conserved than OBPs, with an average of about 40% of common residues, even between very distant species. In apparent constrast with this charactersistics, polyclonal antibodies raised agaist CSPs of different species do not cross-react. Two pairs of nearly adjacent conserved cysteines are linked by two disulphide bridges, that apparently do not contribute to the three-dimensional structure of the molecules. Immunocytochemical experiments have indicated that they are highly concentrated in the sensillar limph of chemosensory hairs. Members of this family have been identified in the ejaculatory bulb of Drosophila, the same organ secreting the sex pheromone, and in subcuticular cells. The protein extracted from this latter source contains, as endogenous ligands, long-chain hydrocarbons. These observations seem to indicate that CSPs might be involved, not only in the perception, but also in the release of pheromones. We have expressed one of the CSPs of Schistocerca gregaria and the Drosophila OS-D in bacterial systems, in order to produce antibodies for immunocytochemical localisation and to obtain enough protein for structural studies. The CSP of S. gregaria has also been obtained in N-15 enriched form and used in NMR conformational studies. We report preliminary data on the three-dimensional structure of this protein, composed of six alpha-helices. A9: XXI International Congress of Entomology, Foz do Iguassu (Brazil), 20-26 August 2000 Chemosensory proteins in Schistocerca gregaria Angeli S. Scuola Superiore S. Anna, 56124 Pisa, Italy The desert locust Schistocerca gregaria is one of the major crop pest in Africa and the Middle East. Recently, chemoreception of the species has been investigated with regard to ecological, behavioral and physiological aspects. An aggregation-maturation pheromone and an egg-pod attraction pheromone have been isolated and characterized and the fine-structure of antennal sensilla was studied. Here we describe the biochemical characterization and the immunocytochemical localization of the first family of chemosensory proteins (CSP) expressed in Orthoptera. Three acidic and soluble proteins of 14 kDa were purified, each from distinct chemosensory organs (antennae, tarsi, and labrum) of crowed-reared locusts. The N-terminal sequences were obtained and were found to be similar to each other. They exhibit significant homology with a sub-class of putative chemoperception proteins, expressed from Phasmids to Diptera. A large quantity of the tarsi-specific protein was purified from females and used for chemical characterization, polyclonal antibody preparation and binding assays. In TEM-immunocytochemical of chemosensory organs, the antibody labelled specifically the receptor lymph of contact, terminal pore sensilla. Therefore we designed degenerated primers from the N-sequence and were able to amplify, by RT-PCR, the nucleotide sequences from mRNA isolated from the tarsi of one female. After cloning and sequencing, the sequence analysis showed five isoforms, all encoding for proteins 109 amino acids long. Comparison of peaks of the mass-spectrometry analysis of the native protein with the theoretically calculated molecular mass from the five cDNAs, showed a completed overlap, suggesting that this isoforms are all expressed in single animals without any post-translaction modifications. Therefore, we decided to express an isoform in E. coli. The chemical analysis of all soluble recombinant protein showed no differences with the native protein in terms of PAGE analysis, mass-spectrometry, disulfide bridges dispositions and circular dichroism. Crystallization experiments and ligand-binding assays of the recombinant from are now in progress. Although the specific function of these proteins is still unknown, their high degree of homology with the 4 conserved cysteines, and their expression in chemosensory organs of large number of species have suggested a role in chemoreception. Recently a member of this family was found to bind the Drosophila pheromone vaccenyl acetate. In Orthoptera, our experimental data suggest a role for these proteins in contact chemoreception, for instance in response to chemicals on surfaces like cuticular hydrocarbons. No binding of radioactively labelled glucose or bicarbonate was detected. Index terms: Schistocerca gregaria , chemoperception, chemosensory proteins. A8: Proc. of VI European Symposium on Insect Taste and Olfaction, Tutzing (Germany), 5-10 September 1999 Immunocytochemical distribution of a chemosensory protein of Carausius morosus Minnocci A., Angeli S., Monteforti G., and Petacchi R. BioLabs - Scuola Superiore Sant'Anna, Via Carducci 40 I- 56127 Pisa (Italy) Isolation of soluble low molecular weight proteins in antennae, tarsi and labrum of S. gregaria, clearly showed the existence of a new class of Chemosensory protein (Angeli et al., 1999) likely to be involved in chemical communication. Similar proteins was also demonstrate to be present in Phasmids (Mameli et al., 1996), in particular in Carausius morosus (Tuccini et al., 1996). These proteins belong to a family, whose first member was identified in the antennae of D. melanogaster and named OS-D-like, described in different Orders of insects, from Phasmids to Lepidoptera and relatively well conserved across evolution. Polyclonal antibodies used to immunodetect these proteins in Schistocerca gregaria, showing that it is localized in contact chemosensory hairs of tarsi, antennae and labrum nor in olfactory sensilla.  