ABSTRACTS Iguassu Falls 04-08 July 2016 GENERAL COORDINATION Paulo Henrique Gorgatti Zarbin – UFPR,

LOCAL ORGANIZING COMMITTEE (BRAZIL) Antonio Euzébio Goulart Santana – Universidade Federal de Alagoas, Brazil Camila Borges da Cruz Martins - Universidade Federal do Paraná, Brazil Carla Fernanda Fávaro - Universidade Estadual de Santa Cruz, Brazil José Mauricio Simões Bento – ESALQ/Universidade de São Paulo, Brazil Maria Fátima das G. F. da Silva – Universidade Federal de São Carlos, Brazil Miryan Denise Araújo Coracini - Universidade Estadual do Oeste do Paraná, Brazil

ISCE REPRESENTATIVE Ann Marie Ray - Xavier University, USA Kenneth F. Haynes - University of Kentucky, USA Walter Soares Leal, University of California, USA

ALAEQ REPRESENTATIVE Jan Bergmann - Pontificia Universidad Católica de Valparaiso, Chile Marcelo Gustavo Lorenzo - Fundação Oswaldo Cruz, Brazil Pablo Guerenstein - Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina

SCIENTIFIC COMMITTEE Andrés Gonzáles Ritzel - Facultad de Química, Uruguay Angel Guerrero - Consejo Superior de Investigaciones Cientificas, Spain Alvin Kah Wei Hee - Universiti Putra Malaysia, Malaysia Baldwyn Torto - International Centre of Physiology and Ecology, Kenya Christer Löfstedt - Lund University, Sweden Eraldo Rodrigues de Lima - Universidade Federal de Viçosa, Brazil Jeffrey Aldrich - University of California, USA Paulo Henrique Gorgatti Zarbin - UFPR, Brazil Stefano Colazza - Universita degli Studi di Palermo, Italy Stefan Schulz - Technische Universitat Braunschweig, Alemanha Tetsuo Ando - Tokyo University of Agriculture and Technology, Japan Thomas C. Baker - Pennsylvania State University, USA

OBSERVATION

The writings, concepts and opinions expressed in the abstracts of this publication are responsibility of their authors. The Organizing Committee doesn’t assume the responsibility for data and conclusions punctuated by the authors. SUMMARY

APPLIED USES OF SEMIOCHEMICALS A METHOD FOR CREATING HIGHLY CONCENTRATED ODOR PLUMES PASSIVELY EMANATING FROM ODOR DISPENSERS ...... 19 ATTRACTION OF SPODOPTERA FRUGIPERDA MALES FROM DIFFERENT REGIONS OF ARGENTINA TOWARDS DIFFERENT FORMULATIONS OF THE FEMALE SEX PHEROMONE, IN THE LABORATORY AND IN THE FIELD ...... 20 BRINGING NOVEL SEMIOCHEMICAL FORMULATIONS TO THE MARKET ...... 21 DISCRIMINATION OF INFESTED COTTON SQUARES BY FEMALES OF ANTHONOMUS GRANDIS BOHEMAN (COLEOPTERA: CURCULIONIDAE)...... 22 EVALUATION OF SEX ATTRACTANT PHEROMONE FROM RICE STALK STINK BUG TIBRACA LIMBATIVENTRIS IN LABORATORY AND FIELD ...... 23 EAVESDROPPING ON THE VICTIM: A GENERALIST PREDATOR UTILIZES AN ALARM PHEROMONE TO LOCATE AN INVASIVE PEST ...... 24 FROM GENE DISCOVERY TO METABOLIC ENGINEERING: PRODUCTION OF PHEROMONES IN AND CELL FACTORIES ...... 25 IDENTIFICATION OF VOLATILE COMPOUNDS OBTAINED FROM LARVAE- INFESTED AND ROTTEN FRUIT ATTRACTIVE FOR THE FRUIT FLY PARASITOID DIACHASMIMORPHA LONGICAUDATA ASHMEAD (: BRACONIDAE) ...... 26 MASS-TRAPPING OF CODLING MOTH MATED FEMALES IN DISRUPTED ORCHARDS ...... 27 MATING DISRUPTION 50 YEARS ON: PHEROMONES COMING OF AGE - FINALLY!...... 28 MAXIMIZING INFORMATION YIELD AND IMPROVING THE PRECISION OF PHEROMONE-BAITED MONITORING TRAPS ...... 29 NANOFIBERS ARE EXCELLENT DISPENSERS FOR GRAPHOLITA MOLESTA SEX PHEROMONES SUITABLE AS ATTRACTANTS IN BRAZILIAN PEACH ORCHARDS ...... 30 PROXIMATE MECHANISMS OF JASMONIC ACID-INDUCED DEFENSES IN SUGARCANE AGAINST A GENERALIST AND SPECIALIST HERBIVORE ...... 31 REDUCE AN APPLICATION OF INSECTICIDES FOR THE USE OF PHEROMONES IN SOYBEANS ...... 32 SEMIOCHEMICALS INFLUENCING THE BEHAVIOR OF THE LADY TENUISVALVAE NOTATA (MULSANT) (COLEOPTERA: COCCINELLIDAE) ...... 33 SUSTAINABLE CHEMICAL TOOLS TO IMPROVE HONEYBEE FORAGING EFFICIENCY IN A POLLINATOR-DEPENDENT CROP ...... 34 SYNTHESIS OF SEX PHEROMONES AND THEIR APPLICATION IN FIELD MATING DISRUPTION TRIALS ...... 35 SEXUAL BEHAVIOUR OF EPICAUTA ATOMARIA (COLEOPTERA: MELOIDAE), A PEST OF HORTICULTURAL CROPS, A PRELIMINARY STUDY ...... 36 TESTING AN ‘ATTRACT-AND-REWARD’ APPROACH TO INCREASE ECOSYSTEM SERVICES FROM NATURAL ENEMIES IN AGRICULTURE ...... 37 TRACKING THE EUROPEAN CORN BORER OSTRINIA NUBILALIS NORTHWARD EXPANSION IN SCANDINAVIA USING PHEROMONE TRAPS...... 38 USE OF SEMIOCHEMICALS FOR AEDESAEGYPTI SURVEILLANCE AND CONTROL...... 39 USE OF KAIROMONES TO ENHANCE THE BISEXUAL CATCH OF CODLING MOTH (CYDIA POMONELLA) AND ORIENTAL FRUIT MOTH (GRAPHOLITA MOLESTA) (: TORTRICIDAE) IN POME AND STONE FRUIT ORCHARDS ...... 40 USE OF SEX PHEROMONE OF PROEULIA AURARIA (LEPIDOPTERA: TORTRICIDAE) FOR MONITORING IN ORCHARDS ...... 41 USE OF SYNTHETIC PHEROMONES TO DETERMINE THE POPULATION OF TACHINIDS IN AN SOYBEAN CULTIVE...... 42

CHEMICAL ECOLOGY OF AFFECTING PEOPLE A NEW TYPE OF SYNERGISM BY BEHAVIOR MODIFICATION IN BLOOD- SUCKING BUGS SIMULTANEOULY EXPOSED TO EUGENOL AND AZAMETHIPHOS ...... 44 AGGREGATION BEHAVIOR IN SHELTERS IN RHODNIUS PROLIXUS AND RHODNIUS ECUADORIENSIS ...... 45 ARRESTANT EFFECT OF HUMAN SCALP COMPOUNDS ON PEDICULUS HUMANUS CAPITIS BEHAVIOUR ...... 46 BED BUGS (:CIMICIDAE) RESISTANT TO INSECTICIDES ALSO SHOW A REDUCED BEHAVIORAL RESPONSE TO REPELLENTS ...... 47 BEHAVIORAL RESPONSE OF ANOPHELES PSEUDOPUCTIPENNIS AND AEDES AEGYPTI LARVAE TO ATTRACTANT AND REPELLENT SUBSTANCES: A VIDEO- TRACKING ANALYSIS ...... 48 DEETS ON MOSQUITO REPELLENTS FOR PREVENTION OF ZIKA INFECTION 49 EFFECTS OF ALKALOIDS ON FEEDING BEHAVIOR AND METABOLISM OF RHODNIUS PROLIXUS, VECTOR OF CHAGAS DISEASE ...... 50 EVALUATION OF THE ACTIVITY OF THE ESSENTIAL OIL FROM AN ORNAMENTAL FLOWER AGAINST AEDES AEGYPTI: ELECTROPHYSIOLOGY, MOLECULAR DYNAMICS AND BEHAVIORAL ASSAYS ...... 51 HOW TO FIND HOST LARVAE IN A MILL: LONG-RANGE HABITAT AND HOST ATTRACTANTS AND CONTACT HOST KAIROMONES SHAPE THE FORAGING BEHAVIOR OF A LARVAL ECTOPARASITOID OF TRIBOLIUM ...... 52 INDEPENDENT AND JOINT ACTION OF THE REPELLENTS DEET E IR3535 ON TRIATOMA INFESTANS ...... 53 OLFACTORY AND GUSTATORY ADAPTATIONS IN ANTHROPOPHILIC INSECTS ...... 54 OVIPOSITIONAL RESPONSES OF CULEX TARSALIS TO FISH-ASSOCIATED SEMIOCHEMICALS IN LABORATORY BIOASSAYS ...... 55 RESPONSE OF HEAD LICE PEDICULUS HUMANUS CAPITIS TO VOLATILES FROM HUMAN SCALP AND CHEMICAL IDENTIFICATION ...... 56 SEMIOCHEMICALLY MEDIATED INTERACTIONS BETWEEN MOTHER AND OFFSPRING IN THE BED BUG, CIMEX LECTULARIUS ...... 57 THE LUTZOMYIA LONGIPALPIS COMPLEX: A BRIEF NATURAL HISTORY OF AGGREGATION-SEX PHEROMONE COMMUNICATION...... 58 TARSAL GLANDS AS A SOURCE OF ARRESTING PHEROMONES IN TRIATOMA INFESTANS ...... 59 THE FIRST RECEPTOR FOR COMPONENTS OF A CLASSICAL SEX PHEROMONE IN DIPTERA ...... 60 USE OF AEDES AEGYPTI LARVAE ATTRACTANTS TO ENHANCE THE EFFECTIVENESS OF LARVICIDES ...... 61

CHEMICAL ECOLOGY OF PHYTOPHAGOUS FLIES ANALYSIS OF CUTICULAR HYDROCARBON PROFILES IN BACTROCERA DORSALIS COMPLEX (DIPTERA: TEPHRITIDAE) ...... 63 ATTRACTION OF MALE AND FEMALE ORIENTAL FRUIT FLY TO A NOVEL TERPENOID COMPOUND ...... 64 CHEMICAL CUES USED FOR HOST INTERACTIONS AMONG VARIOUS FLIES . 65 CHEMICAL ECOLOGY OF DROSOPHILA SUZUKII: ATTRACTION TO FRUIT VOLATILES ...... 66 DANGEROUS DUNG DETER DROSOPHILA ...... 67 EFFECT OF PLANT CHEMICALS ON THE SEXUAL BEHAVIOR OF A PHYTOPHAGOUS TEPHRITIDAE FLY ...... 68 GUAVA VOLATILES ENHANCE THE SEXUAL BEHAVIOR OF SUDAMERICAN FRUIT FLY (DIPTERA: TEPHRITIDAE) MALES WITH NO EVIDENT CHANGES IN THEIR PHEROMONE AND CUTICLE CHEMICAL PROFILES ...... 69 IDENTIFICATION OF ATTRACTANT FOR FRUIT FLY, SPOTTED WING DROSOPHILA ...... 70 MALE LURES AND THE SEXUAL BEHAVIOR OF TEPHRITID FRUIT FLIES ...... 71 PARALLEL EVOLUTION OF TOXIN RESISTANCE IN LEAF-MINER FLIES (DIPTERA: AGROMYZIDAE) ...... 72 POTENTIAL ATTRACTANTS FOR THE FRUIT FLIES ANASTREPHA FRATERCULUS AND A. OBLIQUA (DIPTERA: TEPHRITIDAE) ...... 73 TRACKING THE OBJECTS OF FLIES' AFFECTIONS ACROSS SPECIES AND CONTINENTS ...... 74 THE RESPONSES OF WHITEFLIES TO GLUCOSINOLATES WITHIN A PHYLOGENETIC CONTEXT ...... 75

CHEMICAL ECOLOGY OF VECTOR-HOST INTERACTIONS AN INSIGHT INTO VARROA OLFACTION AND HOST FINDING MECHANISM ... 77 ANTENNAL RESPONSES OF RHODNIUS PROLIXUS (REDUVIIDAE: TRIATOMINAE) TO VOLATILES PRODUCED IN VITRO BY BACTERIA ISOLATED FROM HUMAN SKIN FACES ...... 78 ALTERATION OF PLANT PHENOTYPES BY MICROBES: IMPLICATIONS FOR VECTOR-BORNE DISEASE TRANSMISSION AND MULTI-TROPHIC PLANT- INSECT INTERACTIONS ...... 79 APHID RESISTANCE IN BRAZILIAN HEXAPLOID WHEAT DOES NOT CORRELATE WITH LEVELS OF THE BENZOXAZINOID NATURAL PRODUCT DIMBOA ...... 80 CHANGES IN HUMAN SKIN ODORS ASSOCIATED WITH MALARIA INFECTIOUS STAGES ...... 81 EXPERIENCE-DEPENDENT BEHAVIORAL PLASTICITY: WHAT CAN KISSING BUGS LEARN ABOUT THEIR HOSTS? ...... 82 INFLUENCE OF AEDES AEGYPTI LARVAE EXTRACTS IN THE BEHAVIOR OF INTRA-SPECIFIC OVIPOSITION ...... 83 -HOST SEMIOCHEMICALS DELIVERED TO SUSCEPTIBLE DOGS USING SLOW-RELEASE FORMULATIONS REPEL RHIPICEPHALUS SANGUINEUS SENSU LATO TICKS ...... 84 TASTE AVERSION FOR SALTS AND ALKALOIDS: CAN KISSING BUGS DISCRIMINATE THEM? ...... 85 THE ROLE OF TRYPANOSOMA RANGELI TEJERA INFECTION OF RHODINIUS PROLIXUS STAHL ON THE MATING BEHAVIOR AND METASTERNAL GLAND PROTEASE ACTIVITY ...... 86 THE LABORATORY-FIELD EXPERIMENTAL INTERFACE: TESTING AND MEASURING ATTRACTION TO ODORANTS IN THE LABORATORY AIMING AT DEVELOPING AN ODOR-LURE, A STUDY ON KISSING BUGS ...... 87 VECTOR-HOST INTERACTIONS: NEW OPPORTUNITIES FOR PROTECTION OF AND VERTEBRATE AGAINST VECTORED PATHOGENS BY EXPLOITED STRESS RELATED SIGNALLING ...... 88

CHEMICAL INTERACTIONS MEDIATED BY MICROORGANISMS ANTIFUNGAL COMPOUNDS PRODUCED BY PAENIBACILLUS POLYMYXA ALLI- 03-01 ISOLATED FROM THE STINGLESS BEE MELIPONA SCUTELLARIS ...... 90 BIO- AND CHEMODIVERSITY OF THE BACTERIAL CUTICULAR MICROBIOTA OF ANTS IN FRENCH GUIANA ...... 91 FRAGRANCE AND MICROBIAL DIVERSITY IN CECIDOMYIIDAE (DIPTERA)- INDUCED HAPLOPAPPUS FOLIOSUS () APICAL GALLS ...... 93 FUNGAL STRAINS ASSOCIATED TO THE AMBROSIA BEETLE MEGAPLATYPUS MUTATUS AND THEIR VOLATILE EMISSIONS ...... 94 GLYCEOLLIN INDUCTION AFTER PHAKOPSORA PACHYRHIZI INFECTION AND ITS INTERACTION WITH MEVALONATE PATHWAY IN SOYBEAN ...... 95 INFLUENCE OF CULTURE CONDITIONS ON ANTIBIOTIC PRODUCTION BY KRASILNIKOVIA SP. T082, A RARE ACTINOBACTERIUM...... 96 LIPOPEPTIDES FROM MARINE CYANOBACTERIA INDUCING BEHAVIORS ALONG A TROPHIC CASCADE ...... 97 MOLECULAR AND GENETIC EXCHANGES IN THE FUNGUS-GROWING ANT SYSTEM ...... 98 NATURAL PRODUCTS FROM ENTOMOPATHOGENIC BACTERIA - FROM CHEMICAL ECOLOGY TO SYNTHETIC BIOLOGY ...... 99 PRODUCTION OF INDOLE-3-ACETIC ACID BY ATTA SEXDENS RUBROPILOSA ASSOCIATED PSEUDONOCARDIA SP ...... 100 PLAGUE INSECTS’ GUT BACTERIA AS A RICH SOURCE FOR BACTERIAL POLYHYDROXYALKANOATE PRODUCERS ...... 101 THE ROLES OF STINGLESS BEES AND BACILLUS SPP. INTERACTIONS ...... 102 THE A-FACTOR GOES VOLATILE? NEW BUTYROLACTONES FROM MARINE SALINISPORA BACTERIA...... 103 THE COMBINATION OF PLANT GROWTH PROMOTING RHIZOBACTERIA AND METHYL JASMONATE PRODUCE POSITIVE AND SYNERGETIC EFFECTS ON PHYTOCHEMICAL AND MORPHOLOGICAL CHANGES IN MENTHA X PIPERITA L...... 104 VOC BIOMARKER AND INSECT ANTENNA-BASED BIOSENSOR FOR DETECTION OF FUNGAL CONTAMINATION IN GRAIN ...... 105 VOLATILES OF THE HUMAN SKIN MICROBIOME ACT AS QUORUM QUENCHER ...... 106 YEAST VIS-À-VIS CHEMICAL ECOLOGY OF GREEN LACEWINGS (NEUROPTERA: CHRYSOPIDAE) ...... 107

GENERAL CHEMICAL ECOLOGY A MULTIDISCIPLINARY APPROACH TOWARDS UNDERSTANDING TRITERPENOID METABOLISM AND SIGNALING IN PLANTS ...... 109 ALDEHYDE OXIDASE FOUND IN HALYOMORPHA HALYS (STAL) (HETEROPTERA: ) AND DEGRADATION OF DEFENSIVE ALDEHYDES ...... 110 AN UNUSUAL PATTERN OF SEX PHEROMONE PRODUCTION AND CALLING BEHAVIOR IN HELICOVERPA ARMIGERA (LEPIDOPTERA: NOCTUIDAE) ...... 111 ANTENNAL ELECTROPHYSIOLOGICAL RESPONSES OF WORKERS TO QUEEN CUTICULAR HYDROCARBONS IN A HIGHLY EUSOCIAL BEE, TETRAGONISCA ANGUSTULA (: MELIPONINI)...... 112 BEHAVIORAL RESPONSES OF BT -RESISTANT AND -SUSCEPTIBLE SPODOPTERA FRUGIPERDA MALES TO FEMALE SEX PHEROMONE ...... 113 BIOSYNTHESIS, CHEMICAL SYNTHESIS AND OCCURRENCE OF HEMOLYMPH TOXINS IN CHRYSOMELINA ...... 114 CHARACTERIZATION OF THE PHEROMONE COMMUNICATION CHANNEL IN MAMESTRA CONFIGURATA ...... 115 CHEMICAL ECOLOGY OF THE BEARDED PALM WEEVIL RHINOSTOMUS BARBIROSTRIS (COLEOPTERA: CURCULIONIDAE) ...... 116 CONTACT CUES DETERMINE OVIPOSITION OF THE SPECIALIST WILLOW SAWFLY NEMATUS OLIGOSPILUS ...... 117 CHANGES IN PHEROMONE EMISSION BY A SEED-FEEDING HEMIPTERAN CORRESPOND TO HOST PLANT QUALITY ...... 118 CLIMATE CHANGE: ATMOSPHERIC CO2 CONCENTRATION AFFECT APHID ALARM SIGNALING ...... 119 COULD PROSTERNAL INDENTATIONS BE ASSOCIATED WITH PRODUCTION OF VOLATILE PHEROMONES IN THE CERAMBYCID AGLAOSCHEMA CONCOLOR AND ORTHOSTOMA ABDOMINALE? ...... 120 CROTALARIA SPECTABILIS SEEDS EXTRACT AND ASSOCIATED ADJUVANTS: NEMATICIDE EFFECT ON THE PRATYLENCHUS BRACHYURUS JUVENILES .... 121 EPIDERMAL GLANDS IN THE FOOD ROLLING BEETLE, DELTOCHILUM FURCATUM (COLEOPTERA: SCARABAEINAE) ...... 122 EFFECT OF LEAF DAMAGE ON FLORAL HEADSPACE AND VISITATION RATES OF A TROPICAL SHRUB...... 123 EFFECT OF SINGLE AND MULTIPLE HERBIVORY IN ATTRACTION OF THE GENERALIST PREDATOR ORIUS INSIDIOSUS (SAY, 1832) TO ROSE PLANTS ... 124 EVOLUTION OF PHEROMONE RECEPTORS IN A PRIMITIVE MOTH, ERIOCRANIA SEMIPURPURELLA (ERIOCRANIIDAE: LEPIDOPTERA) ...... 125 FRIENDS AND FOES ATTRACTED BY THE CONSPICUOUS CHEMICAL FLORAL ADVERTISEMENT OF THE BOCAIUVA PALM (ACROCOMIA TOTAI; ARECACEAE) ...... 126 GENISTEIN (4’, 5, 7-TRIHYDROXYISOFLAVONE): A CONSTITUTIVE NON INDUCIBLE SOYBEAN SEEDS RESPONSE AT HIGH SOWING DENSITY AND CONSTITUTIVE INDUCIBLE AT LOW SOWING DENSITY ...... 127 HEAD LICE-HOST INTERACTION: HUMAN SCALP EXTRACTS MODIFY THE BEHAVIOUR OF PEDICULUS HUMANUS CAPITIS ...... 128 HOST LOCATION CUES OF SPINTHERUS DUBIUS (NEES) (HYMENOPTERA: PTEROMALIDAE), THE CLOVER WEEVIL PARASITOID...... 129 INJURIES OF HELICOVERPA ARMIGERA AND ITS IMPACT ON ISOFLAVONES CONTENT IN SOYBEAN GENOTYPES ...... 130 IDENTIFICATION OF 2,5-DIKETOPIPERAZINES IN XYLELLA FASTIDIOSA AND CULTURE MEDIUM PW ...... 131 INTERACTION DIVERSITY AMONG SPECIES AND THEIR ASSOCIATED FAUNA ...... 132 INVESTIGATING THE PHEROMONE COMPOSITION OF CYDIA ARAUCARIAE (LEPIDOPTERA, TORTRICIDAE): A SEED BORER OF THE ENDANGERED TREE SPECIES ARAUCARIA ANGUSTIFOLIA ...... 133 INVESTIGATING VOLATILE EMISSION PATTERNS TO ELUCIDATE THEIR ROLE IN ECOLOGICAL INTERACTIONS ...... 134 J.H. FABRE (1823-1915) AND T. EISNER (1929-2011) - TWO EXCEPTIONAL CHARACTERS IN CHEMICAL ECOLOGY OF THE 19TH AND 20TH CENTURY . 135 LUCIFERIN SULFATE IS BIOSYNTHESIZED BY A SPECIALIZED LUCIFERIN SULFOTRANSFERASE IN FIREFLIES ...... 136 LUPANINE DETERMINES THE LEVEL OF ACCEPTABILITY AND PALATABILITY OF LUPINE LUPINUS SPP. VARIETIES TO THE PEA APHID ACYRTHOSIPHON PISUM HARRIS ...... 137 METHOD FOR EXTRACTION OF VOLATILE ORGANIC COMPOUNDS IN NESTS OF THE BEE CENTRIS ANALIS.KEY WORDS: HS-SPME-GC-MS, NESTING, VOLATILE ORGANIC COMPOUNDS, CENTRIS ANALIS ...... 138 METHOXYLATED PYRAZINES AND THE ATTRACTION OF FLORIVOROUS CYCLOCEPHALINE SCARABS (MELOLONTHIDAE, DYNASTINAE) ASSOCIATED TO SOUTH AMERICAN PALMS (ARECAEAE) ...... 139 OLFACTORY-MEDIATED REPELLENCY IS OBSERVED IN BLATTELLA GERMANICA EXPOSED TO PYRETHROIDS ...... 140 OLFATOMETRY OPTIMIZATION FOR TUTA ABSOLUTA LARVAE ...... 141 OPTIMISING THE MALE-PRODUCED SEX PHEROMONE OF GALLERIAMELLONELLA (PYRALIDAE): ELECTROPHYSIOLOGICAL AND BEHAVIOURAL ANALYSES OF STEREOISOMERS OF 5,11- DIMETHYLPENTACOSANE ...... 142 PREFERENCE-PERFORMANCE CORRELATION IN A MONOPHAGOUS SAWFLY ...... 143 PHYTOCHEMISTRY DIVERSITY AT THE INTERFACE OF TRI-TROPHIC INTERACTIONS -LEPIDOPTERA-PARASITOIDS ...... 144 REPELLENT AND ANTIFEEDANT PROPERTIES OF CRUDE EXTRACTS AGAINST EPICAUTA (COLEOPTERA: MELOIDAE) ...... 145 RESPONSE OF GRAVID FEMALES OF CODLING MOTH (CYDIA POMONELLA) TO VOLATILES OF FOUR VARIETIES OF QUINCE (CYDONIA OBLONGA) IN A Y- TUBE OLFACTOMETER...... 146 SECONDARY METABOLITES EXTRACTED FROM SPODOPTERA FRUGIPERDA ADULT BODIES AS INDICATORS OF SOYBEAN FEEDING AT LARVAL STAGE ...... 147 SOYBEAN ISOFLAVONOIDS AS CHRYSODEIXIS INCLUDENS FEEDING INDICATOR ...... 148 SEARCH FOR BUMBLEBEE FATTY ACYL REDUCTASES, WHICH UNDERLAY SPECIES-SPECIFIC COMPOSITION OF MALE MARKING PHEROMONE ...... 149 SECRETIONS FROM A SPINNERET OF A SILKWORM (BOMBYX MORI) SUPPRESS GREEN LEAF VOLATILE PRODUCTION IN MULBERRY LEAVES ... 150 SOME THINGS OLD, SOME THINGS NEW: AREAS OF CHEMICAL ECOLOGY THAT MERIT ATTENTION ...... 151 SPINOCHROMES: KAIROMONES INVOLVED IN THE SYMBIOSIS BETWEEN THE SHRIMP TULEARIOCARIS HOLTHUISI AND ECHINOMETRA MATHAEI . 152 SUB-FUNCTIONALIZATION OF A NICOTIANA SPECIFIC COI1 HOMOLOG IS REQUIRED FOR JASMONATE-DEPENDENT REGULATION OF FLORAL MATURATION AND DEFENSIVE ROOT METABOLISM ...... 153 TERGAL GLAND SECRETION OF THE ALEOCHARA PSEUDOCHRYSORROA CARON, MISE & KLIMASZEWSKI (STAPHYLINIDAE: COLEOPTERA) ...... 154 TETRODOTOXIN IN THREE NEW SPECIES OF CUTTLEFISH: METASEPIA PFEFFERI, SEPIA BANDENSIS, AND SEPIA OFFICINALIS ...... 155 THE MALE-PRODUCED SEXUAL PHEROMONE OF THE STRAWBERRY SAP- BEETLE LOBIOPA INSULARIS (CASTELNAU, 1840) (COLEOPTERA: NITIDULIDAE) ...... 156 TOWARDS THE IDENTIFICATION OF THE SEX PHEROMONE OF H. ERATO PHYLLIS (FABRICIUS, 1775) (NYMPHALIDAE, HELICONIINAE) ...... 157 THE ROLE OF CHEMICAL SIGNALS IN THE ADAPTIVE RADIATION OF CLEARWING BUTTERFLIES ...... 158 THE ROLE OF VOLATILE COMPOUNDS IN THE INTERACTION DIVERSITY OF PIPER MALACOPHYLLUM AND P. GAUDICHAUDIANUM ...... 159 TO GROW AND TO DEFEND: FAR-RED MODULATES NICOTIANA ATTENUATA'S SHADE-AVOIDANCE GROWTH, WITH NO COMPROMISES IN MEJA-INDUCED PLANT DEFENSES ...... 160 UNCOVERING THE MOLECULAR BASIS OF BUMBLEBEE MALE MARKING PHEROMONE DIVERSITY VIA RNA SEQUENCING ...... 161 VOCS EMITTED BY ILEX PARAGUARIENSIS PLANTS ARE AFFECTED BY HERBIVORY OF THE LEPIDOPTERAN THELOSIA CAMINA (SCHAUS, 1920) AND THE COLEOPTERAN HEDYPATHES BETULINUS (KLUG, 1825) ...... 162 WHAT DOESN’T KILL YOU MAKES YOU STRONGER? THE COMPARATIVE EFFECT OF ALIPHATIC AND INDOLIC GLUCOSINOLATES ON LEPIDOPTERAN DEVELOPMENT ...... 163