We are currently investigating the ultrastructural localization of a CSP in the different sensory organs of this stick insect. Angeli S., Ceron F., Scaloni A., Monteforti G., Minnocci A., Petacchi R. and Pelosi P. 1999. Structural characterisation, cloning and immunocytochemical localisation of Chemoreception proteins from Schistocerca gregaria. (in press) Mameli M., Tuccini A., Mazza M., Petacchi R., Pelosi P., 1996. Soluble proteins in chemosensory organs of Phasmids. Insect Biochem. Mol. Biol. 26: 875-882. Tuccini A., Maida R., Rovero P., Mazza M., Pelosi P.,1996. Putative odorant-binding proteins in antennae and legs of Carausius morosus. Insect Biochem. Mol. Biol. 26: 19-24. A7: Proc. of the VI European Symposium on Insect Taste and Olfaction, Tutzing (Germany), 5-10 September 1999 Major chemosensory proteins of Phasmids 1Marchese S., 1Brandazza A., 1Ceron F., 2Angeli S., 2Mionnocci A., 3Mazza M. and 1Pelosi P. 1 Dip. Chimica e Biotecnologie Agrarie, via S. Michele 4, 56124 Pisa, Italy 2 Scuola Superiore S. Anna;, 56124 Pisa, Italy 3 Dip. Scienze Uomo Ambiente, Pisa; Italy The chemosensory organs of Phasmids express very high levels of soluble proteins of the class referred to as OS-D like or CSP (Chemosensory Proteins). Proteins of this class are also expressed in other orders of insects, such as Lepidoptera, Diptera, Imenoptera, Orthoptera and Blattoidea.  Here we describe the cloning and sequencing of some members of this class of proteins in the Phasmid species Eurycantha calcarata and Carausius morosus. Mature polypeptides are 100 to 117 residue long and contain four conserved cysteines. The same proteins or similar ones appear to be expressed in the antennae, in the tarsi and in other parts of the body. Polyclonal antibodies prepared against purified proteins from both species, as well as against a similar protein of S. gregaria, indicated no cross-reactivity, despite the sequence similarities between the three proteins. Each antiserum, however, stained bands of similar molecular weight in different chemosensory organs of the same species. A comparison between all the known sequences of CSPs in different orders of insects is also presented and discussed. A6: Proc. of the VI European Symposium on Insect Taste and Olfaction, Tutzing (Germany), 5-10 September 1999 Bacterial expression of a chemosensory protein (CSP) of Schistocerca gregaria 1Angeli S., 2Marchese S., 2Brandazza A., 1Petacchi R. and 2Pelosi P. 1 Scuola Superiore Sant'Anna 56124 Pisa, Italy 2 Dip. Chimica e Biotecnologie Agrarie Via S. Michele 4, 56124 Pisa, Italy We have recently isolated from chemosensory organs of Schistocerca gregaria, several polypeptides of low molecular weight (around 14 kDa) and acidic nature. Their amino acid sequences revealed that all the isolated proteins belong to the same sub-family, whose first member was identified in Drosophila melanogaster and named OS-D. The five gene products, identified in both sexes, contain 109 amino acids, are very similar to each other and present four cysteine residues involved in two disulphide bridges, as demonstrated by mass spectrometry. This technique also confirmed the calculated molecular weights and excluded the presence of additional post-translational modifications.  Here we report the cloning of one of these genes into a pET-5 vector and the expression, purification and characterisation of the recombinant protein. The protein is expressed with an additional residue of methionine at the N-terminus, as the sole difference with the native polypeptide. Yields were in the range of 50-70 mg of soluble protein per litre of bacterial colture. Purification was accomplished by standard methods, involving ammonium sulphate fractionation, ion-exchange chromatography and gel filtration. The recombinant protein showed chemical properties identical with those of a native sample. A5: XIII Congress European Chemoreception Research Organization (XIII ECRO), Siena, Italy, 8-12 September 1998 Cloning of soluble chemo-sensory proteins in Phasmids 1Marchese S., 1Ceron F., 1Angeli S., 2Mazza M., 3Picimbon J.F., 3Leal W.S. and 1Pelosi P. 1 Dip. Chimica e Biotecnologie Agrarie, Via S. Michele 4, 56124 Pisa, Italy 2 Dip. Scienze Uomo Ambiente, Pisa, Italy 3 NISES, Tsukuba, Japan Based on our previously reported N-terminal sequences, we have amplified by PCR, cloned and sequenced genes encoding soluble proteins in chemosensory organs of two species of Phasmids, Eurycantha calcarata and Carausius morosus. Several related sequences have been amplified in both species. Here we report the complete amino acid sequences of three proteins expressed in the antennae of E. calcarata and one in the tarsi of C. morosus. They consist of polypeptides of 100-117 residues, bearing four conserved cysteines. All exhibit significant similarity with a recently discovered class of chemosensory proteins, including Drosophila melanogaster OS-D, Cactoblastis cactorum CLP-1 and five members of Schistocerca gregaria (Angeli et al., ECRO XIII, 1998).  