INTERNATIONAL COLLABORATIVE RESEARCH ON CHEMICAL ECOLOGY IN LATIN AMERICA EFFECTS OF DEFENSES OF DEVELOPING SEEDS OF FIELD-GROWN SOYBEAN ON STINK BUGS (NEZARA VIRIDULA) ...... 165 FLORAL SCENTS IN THE ORCHID CATASETUM: DO SCENT PATTERNS PREDICT POLLINATING EUGLOSSINE BEE GENERA? ...... 166 FIRST REPORT OF SCENT MIMICRY IN FOOD DECEPTIVE FLOWERS: THE CASE OF CEPHALANTHERA RUBRA (ORCHIDACEAE) ...... 167 IDENTIFICATION OF EUSCHISTUS CONSPERSUS EGG VOLATILES AND KAIROMONAL ACTIVITY BY THE PARASITOID PODISI ...... 168 IDENTIFICATION OF HALYOMORPHA HALYS EGG VOLATILES AND KAIROMONAL EFFECT ON THE PARASITOID TELENOMUS PODISI ...... 169 LESSONS FROM THE FIRST INTERNATIONAL COOPERATIVE BIODIVERSITY GROUP (ICBG) IN BRAZIL ON MICROBIAL SYMBIONTS OF INSECTS ...... 170 THE PROTECTIVE ROLE OF FOLIAR ENDOPHYTIC FUNGI IN EMBOTHRIUM COCCINEUM (PROTEACEAE) ...... 171 TRANSGENERATIONAL EFFECTS OF TUBER HERBIVORY ON POTATO COMPENSATION AND RESISTANCE ...... 172 THE SEX PHEROMONE OF THE CITROPHILUS MEALYBUG: FROM IDENTIFICATION TO THE DEVELOPMENT OF APPLICATIONS IN PEST MANAGEMENT ...... 173 ONTOGENETIC AND GEOGRAPHIC VARIATION IN CYANOGENIC DEFENSES AGAINST HERBIVORES OF TURNERA VELUTINA ...... 174

OTHER A PORTABLE SYSTEM FOR PLANT VOLATILES COLLECTION ...... 176 A NEW APPROACH TO INVESTIGATING DIVERSE PHYTOCHEMICAL PROFILES AND ECOLOGICAL INTERATIONS ...... 177 BEHAVIOURAL CONSIDERATIONS ON THE IDENTIFICATION OF COPITARSIA UNCILATA UNDER LABORATORY CONDITIONS ...... 178 CITRUS VOLATILE COMPOUNDS AS PUTATIVE KAIROMONES MEDIATING DIAPHORINA CITRI OVIPOSITION ...... 179 CARDENOLIDES AS A MODEL FOR THE EVOLUTION OF MULTITROPHIC INSECT-PLANT INTERACTIONS ...... 180 CHEMICAL ECOLOGY AS PLANETARY MEDICINE ...... 181 COMBINED USE OF HERBIVORE-INDUCED PLANT VOLATILES AND SEX PHEROMONES FOR MATE LOCATION IN BRACONID PARASITOIDS ...... 182 COMMUNICATION INTERFERENCE BETWEEN NON-RELATED SYMPATRICALLY OCCURRING NOCTUIDAE MOTH SPECIES ...... 183 CONTEXTUAL PERCEPTION AND RECOGNITION OF HOST-RELATED ODOR BLENDS - A CASE STUDY OF OLFACTORY RESPONSE IN THE PARASITOID, MICROPLITIS CROCEIPES ...... 184 DIFFERENCES IN SEX PHEROMONE PRODUCTION BY SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) FEMALES RESISTANT AND SUSCEPTIBLE TO BT CORN ...... 185 DYNAMICS AND ORIGIN OF CYTOKININS INVOLVED IN PLANT MANIPULATION BY A LEAF MINING INSECT ...... 186 EFFECT OF SILICON AND METHYL JASMONATE ON LARVAL PERFORMANCE OF SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) IN RICE PLANTS ...... 187 ESSENTIAL OILS FROM POLYGAMA AND BACCHARIS SPARTIOIDES ENHANCE MATING COMPETITIVENESS OF ANASTREPHA FRATERCULUS MALES...... 188 EFFECT OF STARVATION ON THE OLFACTORY RESPONSES OF THE GRAPE WEEVIL, NAUPACTUS XANTHOGRAPHUS (COLEOPTERA: CURCULIONIDAE) ...... 189 EXTRAFLORAL NECTAR VOLATILES AFFECT ANT CHOICE BEHAVIOR ...... 190 GENERALIST PIERCING-SUCKING INSECT DAMAGE CHANGE VOLATILE PROFILE IN CORN SEEDLINGS ...... 191 HOW TO MAKE AN EFFICIENT ANT-PLANT MUTUALISM: PARTNER MANIPULATION, PHENOTYPIC ADJUSTMENTS AND HONEST SIGNALLING .. 192 HYDROCARBONS INCORPORATED INTO THE CUTICULAR PROFILE THROUGH DIET DO NOT GENERATE AGGRESSION IN ARGENTINE ANTS (LINEPITHEMA HUMILE MAYR) ...... 193 INDUCED CHANGES IN THE BEHAVIOR OF RODHNIUS PROLIXUS AS AN INDICATOR OF SENSITIVITY TO DIFFERENT BITTER COMPOUNDS ...... 194 INDUCIBLE DE NOVO BIOSYNTHESIS OF ISOFLAVONOIDS IN SOYBEAN LEAVES BY SPODOPTERA LITURA (LEPIDOPTERA: NOCTUIDAE) DERIVED ELICITORS: TRACER TECHNIQUES AIDED BY HIGH RESOLUTION LCMS ..... 195 INSECTS’ LITTLE HELPERS: SYMBIONT-PRODUCED SECONDARY METABOLITES FOR PATHOGEN DEFENSE ...... 196 ISOLATION AND IDENTIFICATION OF A MALE-PRODUCED ATTRACTANT PHEROMONE FOR THE INVASIVE VELVET LONGHORNED BEETLE, TRICHOFERUS CAMPESTRIS (: HESPEROPHANINI) ...... 197 METHYL JASMONATE APPLICATION IN TOMATO REDUCES INFESTATION OF TETRANYCHUS URTICAE KOCH, 1836 (TETRANYCHIDAE)...... 198 MS/MS MOLECULAR NETWORKING AS A DEREPLICATION STRATEGY APPLIED TO FUSARIUM SP EXTRACTS ISOLATED FROM SENNA SPECTABILIS’S RIZHOSOHERE ...... 199 MOLECULAR NETWORKING ON NMR - IDENTIFICATION OF COMPOUNDS CLASSES USING 1H, 13C E 13C-HSQC ...... 200 NON-TARGETED AND TARGETED METABOLOMIC APPROACHES REVEAL DIFFERENCES IN LEGUME CHEMISTRY BEFORE AND AFTER INFESTATION WITH PEA APHID HOST RACES ...... 201 ONE PATHWAY IS NOT ENOUGH: GLUCOSINOLATE METABOLISM IN THE CABBAGE STEM FLEA BEETLE PSYLLIODES CHRYSOCEPHALA ...... 202 OVIPOSITION CHOICES OF THE WILLOW SAWFLY NEMATUS OLIGOSPILUS IS AFFECTED BY CONSPECIFIC HERBIVORY IN THE NATIVE WILLOW SALIX HUMBOLDTIANA ...... 203 PHLOEM SAP INGESTION-RELATED PLANT - APHID TRANSFER OF CYCLITOLS AND THEIR EFFECT ON THE PEA APHID ACYRTHOSIPHON PISUM HARRIS PROBING BEHAVIOUR ...... 204 PLANT LITTER VOLATILES AND THEIR INTERACTION WITH DECOMPOSER ORGANISMS ...... 205 RESISTANCE TO CARDENOLIDES CARDENOLIDES – GENETIC BACKGROUNDS MATTER! ...... 206 SHEDDING LIGHT ON PHYTOCHEMICAL DIVERSITY ...... 207 SPECIFICITY OF PLANT RESPONSES TOWARD HERBIVORE’S TOUCH, FEEDING AND OVIPOSITION: IMPLICATION FOR EGG PARASITOIDS ...... 208 SYNERGETIC EFFECTS OF CIS-JASMON AND METHYL SALICYLATE IN ATTRACTING DIFFERENT MEMBERS OF A COMPLEX MULTITROPHIC FLOWER-VISITING ARTHROPOD COMMUNITY ...... 209 TWO SIDES OF THE SAME COIN: THE BENEFIT AND BACKLASH OF ISOTHIOCYANATE DETOXIFICATION ...... 210 THE CHEMISTRY OF MALE DOMINANCE: SECRETION OF 3-METHYL-2- BUTENYL BUTYRATE CORRELATES WITH BEHAVIOURAL STATUS IN THE BRONZE BUG, THAUMASTOCORIS PEREGRINUS ...... 211 TRAPPING LITERATURE FOR FOREST COLOEPTERA: A META-ANALYSIS .... 212 UTILIZING CHLOROPHYLL FLUORESCENCE IMAGING AS A TOOL TO DETERMINE THE CHEMOATTRACTANT POTENTIAL OF GINSENOSIDES IN THE GINSENG – PYTHIUM IRREGULARE PATHOSYSTEM ...... 213

PLANT COMMUNICATION ARBUSCULAR MYCORRHIZAE FUNGI CHANGE THE RESISTANCE OF RICE PLANTS IN RESPONSE TO FEEDING WITH FALL ARMYWORM, SPODOPTERA FRUGIPERDA ...... 215 COMMUNICATING PLANTS: THE CHEMICAL LANGUAGE OF GOLDENRODS (SOLIDAGO ALTISSIMA) AND ITS ECOLOGICAL CONSEQUENCES ...... 216 EFFECTS OF MULTIPLE STRESS ON VOLATILE-MEDIATED TRITROPHIC INTERACTIONS ...... 217 FLORAL CO2: A BREATH OF FRESH AIR IN PLANT-POLLINATOR COMMUNICATION ...... 218 ORCHIDS’ CHEMICAL IMITATION GAME: FLORAL SCENTS OF CATASETUM CERNUUM AND GONGORA BUFONIA SUGGEST CONVERGENT EVOLUTION TO ATTRACT A UNIQUE POLLINATOR ...... 219 PHYTOTOXITY OF EUCALYPTUS SALIGNA SM ESSENTIAL OIL ON GROWTH OF GRASSES ...... 220 PLANT BETA-GLUCOSIDASES RESIST DIGESTION BY A LEPIDOPTERAN HERBIVORE TO PROMOTE SUSTAINED ACTIVATION OF CHEMICAL DEFENSES ...... 221 POPLAR COMMUNICATIONS: HOW A WOODY PLANT TEXTS ITS HERBIVORES, HERBIVORE ENEMIES AND NEIGHBORS ...... 222 PLANT BIO-WARS: PROTEIN NETWORKS REVEAL TISSUE-SPECIFIC DEFENSE STRATEGIES ...... 223 PLANT COMMUNICATION IN THE RHIZOSPHERE ...... 224 QUANTIFYING CHEMICAL DIVERSITY AND ITS ECOLOGICAL CONSEQUENCES ...... 225 RESPONSE OF TWO MIRID PREDATORS AND COMPARATIVE ANALYSIS OF HERBIVORE-INDUCED PLANT VOLATILES FROM PEST INFESTED AND UNINFESTED TOMATO PLANTS ...... 226 SEQUESTRATION AND TRANSPORT PROTEINS IN LEAF BEETLE LARVAE .... 227 SYMBIOSIS WITH SYSTEMIC FUNGAL ENDOPHYTE CHANGES LEAF LITTER VOLATILES IN LOLIUM MULTIFLORUM ...... 228 WHO’S NEXT? INSECT OVIPOSITION REVEALS THE IDENTITY OF THE UPCOMING HERBIVORES TO NICOTIANA ATTENUATA ...... 229