One of the E. calcarata sequences is identical in its first 33 residues with that determined by direct Edman degradation on the purified protein, while in C. morosus the sequence deduced from the cDNA is markedly different from the N-terminal information obtained on the isolated protein. A comparison between proteins of this class across different orders shows that they are well conserved, when compared with OBPs.  Polyclonal antibodies prepared against purified proteins from both species, as well as against a similar protein of S. gregaria, indicated no cross-reactivity, despite the similarity between the three proteins. Each antiserum, however, stained bands of similar molecular weight in different chemosensory organs of the same species.  To test their hypothesised role in carbon dioxide sensing, we performed binding experiments with radioactive bicarbonate, but results were negative in the conditions employed. Being the proteins of this class probably involved in chemoreception, as suggested by their tissue localisation, we propose the general name of CSP (ChemoSensory Protein). A4: XIII Congress European Chemoreception Research Organization (XIII ECRO), Siena, Italy, 8-12 September 1998 Chemosensory proteins(CSP) of Schistocerca gregaria 1Angeli S., 2Ceron F., 3Scaloni A., 3Monti M., 1Petacchi R., 1Minnocci A., 1Monteforti G. and 2Pelosi P. 1 Scuola Superiore S. Anna, Pisa, Italy 2 Dip. Chimica e Biotecnologie Agrarie, via S. Michele 4, 56124 Pisa, Italy 3 CISESMA - IABBAM, CNR, Napoli, Italy Soluble proteins of chemosensory organs have been described in several orders of insects and include pheromone- and odorant-binding proteins, as well as other polypeptides of similar structure probably involved in gustatory perception. Information of this type is not available for Orthoptera, despite the high economical interest of several species of this order. In our search for OBP-like proteins in Schistocerca gregaria, we have isolated several polypeptides of low molecular weight (around 14 kDa) and acidic nature from chemosensory organs, such as antennae, tarsi and upper labrum. N-terminal sequencing revealed that all the isolated proteins belong to the same sub-family, including Drosophila melanogaster OS-D and Cactoblastis cactorum CLP-1. By using degenerate primers and PCR, we have amplified, cloned and sequenced genes encoding five members of these proteins in tarsi of both sexes. The five gene products contain 109 amino acids, are very similar to each other and present four cysteine residues probably involved in two disulphide bridges. Calculated molecular weights are identical to those measured by mass spectrometry, indicating the absence of other post-translational modifications.  Polyclonal antibodies, raised against the purified protein, have been used in immunocytochemical localisation. Both in antennae and tarsi, single-pore gustatory sensilla are labelled, but not olfactory sensilla. Labelling is limited to the sensillar lymph.  The same polyclonal antibodies have indicated in Western blot experiments that the synthesis of these proteins, both in antennae and tarsi, starts at the second instar and reaches high levels of expression at the third instar. A role in carbon dioxide sensing has been suggested for the homologous protein of C. cactorum. Binding of labelled bicarbonate, however, has been unsuccessful with the proteins of S. gregaria, while other prospective ligands are being tested. Key-words: Taste, Sequences, Sensilla, Binding, Immunocytochemistry. A3: XVIII Congresso Nazionale Italiano di Entomologia (XVIII CNIE), Maratea (Italy), 21-26 June 1998, spoken presentation Studi sulla percezione degli odori in Schistocerca gregaria (Orthoptera: Acrididae) 1Angeli S., 1Minnocci A., 1Petacchi R., 2Monti M., 2Scaloni A., and 3Pelosi P. 1 Scuola Superiore di Studi Universitari e di Perfezionamento S.Anna, Pisa 2 Centro Internazionale Servizi di Spettrometria di Massa, CNR, Napoli 3 Dipartimento di Chimica e Biotecnologie Agrarie, Università di Pisa La locusta del deserto Schistocerca gregaria è un insetto polimorfico che presenta due fasi: quella solitaria e quella gregaria,quest'ultima particolarmente dannosa in agricoltura. Il passaggio fra le due fasi è reversibile in ogni stadio di sviluppo ed è mediato da sostanze chimiche generalmente volatili appartenenti alla classe dei feromoni. Altri