ROLE OF CHEMICAL ECOLOGY IN SOCIAL BEHAVIOR, AND VICE VERSA CUTICULAR CHEMISTRY OF THE STINGLESS BEE TETRAGONISCA ANGUSTULA ...... 231 CHEMICAL ECOLOGY OF POLLINATION SYSTEMS MEDIATED BY NOCTURNAL BEES: A BRAZILIAN-AUSTRIAN INITIATIVE ...... 232 CHEMICALLY MIMETIC SOCIAL PRODUCES NON-MIMETIC BROOD ...... 233 DO QUEENS OF PRIMITIVELY EUSOCIAL INSECTS USE PHEROMONES TO DOWNREGULATE WORKER REPRODUCTION? ...... 234 EFFECT OF THE ESSENTIAL OIL OF EUPATORIUM BUNIIFOLIUM IN THE PRODUCTION OF THE HONEY BEE PHEROMONE, ETHYL OLEATE ...... 235 HOME IS WHERE THE POOP IS: DEVELOPMENT OF SHELTER-SEEKING BEHAVIOR IN ORIENTAL COCKROACH ...... 236 HOSTS AND PARASITES: CHEMICAL COMMUNICATION AND SIGNAL INTERCEPTION IN TWO POLISTES SOCIAL WASPS ...... 237 IMPACT OF A CUTICULAR COMPOUND CORRELATED WITH FERTILITY IN THE ESTABLISHMENT OF HIERARCHIES IN NEOPONERA APICALIS (HYMENOPTERA: FORMICIDAE) ...... 238 INNOVATIVE APPROACHES TO CONTROLLING A THREAT TO HONEYBEES IN THE SUB-TROPICS ...... 239 MODULATION OF FEEDING BEHAVIOR FOLLOWING STARVATION IN ADULT WORKER HONEY BEES ...... 240 MANDIBULAR GLAND PHEROMONES FROM AFRICAN HONEYBEES: NEW INSIGHTS AND PROSPECTS AS A COMPLEMENTARY TAXONOMIC TOOL ..... 241 SOLENOPSIS INVICTA AND SOLENOPSIS RICHTERI HYBRIDIZATION IN THE USA AND SOUTH AMERICA – PAST AND PRESENT ...... 242 STATE OF THE ART IN HONEY BEE CHEMICAL ECOLOGY...... 243 THE EVOLUTIONARY ORIGINS OF SOCIAL INSECT QUEEN PHEROMONES: HONESTY AND DYNAMICS OF FERTILITY SIGNAL PRODUCTION IN A SOCIALLY POLYPHENIC HALICTID BEE ...... 244 VOLATILE COMPOUNDS FROM V. DESTRUCTOR-INFESTED PUPAE ELICITED HYGIENE BEHAVIOR IN A. MELLIFERA WORKERS ...... 245 WASTE TRAFFIC CONTROL IN ATTA SEXDENS RUBROPILOSA ...... 246

THE CHEMISTRY OF CHEMICAL ECOLOGY CIRCADIAN RHYTHM IN P. SCHEIDEANUM ...... 248 FROM NATURAL PRODUCT TO SEXUAL PHEROMONE MIMIC – CHALLENGES BEYOND STRUCTURAL ELUCIDATION IN THE CHEMISTRY OF SEXUAL DECEPTION ...... 249 GC/MS-BASED SELECTIVE ASCAROSIDE SCREENING OF NEMATODE METABOLOMES ...... 250 GLUTATHIONE-DEPENDENT DETOXIFICATION OF SINALBIN IN SPODOPTERA LITTORALIS ...... 251 HIGH SOWING DENSITY INHIBITS THE INDUCTION OF DEFENSIVE COMPOUNDS DAIDZEIN (4’, 7-DIHYDROXYIFLAVONE) AND GENISTEIN (4’, 5, 7- TRIHYDROXYISOFLAVONE) IN SOYBEAN LEAVES UNDER ANTICARSIA GEMMATALIS H. HERBIVORY ...... 252 IDENTIFICATION AND SYNTHESIS OF MALE-SPECIFIC COMPOUNDS OF CHAULIOGNATUS IGNACIOUS ...... 253 IDENTIFICATION OF A NOVEL PHEROMONE COMPOUND FROM CHILECOMADIA VALDIVIANA (PHILIPPI) (LEPIDOPTERA: COSSIDAE)...... 254 IDENTIFICATION AND SYNTHESIS OF THE SEX PHEROMONE OF THELOSIA CAMINA (LEPIDOPTERA:EUPTEROTIDAE) ...... 255 IDENTIFICATION OF NEW SEMIOCHEMICALS - ACCIDENTS, PITFALLS, AND SUCCESS ...... 256 IDENTIFICATION OF THE AGGREGATION PHEROMONE OF HOMALINOTUS VALIDUS (COLEOPTERA: CURCULIONIDAE) ...... 257 IDENTIFICATION OF THE HOST MARKING PHEROMONE IN THE AFRICAN INDIGENOUS FRUIT FLY CERATITIS COSYRA ...... 258 KETONES AS PHEROMONE ANTAGONISTS: LIGHTS AND SHADOWS ...... 259 METABOLISM OF COMPOUNDS FROM GLYCINE MAX (L.) MERRILL LEAVES BY ANTICARSIA GEMMATALIS (HÜBNER) ...... 260 PHEROMONES OUTSIDE THE BOX: UNEXPECTED PHEROMONES FROM CERAMBYCID BEETLES ...... 261 PYRANOCOUMARINS PRODUCTION EVALUATION IN DIFFERENT GENOTYPES OF NAVEL ORANGE (CITRUS SINENSIS (L). OSBECK) AND RANGPUR LIME (CITRUS LIMONIA OSBECK) ...... 262 RECENT RESULTS IN PHEROMONE SYNTHESIS ...... 263 SEXUAL COMMUNICATION OF PAYSANDISIA ARCHON. AN INTERESTING CASE OF EVOLUTIONARY CONVERGENCE ...... 264 SHEDDING LIGHT ON PHYTOCHEMICAL AND INSECT DIVERSITY: EXPLORING THE PHOTOCHROMISM OF CHROMENES ISOLATED FROM PIPER KELLEYI ...... 265 STUDIES ON THE PHEROMONE CHEMISTRY OF THE CITRUS BORER, ROTUNDICOLLE (COLEOPTERA: CERAMBYCIDAE) ...... 266 SYNTHESIS OF FOUR STEREOISOMERS OF METHYL 4,8,12- TRIMETHYLPENTADECANOATE, AIMING TO DETERMINE THE ABSOLUTE CONFIGURATION OF THE MAJOR COMPONENT OF THE SEX PHEROMONE OF EDESSA MEDITABUNDA (HEMIPTERA: PENTATOMIDAE) ...... 267 SYNTHESIS AND BIOLOGICAL EVALUATION OF JASMONOIDS ...... 268 TENUAZONIC ACID PRODUCED BY A FUNGUS ISOLATED FROM COLONY OF THE STINGLEES BEE FRIESEOMELITTA VARIA...... 269 THE CHEMICAL ANALYSIS OF BUFADIENOLIDES IN DEFENSIVE GLANDS OF THE ASIAN RHABDOPHIS SNAKES ...... 270 THE RELATIONSHIP BETWEEN ENDOPHYTES AND OXYLIPINS IN A. BRASILIANA AND A NEW WOUNDING SIGNALLING MOLECULE ...... 271 TRAPPING CAPACITY AND BREAK-THROUGH VOLUMES OF AERATION COLUMNS ...... 272 Z-SELECTIVE OLEFIN CROSS-METATHESIS IN THE SYNTHESIS OF ECOLOGICALLY RELEVANT COMPOUNDS: JASMONATES AS A CASE STUDY ...... 273

APPLIED USES OF SEMIOCHEMICALS

A METHOD FOR CREATING HIGHLY CONCENTRATED ODOR PLUMES PASSIVELY EMANATING FROM ODOR DISPENSERS.

Thomas BAKER1*

1Penn State University, University Park, PA, USA

Male flying in wind respond with upwind surges to individual strands of pheromone emanating from point source dispensers and they respond to the pockets of cleaner air between the pheromone strands by initiating cross-wind casting flight. Time- averaged emission rates from pheromone mating disruption dispensers are instructive for calculating field longevity of dispensers and understanding emission levels field-wide that produce desired levels of mating impairment and damage reduction. However, such dispenser emission rate measurements are not relevant to optimizing the ability of each dispenser to manipulate male moth behavior from as far downwind as possible. Widely spaced, high-emission-rate pheromone mating disruption dispensers should be designed so that they can have the farthest downwind “reach”, e.g., with the strongest possible downwind strands so that each dispenser can get males to “lock on” to a pheromone plume from far away and begin dosing and habituating themselves with pheromone strands as they fly upwind in the plume. Here we show with measurements taken from commercial, high-emission-rate pheromone dispensers, how the plume-strand pheromone flux and therefore the corresponding downwind reach of their emissions can be optimized by simple procedures without changing the dispensers’ loadings or their overall time- averaged emission rates.

19

ATTRACTION OF SPODOPTERA FRUGIPERDA MALES FROM DIFFERENT REGIONS OF ARGENTINA TOWARDS DIFFERENT FORMULATIONS OF THE FEMALE SEX PHEROMONE, IN THE LABORATORY AND IN THE FIELD.

Celina BRATOVICH1; Adriana SALUSO2; Gabriela MURÚA3; Pablo GUERENSTEIN4*

1CICyTTP-CONICET and Facultad de Ingeniería, UNER, Diamante/Oro Verde, Entre Ríos, ARGENTINA 2EEAParana-INTA,, Oro Verde, Entre Ríos, ARGENTINA 3ITANOA-EEAOC-CONICET, Las Talitas, Tucumán, ARGENTINA 4CICyTTP-CONICET and Facultad de Ingeniería, UNER, Diamante/Oro Verde, Entre Ríos, ARGENTINA

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is an important pest of corn. It is known that the chemical composition of the female sex pheromone of this species varies between populations of different geographical areas. While the pheromonal composition of populations from North and Central America and Brazil is known, the pheromone composition of females from Argentina is unknown. There are different commercial versions of synthetic sex pheromone of this lepidopteran. We studied the attraction of male S. frugiperda from different geographic areas of Argentina to two commercial versions of synthetic sex pheromone, in the laboratory and in two distant locations in the field. Five geographically different argentine populations were used for laboratory tests. The commercial versions of S. frugiperda pheromone evaluated were those from: a) Great Lakes® Hercon® (GL) and b) ChemTica® (CH). A negative control was also tested. In the laboratory, tests were conducted in a wind-tunnel. The behaviors recorded were mainly: takeoff, flight halfway through the tunnel, approaching the source at a radius <10 cm, and contact. Usually at least 80% of the moths tested took off and at least 70% flew halfway through the tunnel. GL evoked approach in 5-50% of the moths tested depending on the population, whereas the % approach for CH was somewhat lower (however, an experimental, non-commercial formulation from ChemTica® performed similarly to GL in preliminary tests). In corn fields, data from Unitrap traps were collected during 3 months. The number of S. frugiperda males collected was 51 and 48 with GL 15 and 2 with CH, and 6 and 0 with control, in Tucumán and Paraná, respectively. Even when the pheromone blends tested showed attraction, the formulation should be improved.

20

BRINGING NOVEL SEMIOCHEMICAL FORMULATIONS TO THE MARKET.

Agenor MAFRA-NETO1*; Christopher FETTIG2; Steve MUNSON3; Romeno FALEIRO4; Abdallah ben ABDALLAH4; Cesar RODRIGUEZ-SAONA5; Márcio Fernandes PEIXOTO6; Robert HOLDCRAFT7; Carmem BERNARDI8; William URRUTIA8; Rodrigo SILVA8;

1ISCA Technologies, Inc, Riverside, Californnia, USA 2Forest Service, Pacific Southwest Research Station, Davis, California, USA 3Forest Service, Intermountain Region - FHP, Ogden, Utah, USA 4FAO / Al-Hassa Date Palms & Dates Center, Al-Hassa, Al-Hassa, Saudi Arabia 5Rutgers University Philip E. Marucci Blueberry and Cranberry Research Center, Chatsworth, NJ, USA 6Instituto Federal Goiano, Rio Verde, Goiás, Beasil 7Rutgers University Philip E. Marucci Blueberry and Cranberry Research Center, Chatsworth, NJ, USA 8ISCA Technologies, Inc, Riverside, California, USA

SPLAT® (Specialized Pheromone and Lure Application Technology) matrix is a unique controlled-release technology that can be adapted to dispense and protect a wide variety of compounds from degradation, including semiochemicals, pesticides, and phagostimulants, in diverse environments. ISCA Technologies in collaboration with colleagues in academia, government, and industry, has been developing SPLAT®-based insect control products for over a decade. We will provide an overview of SPLAT® technology and existing commercial formulations and describe ongoing efforts to develop new SPLAT® mating disruption, attract-and-kill, and repellent products for pest control in agricultural and forest environments.

21

DISCRIMINATION OF INFESTED COTTON SQUARES BY FEMALES OF ANTHONOMUS GRANDIS BOHEMAN (COLEOPTERA: CURCULIONIDAE).

Ana NUSSENBAUM1*; Francisco DEVESCOVI1; Pablo SCHIAVO2; Mariela FOGAR3; Maria SIMONELLA4; Ivan BONACIC4; Juan SALERNO5; Jorge CLADERA5; Diego SEGURA5

1Instituto de Genética E.A. Favret, Instituto Nacional de Tecnología Agropecuaria (INTA) / CONICET, Hurlingham, Buenos Aires, Argentina 2FACULTAD DE CIENCIAS EXACTAS Y NATURALES, UBA, C.A.B.A, BUENOS AIRES, ARGENTINA 3Estación Experimental Agropecuaria Saenz Peña, Saenz Peña, Chaco, Argentina 4ESTACIÓN EXPERIMENTAL AGROPECUARIA SAENZ PEÑA, SAENZ PEÑA, CHACO, ARGENTINA 5Instituto de Genética E.A. Favret, Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Buenos Aires, Argentina

The choice of an oviposition site has a direct impact on the reproductive success of insects, particularly for species that developed in confined patches. Females of Anthonomus grandis, an important pest of cotton in Argentina, seal the oviposition punctures with secretions that are hipothesized to act as a chemical signal that deters other females from ovipositing. In this work, we evaluated the behaviour of females to oviposit in previously infested squares (flower buds of cotton). For this, preference for oviposition in infested vs non-infested squares was studied in choice tests in the laboratory, and the distribution of ovipositions by A. grandis in field collected squares from plants and fallen squares (abscised) were assessed in cotton plots with different infestation levels.In the laboratory, the number of oviposition in infested vs non-infested squares was significantly higher in non-infested squares (t (41) = 2.19; p = 0.03). Furthermore, the rate of acceptance of squares as oviposition substrate was higher for non-infested squares than for infested squares (Chi-square (51) = 6.74; p = 0.03). In the field, a pattern indicating avoidance of infested squares was found in fallen squares sampled in plots with high infestation. In these, a deviation from the Poisson distribution was found, where the trend was to avoid previously infested squares. On the other hand, squares sampled directly from the plant fitted a random distribution in some plots and the avoidance pattern in others. We never found a distribution that deviates towards an aggregated distribution. The same results were found in two consecutive years and in two different provinces.Our results suggest the possible presence of a host marking pheromone affecting female behavior by deterring the oviposition. Knowledge of the behaviour of females and identification of these signals could provide a basis to develop control methods against this important pest.

22

EVALUATION OF SEX ATTRACTANT PHEROMONE FROM RICE STALK STINK BUG TIBRACA LIMBATIVENTRIS IN LABORATORY AND FIELD.

Thais Fernanda Stella de FREITAS1*; Josué SANT'ANA1; Maria Carolina BLASSIOLI-MORAES2; Raul Alberto LAUMANN2; Miguel BORGES2; Ashot KHRIMIAN3

1PPG-Fitotecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil 2EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DISTRITO FEDERAL, BRASIL 3USDA-ARS, Beltsville, MD, USA

Tibraca limbativentris (Hemiptera: Penatomidae) is an important pest in several rice producer countries in South America. Zingiberenol was identified as a sexual pheromone of the species. We registered in laboratory and field bioassayschemotactic responses of rice stalk stink bug (RSSB) by using rubber septa baited with zingiberenol mixtures (1 mg/septum of each diastereoisomeric pair). Treatments were: Zingiberenol I (3S,6S,7R + 3R,6R,7R); Zingiberenol II (3R,6S,7R + 3S,6R,7R); Zingiberenol III (3S,6S,7S + 3R,6R,7S); Zingiberenol IV (3R,6S,7S + 3S,6R,7S) and control (hexane). Laboratory bioactivity of virgin females was evaluated in Y-tube olfactometer. Females underwent the contrast the odor from septa baited with each pheromone treatment and hexane (control). Olfactometer data were analyzed by χ2-tests (p<0.05) (n=40). Field experiment was carried out on a rice crop in Eldorado do Sul, RS, Brazil, in 2013/2014 during the growing seasons. Rubber septa were placed in PET bottles traps, in randomized block design with four replications. Traps were checked every week and the number of caught insects was compared by Duncan (p<0.05). RSSB occurrence was estimated by sampling 1m2 area units in 16 points every week. The number of sampled and pheromone trapped insects was not different (T-test, p=0.325). In both olfactometer (p=0.027) and field experiments (p<0.001) females were more attracted to Zingiberenol I. Researches might enhance sustainable pest management by using this pheromone composition in agricultural systems.

23

EAVESDROPPING ON THE VICTIM: A GENERALIST PREDATOR UTILIZES AN ALARM PHEROMONE TO LOCATE AN INVASIVE PEST.

Diego F FRAGA1*; Cesar RODRIGUEZ-SAONA2; George C HAMILTON2; Anne L NIELSEN2; Antonio C BUSOLI2

1UNESP, Jaboticabal, São Paulo, BRAZIL 2Rutgers University, NEW BRUNSWICK, New Jersey, United States

The brown marmorated stink bug (BMSB), Halyomorpha halys, is an invasive pest of vegetable and fruit crops in North America. Generalist insect predators utilize BMSB as prey; however, the chemical cues used by these natural enemies in host location remain unknown. I tested the hypothesis that tridecane, a BMSB-associated volatile, acts as a kaironome for Orius insidiosus during prey location. Experiments were conducted in 2014 at two research facilities, Rutgers University, NJ (USA). In the laboratory, a four- arm olfactometer was used to record the behavioral response of O. insidiosus males and females to tridecane (1, 10 and 100 ng/ml). Olfactometer studies showed that O. insidiosus adults were attracted and arrested by tridecane. In the greenhouse, the effects of tridecane on O. insidiosus attraction and egg predation were assessed in cages. These studies showed that O. insidiosus was attracted to plants baited with tridecane, but egg predation was not affected. Finally, the attraction of Orius spp. to tridecane-baited traps was tested in peach, blueberry, and sunflower fields as well as its effect on egg predation. Traps and sentinel eggs were placed near dispensers containing either 1, 2, or 3 ml of tridecane; 3 or 5 ml of methyl salicylate (a plant volatile); or nothing. Field studies demonstrated that Orius spp. were attracted to tridecane-baited traps; however, this attraction did not lead to greater egg predation. In conclusion, although O. insidiosus is attracted to tridecane, this attraction did not lead to higher egg predation likely because this kairomone acts as an arrestant.

24

FROM GENE DISCOVERY TO METABOLIC ENGINEERING: PRODUCTION OF MOTH PHEROMONES IN PLANT AND CELL FACTORIES.

Christer LÖFSTEDT1*

1Department of Biology, Lund University, Lund, N/A, SWEDEN

The use of pheromones for control of pest insects has many advantages over the use of traditional pesticides. The global market for pheromone-based control products is currently estimated to approximately $200 millions and tons of synthetic pheromones are produced commercially for this purpose. We currently explore two “green chemistry” alternatives to conventional synthetic production of pheromones. One option is the pheromone brewery, a yeast cell factory for pheromone production. Another option is a plant factory, using genetically modified plants for production of pheromones or pheromone precursors. As a proof of the pheromone brewery concept, we co-expressed a ∆11 desaturase and a FAR in the Brewer’s yeast Saccharomyces cerevisiae and produced (Z)-11-hexadecenol. Metabolic engineering of S. cerevisiae and optimized fermentation conditions has the potential to increase production titres dramatically. Using Nicotiana benthamiana as a plant factory, we produced typical 14C and 16C moth sex pheromone components by transient expression of up to four genes coding for consecutive biosynthetic steps. The fatty alcohol fractions from the genetically modified plants were acetylated and mixed to mimic the respective sex pheromones of the small ermine moths Yponomeuta evonymella and Y. padella. These mixtures were very efficient and specific for trapping of male moths and matched the activity of conventionally produced synthetic pheromones. Semi-synthetic preparation of sex pheromones may be a novel and cost- effective way of producing moderate to large quantities of pheromones with high purity and a minimum of hazardous waste.

Hagström ÅK, Wang H-L, Liénard, MA, Lassance JM, Johansson T, Löfstedt C. 2013. A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co- expression of two biosynthetic genes from a noctuid moth in a yeast cell factory. Microbial Cell Factories 12:125:1-11.

Ding B-J, Hofvander P, Wang H-L, Durrett TP, Stymne S, Löfstedt C. 2014. A plant factory for moth pheromone production. Nature commun. 5:3353

25

IDENTIFICATION OF VOLATILE COMPOUNDS OBTAINED FROM LARVAE-INFESTED AND ROTTEN FRUIT ATTRACTIVE FOR THE FRUIT FLY PARASITOID DIACHASMIMORPHA LONGICAUDATA ASHMEAD (HYMENOPTERA: BRACONIDAE).

Francisco DEVESCOVI1*; Patricia C. FERNÁNDEZ2; Guillermo E. BACHMANN3; Ana L. NUSSENBAUM3; Pablo SCHIAVO4; Diego F. SEGURA5

1Instituto de Genética Ewald A. Favret, Instituto Nacional de Tecnología Agropecuaria (INTA)/CONICET, Castelar, Buenos Aires, Argentina 2Estación Experimental Agropecuaria Delta del Paraná, INTA/CONICET, Buenos Aires, Buenos Aires, Argentina 3INSTITUTO DE GENÉTICA EWALD A. FAVRET, INSTITUTO NACIONAL DE TECNOLOGÍA AGROPECUARIA (INTA)/CONICET, Hurlingham, Buenos Aires, Argentina 4Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, CABA, Buenos Aires, Argentina 5INSTITUTO DE GENÉTICA EWALD A. FAVRET, INSTITUTO NACIONAL DE TECNOLOGÍA AGROPECUARIA (INTA)/CONICET, Hurlingham, Buenos Aires, Argentina

Females of the fruit fly parasitoid, Diachasmimorpha longicaudata, are able to locate their host habitat using chemical information. Previous studies have shown that volatiles released by fruit infested by Ceratitis capitata Wiedemann (Diptera: Tephritidae) larvae, as well as rotten (but non-infested) oranges (Citrus sinensis Osbeck var. navel) attract females in laboratory arenas with no air flux. The aim of this study was to confirm the chemical basis of the orientation behavior of D. longicaudata towards its host using a dynamic olfactometer, and determine the candidate compounds associated to that behavior. We evaluated whether parasitoid females are using cues from infested and non- infested, rotten fruit within a Y-tube olfactometer and confirmed those results, suggesting that air borne compounds are responsible for the attractiveness. We then collected the volatile compounds from infested, rotten and intact oranges by dynamic head-space sampling using HayeSep Q traps. After elution with methylene chloride, volatiles were analyzed by GC-MS and were identified using the NIST library. Significant quali- and quantitative differences were found between the GC profiles of infested and rotten fruit when compared with non-infested fruit. Some compounds [such as α- pinene; β-pinene, and D-limonene] were found in larger amount both in infested and rotten oranges compared with control oranges and therefore may be associated to fruit decomposition. On the other hand, several compounds were found only in infested fruit, including ethyl- 2-hexanoate; (-)-Terpinen-4-ol; 2,6-Octadiene, 2,6-dimethyl-; methyleugenol among the most abundant. Likewise, specific compounds were found only in rotten fruit, like (+)-4- Carene; Acetic acid, octyl ester and trans-p-Menthan-3-one. These results constitute a promising base to continue studying the role of specific compounds on the orientation behavior of D. longicaudata females.

26

MASS-TRAPPING OF CODLING MOTH MATED FEMALES IN DISRUPTED ORCHARDS.

Alex ILICHEV1*; David WILLIAMS1

1Biosciences Research, DEDJTR, Tatura, VIC, Australia

Codling moth (CM) Cydia pomonella L. (Lepidoptera: Tortricidae) is an important pest of pome fruit worldwide, and infests commercial apple, pear, quince and nashi orchards in Australia. Sex pheromone mediated mating disruption (MD) successfully controls low populations of CM, but interferes with sex pheromone traps used for CM monitoring. Use of lures with pear-derived kairomone, (E, Z)-2,4-ethyl-decadienoate (pear ester, PE) improved monitoring of CM in apple, but not pear orchards treated with MD. Further research has demonstrated that attractiveness of PE under MD may be synergised by different host-plant volatiles, making the lures even more attractive for CM males and, most importantly, mated females. The real goal of MD is to reduce the female’s ability to reproduce, so attracting CM (mated females or both sexes) into mass trapping devices would be an effective way to compliment MD by reducing the pest populations to levels controllable by MD. Field screening of 24 combinations of host plant volatiles with PE and codlemone identified the most effective mixture for attracting mated CM females in MD treated pears. Further field trials utilised grids of 112 traps in MD treated pear blocks to determine the active radius of attraction of the best performing lure in a standard trap and identify the optimal spatial density of the trap and lure for further mass trapping experiments. Our results demonstrated that the active radius was about 33m for mated female moths and 43m for male moths in MD treated pears. After three consecutive seasons of field trials pear fruit damage was reduced to 0.083% that suggests mass- trapping could significantly reduce CM populations and provides proof of concept for mass-trapping of mated CM females in MD treated pears.