composti volatili, quali l'anisolo e molecole di struttura simile, sono stati riconosciuti come feromoni di aggregazione e sono responsabili della formazione dei grossi sciami devastatori. La comprensione dei meccanismi della comunicazione chimica e la possibilità di modificarla sono al centro di numerose ricerche finalizzate a combattere l'insorgenza di queste periodiche infestazioni. L'indagine biochimica ha portato alla identificazione, purificazione e parziale caratterizzazione di proteine solubili, di basso peso molecolare, probabilmente coinvolte nella percezione di stimoli chimici. In particolare, i tarsi e l'apparato boccale esprimono abbondanti proteine di circa 14 kDa, le cui sequenze amminoacidiche N-terminali risultano simili a proteine della stessa classe da noi precedentemente purificate in alcune specie di Fasmidi, nonché a quelle descritte da altri ricercatori in Ditteri, Imenotteri e Blattoidei. Al fine di chiarire il ruolo di tali proteine nella chemiorecezione, è opportuno studiarne la capacità legante verso gli odori e definirne la localizzazione tissutale. Per affrontare quest'ultimo problema, mediante un approccio di immunoistochimica, sono stati prodotti anticorpi policlonali contro la proteina di 14 kDa purificata dai tarsi. Saggi di immunoblotting, effettuati con tale antisiero parzialmente purificato, hanno confermato la esclusiva espressione della proteina nei tarsi e nell'apparato boccale, come già indicato dalle analisi elettroforetiche e dalle informazioni di sequenza. L'indagine morfologica è stata effettuata attraverso tecniche di microscopia elettronica a scansione e a trasmissione. I risultati finora ottenuti, confermando i dati in letteratura, mostrano diversi tipi di sensilli chemiorecettoriali, in particolare sulle antenne, nella porzione apicale dei palpi mascellari e labiali, sulla superficie interna del labbro superiore e nella porzione ventrale dei pulvilli di ciascun paio di zampe. Questi risultati costituiscono i primi dati sui meccanismi biochimici della chemiorecezione negli Ortotteri e potranno aprire la strada a nuove strategie di lotta contro l'insetto studiato. A2: Proc. of the V European Symposium on Insect Taste and Olfaction (V ESITO), Villasimius (Italy), 20-26 September 1997 Morphological and biochemical investigation of chemosensory organs in Phasmidae 1Mameli M., 2Monteforti G., 2Angeli S., 2Minnocci A., 2Petacchi R., 1Tuccini A., and 1Pelosi P. 1 Dipartimento di Chimica e Biotecnologie Agrarie - Univ. di Pisa - Italy 2 Scuola Superiore di Studi Universitari e Perfezionamento Sant'Anna - Pisa - Italy Phasmids are frequently used as model for many biological experiments in insects. In fact they are simple to rear and their dimensions are usually enough big to allow physiological and biochemistry assays. Moreover they are a very primitive heterometabolous order of insect, in which are known both parthenogenetic and amphigonic species. The chemical communication between sexes has still not been elucidated, but the fact that they have a very long life cycle give suggestion for the presence of a specialized olfactory system. All these characteristics put this order in a different position in respect to the holometabolous order (e.g. Lepidoptera), where chemosensory transduction is usually studied. We have morphologically and biochemically investigated several Phasmid species in order to draw a more complete pattern of information on their chemosensory system and to give some insights on the phylogenetic evolution in odour perception. From the antenna, legs and mouthparts, that are known in insect to be involved in chemosensory processes, we have purified some small, soluble proteins (Mameli et al., 1996) that can be divided mainly in two subclasses and in another species, Sipyloidea sipylus, another protein, antennal specific, that constitutes the only member of a third subclass. In parallel with the biochemistry identification of proteins we have performed scanning and transmission electronic microscopy to study olfactory sensilla structure and function, in the parthenogenetic females of Carausius morosus and both sexes of Eurycantha calcarata and Extratosoma tiaratum. The antenna of these species have in commune a characteristic glandular structure, called bump, pierced by the sectretory ducts of numerous gland cells on dorsal surface and a distribution of sensilla that shows a very marked increasing in the second half of flagellum, in agreement with its huge dimension that can arrive to more than 4 cm. In Carausius morosus antennae we have identified different types of mechanosensitive sensilla (peg-like. Bristle like and campaniform), a small number of poreless thermohygrosensitive and three types of chemosensilla. The fist type is a gustative hair-shaft with fluted aporous walls and spatulate tip. The cross sections of this hair shows a double lumen with five unbranched dendrites inside the innervated one (5x2+1 or 7x2+3). Another