27

MATING DISRUPTION 50 YEARS ON: PHEROMONES COMING OF AGE - FINALLY!.

Don THOMSON1*; Jack JENKINS1

1Pacific Biocontrol, Vancouver, WA, USA

The first report of the successful deployment of a sex pheromone to control an insect pest Trichoplusia ni was published in 1967. Eleven years passed until the US EPA registered the first mating disruption technology, Gossyplure H.F.R, for control of pink bollworm Pectinophora gossypiella in cotton. Over the last 50 years, the commercial deployment of mating disruption has grown substantially with products registered for dozens of insect pests primarily Lepidopteran species in horticultural crops. Pheromone active ingredients are relatively expensive to produce and commercial success of mating disruption has depended on cost-effective controlled release technologies. Passive-release pheromone dispensers applied by hand or with specialized equipment are the most widely used technologies. However with greater adoption, mating disruption products have evolved to improve efficacy, ease-of-use and economics. Recently, active-release dispensers such as aerosols have been successfully developed and increasingly adopted for several important pests. Mating disruption has been very successful when combined with other management technologies and used in large contiguous areas. Some organized area-wide programs have reduced target insect populations to very low and sustainable levels (Cydia pomonella, Grapholita molesta) while others have eradicated the insect from specific geographic areas (Lobesia botrana). Currently, mating disruption is used to manage more than 40 species worldwide with an estimated 750,000 hectares treated. Adoption of mating disruption is increasing especially in the European Union and the United States. Issues related to the adoption of mating disruption such as government regulations, health and environmental concerns and pest management and economic considerations will be reviewed. Fifty years on and finally coming of age, future trends in development and adoption of mating disruption technology will be discussed.

28

MAXIMIZING INFORMATION YIELD AND IMPROVING THE PRECISION OF PHEROMONE-BAITED MONITORING TRAPS.

Larry GUT1*; Christopher ADAMS1; Peter MCGHEE1; James MILLER1

1Department of Entomology Michigan State University, East Lansing, Michigan, USA

Monitoring traps baited with sex pheromones play a critical role in revealing where and when insect pests are active. However, translating the number of individuals captured in a monitoring trap into an accurate estimate of the actual pest density has remained elusive. Using codling moth (CM) as a model organism, we document how novel methods of data analysis can be used to interpret catch data from single-trap multiple-release trapping grid experiments to reveal 1) the attractant plume reach from the trap, 2) the maximum dispersive distance of a population of responders, and 3) the proportion of the population in the trapping area caught. For CM responding to a standard pheromone-baited trap the proportion caught was .01-.02, the dispersive distance was 260 m, the plume reach was only about 2 m, and the trapping area was ca 53 acres. Recognizing the inherent weakness of using a single trap for CM management decisions while seeking to minimize the time and labor required to visit multiple traps distributed throughout an apple block, we conducted field experiments to quantify the benefits of a novel approach to monitoring that we have termed line trapping. The approach entails deploying 5 standard CM traps on 5 adjacent trees at the center of a large orchard block. Field experiments directly comparing the precision of a single trap versus line trapping for CM revealed that mean catch in a line of 5 traps was very similar to that for a single trap. Furthermore, variation in mean catch in the line of traps was substantially reduced over that for a single trap. This fundamental knowledge of how to space traps and interpret catch numbers will enable pest mangers to make more precise projections of damage and therefore more precise and reliable decisions on whether insecticide applications are justified.

29

NANOFIBERS ARE EXCELLENT DISPENSERS FOR GRAPHOLITA MOLESTA SEX PHEROMONES SUITABLE AS ATTRACTANTS IN BRAZILIAN PEACH ORCHARDS.

Bruna Czarnobai DE JORGE1*; Josué SANT'ANA1; Simone.S.LANGNER2; Hans E. HUMMEL2

1UFRGS, Porto Alegre, Rio Grande do Sul, Brazil 2Justus-Liebig-University Giessen, Giessen, Hesse, Germany

In order to address the challenge of controlling insect pests, insecticides still remain the most feasible option in Brazil. Recently, however, pheromones are presented as eco- friendly alternatives for monitoring and controlling insects. Unfortunately, commercially available dispensers still fall short of expectations in terms of costs and durability under field conditions. Peach and apple orchards in Southern Brazil suffer considerable yield production losses caused by the Oriental fruit moth Grapholita molesta (Busck, 1916) (Lepidoptera: Tortricidae). Considering this background, Hummel et al. (1915) conducted studies on implementation and development of novel matrices based on nanotechology in viticultural systems. The intention of our study was to evaluate the capture of G. molesta in peach orchards with polymeric nanofibers (PCL/PEG) containing sex pheromone. We compared it to the commercial product ISCAlure Grafolita (R) and verified the ability of these nanofibers to serve as vehicles for insect pheromone.The result obtained was: Dispensers for pheromones of G.(=Laspeyresia) molesta (Busck) are effective monitoring tools for up to five weeks. We now like to draw the attention of European and North American IPM managers to this effect inG.molesta after previous reports of successful mating disruption in Lobesia botrana (Hummel et al. 2015). SinceL.botrana and G.molesta are related pests belonging to the same genus, it is tempting to compare the management effects obtained under different conditions in two different countries.Details concerning this novel nanofiber technique, specially applied in peach orchards in Brazil in the State of Rio Grande do Sul, will be elaborated and discussed. We anticipate that this technology will soon be used for controlling other insect pests as well.

Reference: Hummel HE, Langner SS, Breuer M (2015). Electrospun mesofibers, a novel biodegradable pheromone dispenser technology, are combined with mechanical deployment for efficient IPM of Lobesia botrana in vineyards. Commun. Agric. Appl.Biol.Sciences, Ghent University 80(3), 331-341.

30

PROXIMATE MECHANISMS OF JASMONIC ACID-INDUCED DEFENSES IN SUGARCANE AGAINST A GENERALIST AND SPECIALIST HERBIVORE.

Patricia Alessandra SANCHES1*; Franciele dos SANTOS1; Maria Fernanda Gomes Villalba PEÑAFLOR2; José Maurício Simões BENTO2

1University of São Paulo, Piracicaba, São Paulo, Brazil 2University of Lavras, Lavras, , BRAZIL

Sugarcane has gained attention in the current scenario because of its potential to produce biofuel. However, sugarcane herbivore-induced defenses are unknown, and its yield is seriously compromised by insect attack. The current study investigated the effects of jasmonic acid (JA) on sugarcane resistance against the specialist herbivore Diatraea saccharalis, and the generalist Spodoptera frugiperda by means of antixenosis and antibiosis. We also measured the temporal dynamics of JA-induced volatile emission and tested the attraction to Cotesia flavipes, a D. saccharalis parasitoid. Results showed that JA-treated sugarcane did not affect mortality or weight gain of the two lepidopterous relative to untreated plant. However, in dual choice assays, both lepidopterous preferred to feed on untreated over JA-treated plant. Leaf colorimetric analyses showed that visual cues are unlikely to be involved on caterpillar preference, whereas olfactometry assays indicated that D. saccharalis prefer JA-induced volatiles over untreated plant. After 48h of treatment, JA-treated plant emitted an attractive volatile blend to C. flavipes, mainly comprised of sesquiterpenes. However, the parasitoid did not discriminate JA-treated over host-damaged plant volatiles. When exposed toJA-treated and JA-treated + host- damaged plants the parasitoid was able to identify host-damaged plant, indicating that minor alcohols are involved in the preference. We conclude that JA promotes antixenosis in sugarcane, but not antibiosis against herbivores. JA-induced volatiles are not involved in antixenosis, which is likely result of gustatory cues; however, they attract the parasitoid. In a scenario where all plants are treated with JA, the parasitoid is likely able to find its host.

31

REDUCE AN APPLICATION OF INSECTICIDES FOR THE USE OF PHEROMONES IN SOYBEANS.

Nilton Cezar BELLIZZI1*; Edgar Estevam de FRANÇA1; Eriki Yuki ITII1; Luiz Carlos Benício BRITO1; Vitor Hugo Malaquias dos Santos ASSUNÇÃO1; Tamires Campos LEAL1

1Universidade Estadual de Goiás - UEG, Palmeiras de Goiás, Goiás, Brasil

The utilization of pheromones in IPM program is the tools that presents the greatest potential for success, because there is not resistance by the insects or effect on mammals, particularly humans. In order to carry out a management application of insecticides in soybean crops, it was made in the 2014/2015 season at Fazenda Bom Sucesso - Property Buritis, located in Palmeiras de Goiás - GO, were monitoring of 200 ha with 60 traps with delta synthetic pheromones four groups of Lepidpteras, 15 pheromone traps for Helicoverpa zea and Helicoverpa armigera (Bio Helicoverpa - (Z) -11-Hexadecenal; (Z) - 9-Hexadecenal - 1.49 g/kg); 15 traps for Heliothis virescens (Bio Heliothis - (Z) -11- Hexadecenal; (Z) -9-Hexadecenal - 0.32 g/kg); 15 traps for Spodoptera frugiperda (Bio Spodoptera - Ethyl (Z) -11-Hexadecenila; Ethyl (Z) -7-Dodecenyl; Ethyl (Z) -9- Tetradecenyl - 0.00197 g/kg) and 15 traps for Chrysodeixis includens (Bio Pseudoplusia - Ethyl (Z) -7-Dodecenyl - 10.45 g/kg). Weekly traps were checked and changed floors, enveloped and stored in a freezer. Pheromones were changed every 30 days. The insects collected in each trap served to define the insecticide application season and the type of pesticide to be applied. The farm staff set to above 5 adults collected per trap per week the area was sprayed places below this number were not pulverizadas.Com this management, property reduced application by 50%, being held on average of 3 insecticide applications while in 2013/2014 season were held between 6 and 8 insecticide applications, depending on the area. In addition to the reduction in the number, the active ingredient of insecticides were also changed, while in the 2013/2014 harvest were applied Thiamethoxan, Lambda-cyhalothrin, Acefate, Flubendiamide and Clorantraniliprole in season 2014/2015 were used products based on Bacillus thuringiensis and Bifenthrin for control of caterpillar, which are less toxic than the former.

32

SEMIOCHEMICALS INFLUENCING THE BEHAVIOR OF THE LADY BEETLE TENUISVALVAE NOTATA (MULSANT) (COLEOPTERA: COCCINELLIDAE).

Elisabete SANTOS1; Christian TORRES1*; Maria MORAES2; Raul LAUMANN2; Miguel BORGES2

1UNIVERSIDADE FEDERAL RURAL DE , RECIFE, PERNAMBUCO, BRASIL 2EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DISTRITO FEDERAL, BRASIL

The lady beetle Tenuisvalvae notata (Mulsant) feeds preferentially on (Pseudococcidae). Recent studies show that its behavior can be mediated by semiochemicals, such as sexual and making pheromones. Thus, we investigated the semiochemicals released by adults T. notata and their effect on its behavior. We collected volatiles from 100 T. notata adults, separated by sex, in glass chambers containing nymphs of Ferrisia dasyrilii (Cockerell), and aerated for 20 consecutive days. The volatiles were collected using the adsorbent Porapak Q and eluted at each 24h. We also conducted aeration of the mealybug to differentiate the compounds released from prey and predator. The compounds present in the footprints of the lady beetles were collected with hexane from Petri dishes impregnated with footprints of groups of 10 beetles (males or females) for a period of 24h. Chemical analyses of compounds obtained from aerations and footprints were analyzed by GC/MS and GC/FID. The results of analyses of aeration extracts of males and females showed the presence of 22 compounds, including a series of carboxylic acids, such octanoic, decanoic and dodecanoic acids, this last one in higher quantity in female extracts; and also showed an specific male nitrogen compound. Meanwhile, in the footprints were found 11 compounds in common to both sexes, from these only five were identified, such as pentacosane, heptacosane and nonacosane, all straight chain and 25 or more carbons. Results of behavioral studies suggest that the extracts obtained both volatile aeration as the footprints (less volatiles) could cause some irritability on insects, marking the substrate to reduce competition between adults. However, more studies are needed to clarify the importance of those semiochemicals in the behavior of T. notata.

33

SUSTAINABLE CHEMICAL TOOLS TO IMPROVE HONEYBEE FORAGING EFFICIENCY IN A POLLINATOR-DEPENDENT CROP.

Cecilia ESTRAVIS BARCALA1; Florencia PALOTTINI1; Walter Marcelo FARINA1*

1Laboratorio de Insectos Sociales, Departamento de Biodiversidad y Biología Experimental, IFIBYNE-CONICET, FCEyN, Universidad de Buenos Aires, Buenos Aires, CABA, ARGENTINA

Honeybees (Apis mellifera Linnaeus) are the most important pollen vectors in agricultural ecosystems. In particular, they are considered essential pollinators in sunflower (Helianthus annuus Linnaeus) crops for hybrid seed production where honeybees have to transfer pollen from male fertile (MF) to male sterile (MS) cultivars. However, this commercial crop presents challenges for insect pollinators such as a variable dimorphism among parental lines that hinders cross-pollinating by honeybee [1]. This commercial crop offers moreover low quality pollen for insects [2]. Our laboratory has already developed a Synthetic Mixture (SM) [3] of VOCs that honeybees cannot discriminate from the natural blend of sunflowers, a fact that improves the activity of the beehives located in the surrounding of these crops [4]. The aim of this study was to evaluate i) the role of this procedure by focusing on the honeybees that foraged on MF and MS cultivars of the crop, and ii) whether a Stimulant Supplement, a mix of antioxidant and natural stimulant compounds (SS), improved the action of the SM on crops. Our results showed that a higher number of bees visited MF and MS cultivars in the surrounding of the SM treated colonies. This number is even higher for the SM+SS treatment. In addition, hives treated with the SM+SS showed higher levels of activity. These results suggest that the synthetic mixture could be used to guide honeybees towards sunflower crops, while the stimulant supplement well complements its role enhancing honeybee foraging behavior.

[1] Susic Martin C, Farina WM (2016). Apidologie, 47:161–170.

[2] Schmidt, L.S., Schmidt, J.O., Rao, H., Wang, W., Xu L. (1995). J. Econ. Entomol. 88, 1591-1595

[3] Farina WM, Díaz PC, Arenas A (2009). Patent application: P 20090104013; INPI.

[4] INNOVAR (2013). Catálogo 2013, 86-89.

This study was supported by funds from CONICET, UBACYT and ANPCYT grants to WMF.

34

SYNTHESIS OF MEALYBUG SEX PHEROMONES AND THEIR APPLICATION IN FIELD MATING DISRUPTION TRIALS.

Takeshi KINSHO1*; Tatsuya FUJII1; Miyoshi YAMASHITA1; Erina OHNO1

1Shin-Etsu Chemical Co., Ltd., Joetsu City, Niigata, Japan

Mealybugs (Hemiptera: Pseudococcidae) are wide-spread sap-sucking pests that attack various plants in tropical, sub-tropical and temperate zones in the world. They are very important pests in agriculture because of their serious direct and indirect damage to crops in various regions. The pest control using conventional insecticides is sometimes very difficult due to powdery wax-covered surface. Pheromone-based mating disruption (MD) has been considered as an alternative eco-friendly pest control measure for these insects. Sex pheromones of some mealybug species - the Vine mealybug (Planococcus ficus SIGNORET), the Japanese mealybug (Planococcus kraunhiae KUWANA), the Passionvine mealybug (Planococcus minor MASKELL) and the Grey pineapple mealybug (Dysmicoccus neobrevipes BEARDSLEY) are irregular monoterpene esters, possessing the common C-10 framework. Synthesis of these pheromones has been studied toward synthetic process suitable for industrial scale-up in order to supply sufficient amounts of the active ingredients for actual application in the field. Polymer tube dispensers were designed and manufactured for controlled release of the synthetic pheromones, and field mating disruption trials for vine mealybug in Europe and South Africa and Japanese mealybug in Japan have been conducted since 2013. MD efficacy was demonstrated through clean trap shutdown of males, suppression of populations, and damage reduction in crops. Pheromone-based mating disruption of mealybugs would be one of the promising tools for Integral Pest Management for these annoying hemipteran pests.

35

SEXUAL BEHAVIOUR OF EPICAUTA ATOMARIA (COLEOPTERA: MELOIDAE), A PEST OF HORTICULTURAL CROPS, A PRELIMINARY STUDY.

Leandro WAGNER1*; Paula CAMPOS-SOLDINI1; Pablo GUERENSTEIN1; Melania SAFENRAITER1; Estrella FERNANDEZ1

1Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción (CICYTTP-CONICET), Diamante, Entre Ríos, Argentina

Epicauta atomaria, which belongs to the E. maculata complex, is an important pest of different horticultural crops, including chard; potato; beetroot. It is found in South America and is abundant in low-lying roadside habitats adjacent to grazed areas or crop. Nothing is known about the sexual behavior of this species although the sexual behavior of some related North American species has been described. An understanding of its sexual behavior could be important for the development of methods of monitoring and control of this pest using synthetic pheromones. We recently started to study the sexual behaviour of E. atomaria. For this, couples of E. atomaria are placed in glass containers under controlled light and temperature conditions. Their behaviour is recorded with a digital camera for at least 10 minutes. Preliminary observations suggest that the sexual courtship of E. atomaria includes a display during which the male walks behind the female and rubs and/or presses the antennas on the elytra of the female. Males rarely used their maxillary palps to contact the female. This display lasts almost a minute and is usually followed by attempts of the male to mount the female. This suggests that contact sex pheromones may exist, as in the case of other meloids. We plan to continue characterizing the sexual behaviour of this meloid so that identification of putative pheromones could be carried out.

36

TESTING AN ‘ATTRACT-AND-REWARD’ APPROACH TO INCREASE ECOSYSTEM SERVICES FROM NATURAL ENEMIES IN AGRICULTURE

Jordano SALAMANCA1*; Cesar RODRIGUEZ-SAONA2; Brígida SOUZA3

1Universidade Federal de Lavras, Lavras, MInas Gerais, Brazil 2Rutgers University, NEW BRUNSWICK, New Jersey, United States 3Universidade Federal de Lavras, Lavras, Minas Gerais, Brazil

Conservation biological control can be improved by the use of multiple tactics to manipulate natural enemy foraging behavior such as the use of synthetic herbivore- induced plant volatiles (HIPVs) and floral resources in an ‘attract-and-reward’ approach. However, whether ‘attract-and-reward’ leads to enhanced ecosystem services provided by natural enemies in agro-ecosystems remains unknown. Here, we tested the novel hypothesis that HIPVs (i.e., attract component), alone or in combination with companion plants (i.e., reward component) reduce pest populations and increase crop yield. Treatments consisted of: 1) common bean (Phaseolus vulgaris) plants alone; 2) bean baited with methyl salicylate (MeSA), an HIPV known to attract insect predators; 3) bean with coriander, as a companion plant; and 4) bean with MeSA and coriander. The experiment was a complete randomized block design, with each treatment replicated 14 times in two growing seasons. Predator and herbivore abundances were measured weekly. At the end of the season, plants from all treatments were harvested to measure crop size and yield. At the predator level, lady beetles, predaceous stink bugs, and syrphids were attracted to MeSA and coriander, either alone or in combination. At the herbivore level, MeSA reduced mite populations in combination with coriander. At the plant level, MeSA also interacted with coriander to reduce crop damage but had no effect on crop size or yield. In conclusion, our study is the first to show that manipulation of natural enemies by combining HIPVs and companion plants in an ‘attract-and-reward’ approach could reduce pest populations and damage in an agricultural crop; however, these results did not translate to an increase in crop size or yield. The implications of these results for conservation biological control will be discussed.

37

TRACKING THE EUROPEAN CORN BORER OSTRINIA NUBILALIS NORTHWARD EXPANSION IN SCANDINAVIA USING PHEROMONE TRAPS.

Olle ANDERBRANT1*; Joana F. MARQUES1; Louise ALDÉN2; Ghita CORDSEN NIELSEN3; Glenn P. SVENSSON4

1Department of Biology, Lund University, Sölvegatan 37, Lund, n/a, Sweden 2Swedish Board of Agriculture, P.O . Box 12, Alnarp, n/a, Sweden 3Seges P/S, Agro Food Park 15, Aarhus, n/a, Denmark 4Department of Biology, Lund University, Sölvegatan 3, Lund, n/a, Sweden

Background: Climate change causes range shifts in many plants and animals. During the last decades, cultivation of some crops has rapidly expanded northward. With the crops follow associated diseases and insects causing damage, if they can adapt to new environments. The agricultural sector calls for more research to be able to monitor, predict, and manage the new situations. Maize production has increased rapidly in Sweden, and in 2010 the European corn borer (ECB), Ostrinia nubilalis, maize host-race (Z pheromone strain) was first documented in this country1. Aim: To determine the occurrence and phenology of the two host races (or pheromone strains E and Z) of ECB in Sweden and Denmark using pheromone traps and to verify strain identity of catches and collected larvae using genetic markers. Methods: Traps baited with E11- and Z11- tetradecenyl acetate ratios mimicking the sex pheromone blend of the two host-races and hybrids were placed in maize fields in Denmark and southern Sweden. The host-race identity of trapped males and larvae collected from maize stalks was determined by using Restriction Fragment Length Polymorphism2. Findings: Genetic analyses confirmed the strain specificity of the lures used. We caught similar numbers of Z- and E-strain males in traps. Catches were low, indicating an early stage of population establishment in these areas. Conclusions: The maize-ECB system in Scandinavia offers an excellent opportunity to study the spread of recently established populations of a pest in relation to climate change and host abundance. We will build a pro-active framework, in collaboration with growers and relevant organizations, which might also be used in other crop-pest systems and for modelling the consequences of management strategies on crop yield.

1 Lehmhus et al. 2012. J Kulturpfl 64:163-167

2 Coates BS et al. 2013. Ecol Evol 3:249-2470

38

USE OF SEMIOCHEMICALS FOR AEDESAEGYPTI SURVEILLANCE AND CONTROL.

Daniel L. KLINE1*

1USDA-ARS, CMAVE, Gainesville, Florida, USA

Aedesaegypti utilizes semiochemicals throughout its life cycle. This presentation will focus on this species' utilization of semiochemicals as adults to locate mates, nectar sources, hosts for blood feeding and oviposition sites. Studies have been conducted in the laboratory (olfactometer studies), semi-field (large outdoor screened cages) and in limited field studies. Major emphasis has been placed on those semiochemicals utilized by adult females to locate human hosts. Over 300 volatile compounds have been identified from human hosts. Many of these have been evaluated in laboratory olfactometer studies. Various combinations have been studied. Several effective three component blends have been identified for attracting host seeking females. Several compounds have also been found that are effective in causing anosmia in host seeking females. Various infusions/chemicals have been evaluated in the laboratory and field as oviposition attractants. Floral volatiles have been evaluated for both males and females that may be utilized to locate nectar sources. Similar studies have been conducted with Aedesalbopictus. In laboratory and field studies flowering plants have been used in oviposition studies in which gravid females seem to increase their activity at a given site. It appears as if both species will forgo their skip oviposition behavior if certain flowering plants are present. The identified semiochemicals are being utilized in the development of new devices as both attractants and repllents. An objective of this research is to develop novel surveillance and population management strategies. One strategy will utilize a combination of these semiochemicals in a push-pull strategy.

39

USE OF KAIROMONES TO ENHANCE THE BISEXUAL CATCH OF CODLING MOTH (CYDIA POMONELLA) AND ORIENTAL FRUIT MOTH (GRAPHOLITA MOLESTA) (LEPIDOPTERA: TORTRICIDAE) IN POME AND STONE FRUIT ORCHARDS.

Eduardo FUENTES-CONTRERAS1*; Liliana CICHÓN2; Esteban BASOALTO3; Wilson BARROS-PARADA3; Valentina MUJICA4; Alan L. KNIGHT5

1Universidad de Talca, Talca, Talca, chile 2INTA-Alto Valle, General Roca, Rio Negro, Argentina 3Universidad Austral de Chile, Valdivia, Los Rios, Chile 4INIA-Las Brujas, Rincón del Colorado, Canelones, Uruguay 5USDA-ARS-Wapato, Yakima, Washington State, USA

Studies were conducted in North and South America during 2012-2016 to evaluate the use of lure combinations of sex pheromones, host plant volatiles, and food baits in delta traps to capture codling moth (Cydia pomonella) and oriental fruit moth (Grapholita molesta) in pome and stone fruit orchards. Studies were conducted with several commercial and experimental lures. Standardized protocols were used in Argentina, Chile, Uruguay and USA. Our results indicate that combining the sex pheromone of both species significantly increased the catch of G. molesta, but affect negatively the captures of C. pomonella. Adding beta ocimene and pear ester significantly increased the activity of the pheromone blend for G. molesta and C. pomonella, respectively. Terpinyl acetate was the only host plant volatile that significantly improved bisexual catches of G. molesta. For C. pomonella the mix of pheromone, acetic acid and the plant volatile dimethyl nonatriene caught greater number of moths, especially females, than the other lure combinations under mating disruption management. Adding microbial derived food attractants, such as acetic acid increased moth catches of both species, especially of females.

40

USE OF SEX PHEROMONE OF PROEULIA AURARIA (LEPIDOPTERA: TORTRICIDAE) FOR MONITORING IN ORCHARDS.

M. F. FLROES1*; T. CURKOVIC2; C. BALLESTEROS2; D. de la TORRE3; A. VALVERDE4

1Pontificia Universidad Católica de Valparaíso, Valparaíso, Valparaíso, Chile 2Universidad de Chile, Santiago, Santiago, Chile 3Pontificia Universidad Católica de Valparaíso, Valparaíso, Valparaíso, Chile 4Universidad de Chile, Santiago, Santiago, Chile

The Chilean fruit leaf roller, Proeulia auraria Clarke (Lepidoptera: Tortricidae), is a polyphagous insect native to Chile. This species is distributed mainly in the central- southern region, and its host plants include economically important species, such as stone and pome fruit trees, grapes, citrus, blueberries, etc. Its sex pheromone was recently identified as a four component mixture that includes tetradecyl acetate (14:OAc), (E)-11- tetradecenyl acetate (E11-14:OAc), (Z)-11-tetradecenyl acetate (Z11-14:OAc), and (E)- 11-tretadecen-1-ol (E11-14:OH).Field tests with synthetic pheromone were carried out in grapes, blueberry, and apple organic and conventional orchards, during two seasons, from September 2014 to April 2016. Lures were prepared with E11-14:OAc and E11-14:OH in a proportion 3:1, dissolved in hexane, loaded in white rubber septa, and placed inside delta traps (Pherocon IV, Trécé Inc). Dose, lure age, and proportion of components were tested under field conditions. The results revealed that P. auraria has two complete generations, observing two flights: the first one, between October-November, where adults come from overwinter larvae, and the second one, between February-March . For pheromone monitoring, is recommendable to use lures with 260 µg of pheromone and replace them every 10 weeks.