is an “hair-like” olfactory sensillum with blunt tipped and smooth walls, perforated by pores and with spoke channels. The number of sensory cells is comprised between four and six. The third type is a “peg-like” sensillum, less abundant and smaller, with a thin cuticular wall and high density pores. The cellular components of all these sensilla consist of a greater and variable (5-22) number of sensory neurons. The dendritic segments are first enclosed in a common sheath and later divided into numerous branches. In this species two soluble proteins have been purified, antennal and leg specific (Tuccini et al., 1996; Mamenli et al., 1996). One is a 19 kDa protein that shares a 30 % amino acid identity with Drosophila melanogaster OS-D or A 10 (McKenna et al., 1994; Pikielny et al., 1994) and belong to the first sub-class of putative OBPs (Odorant Binding Proteins) of Phasmids. The second is a 15 kDa protein that can be grouped in the second sub-class and that has a greater similarity with OS-D or A-10. In the two amphigonic species studied, Eurycantha calcarata and Extratosoma tiaratum, there is a dimorphism between sexes in the flagellum, but the number and the types of sensilla do not differ, implying a similar function in both sexes. In these species the last segments of the antenna, that generally in insects are more involved in the chemosensory perception, reach a density of 1000 sensilla/mm2 and present three different sensilla types in E. calcarata and four in E. tiaratum. In E. calcarata antennae we have identified also the presence of sensilla coeloconica-like and this is interesting because in both species we have purified 14 kDa soluble proteins from antennae and legs that belong to the second sub-class, similar to OS-D or A 10 and this protein, in Drosophila, has been localized in sensilla coeloconica. All the sub-classes of soluble proteins identified and purified in Phasmids don’t show a primary structure similarity with the know Lepidopteran OBPs, and this is probably due to the evolutionary distance between the two orders. A1: Proc. of the V European Symposium on Insect Taste and Olfaction (V ESITO), Villasimius (Italy), 20-26 September 1997 Chemioreception in Schistocerca gregaria (Orthoptera, Acrididae) 1Angeli S., 2Long Z., 1Minnocci A., 1Petacchi R., and 3Pelosi P. 1 Scuola Superiore di Studi Universitari e Perfezionamento Sant'Anna - Pisa - Italy 2 China Agricoltural University - Beijing - PRC 3 Dipartimento di Chimica e Biotecnologie Agrarie - Pisa - Italy The desert locust Schistocerca gregaria is one of the major threats to crops in Africa and Asia. The onset of the gregarious phase is triggered by volatile chemicals. Interphering with the olfactory perception of this species could therefore control aggregation and thus reduce its devastation effects. Biochemical aspects of olfaction have been investigated in several insect species, belonging to the orders of Lepidoptera, Diptera, Hymenoptera and Phasmatodea, but no data have been reported in Orthoptera. Most of the research in other orders has been concerned with soluble proteins, present at high concentration in the lymph of sensory hairs and called OBPs (odorant-binding proteins). These proteins are subdivided in two classes, pheromone-binding proteins (PBPs) and general odorant-binding proteins (GOBP). Here we describe the purification of putative OBPs from sensory parts of S. gregaria. A first screening of soluble proteins in different parts of the body, performed by SDS-PAGE, revealed the presence of low molecular weight bands in all the sensory parts (antenna, legs, mouth), but not in the other extracts. The same extracts, when analyzed by native PAGE, showed to contain fast migrating acidic proteins. No significant differences were observed in the electrophoretic patterns between sexes.  One of the most abundant of them, with an apparent molecular weight of 14 kDa, was purified from the tarsi, using standard chromatographic techniques. Its N-terminal sequence (NH2-EEKYTTKYDNVNLDEILANDRLLNKYVQQLLEXD-) showed significant similarity with Drosophila OS-D (48%) and other proteins o same size purified from several Phasmid species. A very similar protein was also purified from the labrum by preparative electrophoresis. Both proteins migrate on SDS-PAGE with the same apparent molecular weight and show the same isoelectric point (5.1) on a IEF gel in a gradient of Ampholines. An affinity column, prepared with the antiserum against a 15 kDa of Eurycantha calcarata, was effectively used for purifying the proteins of S. gregaria. A specific degenerated primer has been synthesized on the basis of N-terminal sequence and is currently used for amplifying the gene encoding this protein. A the same time, a morphological investigation, performed at the alectron scanning microscope, revealed several types of chemiosensilla in the same organs expressing the 14 kDa protein.

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