Acknowledgments: Fundación para la innovación agraria, FIA grant PYT 0014-2014.

41

USE OF SYNTHETIC PHEROMONES TO DETERMINE THE POPULATION OF TACHINIDS IN AN SOYBEAN CULTIVE.

Nilton Cezar BELLIZZI1*; Alirio Felipe Alves NETTO1; Edgar Estevam de FRANÇA1; Geovana Pires GOMES1; Raniely Ferreira de MEDEIROS1; Selma Divina SOUSA1; Tamires Campos LEAL1

1Universidade Estadual de Goiás - UEG, Palmeiras de Goiás, Goiás, Brasil

Diptera is the fourth largest order of insects, is common in many environments. In agriculture, the Tachinidae family is one of the most abundant and are parasitoids of various insects of the Lepidoptera, Lepidoptera, Coleoptera, Hymenoptera, Heteroptera and Orthoptera. Tachinids may be regarded the economically beneficial when they develop in pests of agriculture. Realizing the management application of insecticides in soybean crops, it was made in the 2014/2015 season at Fazenda Bom Sucesso - Property Buritis, located in Palmeiras de Goiás - GO, in of 200 ha with 60 traps with delta synthetic pheromones four groups of Lepidpteras (Helicoverpa spp, Heliothis virescens, Spodoptera frugiperda and Chrysodeixis includens), using as the main active ingredient (Z) -11-Hexadecenal, (Z) -9-Hexadecenal, Z-7 dodecenyl acetate and its variations. They were observed attracting Tachinids by these traps and collected an average of 10 to 15 adults of Tachinids per cm2, which came to be 50 times greater than the average number of moths collected (0.3 moths per cm2). With this Tachinids population collected in the traps, the caterpillars parasitized ratio in soybean was investigated, verifying that 90% of the caterpillars were collected with one or more eggs Tachinids. These data were used by the farm staff to reduce the use of insecticides in some areas and change the active principle for selective insecticides to these natural enemies, adopting the proportion of 30 to 50 times the population of Tachinids relative to Lepidoptera adult collected, it would not apply insecticides in the area. If this ratio was less than 30 Tachinids by moth, the insecticide to be applied would be specific for Lepidoptera, as Novaluron (Rimon 100 CE) and Triflumuron (Certero).

42 CHEMICAL ECOLOGY OF INSECTS AFFECTING PEOPLE

A NEW TYPE OF SYNERGISM BY BEHAVIOR MODIFICATION IN BLOOD- SUCKING BUGS SIMULTANEOULY EXPOSED TO EUGENOL AND AZAMETHIPHOS.

MM Noel REYNOSO1*; Emilia SECCACINI1; Raúl A ALZOGARAY2; Eduardo N ZERBA2

1Centro de investigaciones de plagas e insecticidas (CITEDEF-CONICET), Villa Martelli, Buenos Aires, Argentina 2Centro de investigaciones de plagas e insecticidas (CITEDEF-CONICET). 3IA- UNSAM., Villa Martelli, Buenos Aires, Argentina

Triatoma infestans is the main vector of Chagas disease in Argentina and other Latin American countries. Due to the emergence of resistance to pyrethroid insecticides in this species, it is necessary to develop innovative tools for controlling it. The objective of this work was to evaluate a possible synergism of eugenol on the toxicity of the organophosphate azamethiphos. Eugenol is a botanical monoterpene that produces hyperkinesis in T. infestans. With this background in mind, we hypothesized that an insect exposed to a surface treated with eugenol and azamethiphos would suffer greater toxicity than if it were exposed to azametiphos alone. The cause of this synergism would be an increase in exposure to the organophosphate caused by the increase in the locomotor activity.First instar nymphs of T. infestans were used in all experiments. Ethovision (XT10) software was used to quantify locomotor activity of insects exposed to filter papers impregnated with azamethiphos (25, 50 or 100 µg/cm²) or eugenol (49, 97, 195 or 390 µg/cm²). Hyperkinesis was observed only in insect exposed to 195 or 390 µg/cm² of eugenol. In another experimental series, insects were exposed to azamethiphos alone (12, 50 or 200 µg/cm²), or mixed with eugenol (400 µg/cm²). Values of Lethal Concentration 50% (LC50) were 53 µg/cm² for azamethiphos alone, and 6.4 µg/cm² for the mixture. This result supports the hypothesis that hyperkinetic insects incorporate more azamethiphos than those with a normal locomotor activity. We will continue exploring this synergism to assess their potential application in controlling T. infestans.

44

AGGREGATION BEHAVIOR IN SHELTERS IN RHODNIUS PROLIXUS AND RHODNIUS ECUADORIENSIS.

Katherine D. MOSQUERA1; Marcelo G. LORENZO1*

1Centro de Pesquisas René Rachou-Fiocruz, BELO HORIZONTE, MINAS GERAIS, Brasil

Chagas disease is caused by the protozoan Trypanosoma cruzi, which is mainly transmitted through infected feces of heteropteran insects belonging to the family Reduviidae, subfamily Triatominae. These hematophagous nocturnal insects leave their shelters at dusk searching for food sources and return to them before dawn, where they spend daylight hours typically motionless in hidden and protected places, aggregated with other members of the population. Ttriatomine aggregation behavior is mediated by chemical signals and thigmotaxis. The existence of aggregation to volatile compounds emitted by feces has been demonstrated for many triatomine species. These signals even promote cross-specific aggregation among several triatomine species. Recently, five compounds commonly emitted by the feces of Triatoma infestans, Panstrongylus megistus and Triatoma brasiliensis have been reported indicating that a synthetic mixture of these compounds actively recruits these insects inside shelters. Given the above, it is important to determine whether this chemical mixture has the potential to promote shelter choice in other triatomine vectors. The aim of our study was to characterize aspects of aggregation in shelters of R. prolixus and R. ecuadoriensis. In this work, a square arena (1 m2) was used to separately test the aggregation of R. prolixus and R. ecuadoriensis to shelters that contained papers impregnated with their respective feces. Furthermore, we tested different doses (16 ng, 160 ng and 1.6 µg) of the five compound blend previously reported to actively recruit insects into shelters for 3 triatomine species. The results obtained with both species show that shelters impregnated with feces, as well as those impregnated with the five substance blend, were not significantly chosen when compared to control ones. The significance of these results in terms of the use of aggregation signals by triatomines will be discussed comparatively.

Funding: FAPEMIG, INCT-EM, CNPq and FIOCRUZ.

45

ARRESTANT EFFECT OF HUMAN SCALP COMPOUNDS ON PEDICULUS HUMANUS CAPITIS BEHAVIOUR.

Isabel ORTEGA INSAURRALDE1*; Ariel Ceferino TOLOZA1; Paola GONZALEZ AUDINO1; María Inés PICOLLO1

1Centro de investigaciones de plagas e insecticidas (CITEDEF-CONICET), Villa Martelli, Buenos Aires, Argentina

The human head louse, Pediculus humanus capitis (Phthiraptera: Pediculidae), is an obligate ectoparasite that causes Pediculosis capitis and has been in contact with humans since the beginning of humankind. Head lice depend exclusively on the human scalp microenvironment which provides them with refuge, food and oviposition sites during their entire life cycle. Unfortunately, there is no information about the chemical communication that occurs when a louse interacts with its host.Host related cues have been widely shown to induce stereotyped behaviour in the parasites and epidemiological reports indicate the presence of differential levels of infestation among humans. Considering it, we proposed the hypothesis that human scalp compounds could modify the behaviour of head lice.In this work, we studied the effect of human scalp compounds on the behaviour of adult head lice. Filter paper segments were rubbed on volunteers scalps during 30 seconds and then were placed in the test arena. The movement of the insects was recorded using the software EthoVision®. Average movement parameters were calculated for the treatments in the bioassays such as total distance, velocity, frequency in the zone transition and time in each zone of the arena.We found that scalp compounds induced head lice to decrease average locomotor activity and to remain arrested on the treated paper. We also observed that head lice, in closeness with human scalp substances, performed behaviour presumably associated to the search for blood on the skin such as stillness, body raising perpendicular to the surface, and head disposition near the surface. Our results showed for the first time the effect of host compounds conditioning head lice behaviour. We discuss the role of these compounds in the dynamics of head lice infestation.

46

BED BUGS (HEMIPTERA:CIMICIDAE) RESISTANT TO INSECTICIDES ALSO SHOW A REDUCED BEHAVIORAL RESPONSE TO REPELLENTS.

Mariano CÁCERES1*; Pablo SANTO ORIHUELA1; Raúl A. ALZOGARAY1; Claudia VASSENA1

1(CIPEIN) UNIDEF - CITEDEF - CONICET | Cidade: VILLA MARTELLI . 3IA- UNSAM., Villa Martelli, Buenos Aires, Argentina

The increasing infestations of bed bugs and resistance to different classes of insecticides represent a new challenge to manage this pest. In particular, repellents are a good option to control populations when they begin to be resistant. In this study, we evaluated the repellence produced by synthetics chemicals on Cimex lectularius from laboratory and field-collected strains.The experiments were performed with the susceptible Harold Harlan strain, and four strains collected from different Argentinian localities and maintained in laboratory conditions. Previously, we identified pyrethroid and carbamates resistance in the field-collected strains. The arena was a circle filter paper divided in two zones with equal areas: an inner circle and an outer ring. A glass ring was placed on the paper to avoid bed bugs to scape. Technical grade chemicals used were N,N-Dietil-meta- toluamida (DEET), ethyl 3-[acetyl(butyl)amino]propanoate (IR3535) and δ- dodecalactone. Acetone was used as solvent. The outer zone of the arena was impregnated with 0.5 µl of repellent solution (1.40 mg/cm2). Arenas treated with acetone alone were used as controls. Five fifth instar nymphs were gently placed in the middle of the arena. The location of individuals was recorded visually each five minutes, during 1 hour. We calculated a Repellence Index for each strain and repellent: [(Nc–Nt)/Nc]x100, where Nc is the number of individuals in the outer zone in the control, and Nt is the number of individuals in the outer zone in the treated paper. Three replicates were carried out for each treatment.DEET and δ-dodecalactone were highly repellent to the Harold Harlan nymphs. However, IR3535 showed a low repellent effect to this strain. In all cases, resistant-strains showed significant lower repellency than the susceptible-strain. Differences in the effectiveness of these repellents and its possible relationship with detoxifying metabolism will be discussed.

47

BEHAVIORAL RESPONSE OF ANOPHELES PSEUDOPUCTIPENNIS AND AEDES AEGYPTI LARVAE TO ATTRACTANT AND REPELLENT SUBSTANCES: A VIDEO-TRACKING ANALYSIS.

Paula GONZALEZ1*; Agustín ALVAREZ COSTA1; Héctor MASUH1

1Centro de Investigaciones de Plagas e Insecticidas (CIPEIN), UNIDEF-CITEDEF- CONICET, Villa Martelli, Buenos Aires, Argentina

Anopheles pseudopunctipennis (Theobald) and Aedes aegypti (L.) are vectors of Malaria and the Dengue Fever in Latin-America. The ability to detect a wide range of sensory cues is essential for the survival and vectorial capacity of mosquitoes. This investigation focuses on the analysis of olfactory-driven behavior in An. pseudopunctipennis and Ae. aegypti larvae to respond to synthetic or natural odorant stimuli. In our laboratory we used the video-tracking software (Etho Vision XT10.1) and established a simple and sensitive behavior assay. Single fourth instar larvae were recorded in an experimental arena, and behavioral traits were analyzed and quantified to a range of two odorant stimuli in both species. The odorant were N, N-diethyl-m-toluamide (Deet), commercial insect repellent, and yeast, a normal component of larval food.In both species, the results were consistent with the exito-repellency hypothesis. When larvae were exposed to a repellent compound, such as DEET, they exhibit an increase in overall movement and velocity. In contrast, yeast, a known attractant, leads to a reduction in the movement and velocity of larvae, while the average resting time is increased. However, the intensity of the effects of these compounds were weaker to Anopheles than Aedes, because Anopheles larvae have few movements and they were abrupt in comparison with the movements of the other specie that were more constant in an ondulating way. The study of the behavior of attraction and repellency is very important, as it could contribute to the understanding of the mechanism of olfaction in An. pseudopuctipennis and Ae. aegypti larvae and lay the foundation to advance in alternatives to mosquito control strategies focused on adult life stages. This work was supported by the Agencia Nacional de Promoción Científica y Técnica (PICT 2012- 1857).

48

DEETS ON MOSQUITO REPELLENTS FOR PREVENTION OF ZIKA INFECTION.

Fang Fang ZENG1; Kaiming TAN1; Pingxi XU1; Young-Moo CHOO1; Rosângela M. R. BARBOSA2; Constância F. J. AYRES2; Walter S. LEAL3*

1Department of Molecular and Cellular Biology, University of California-Davis, Davis, California, USA 2Department of Entomology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Pernambuco, Recife, Brazil 3Department of Molecular and Cellular Biology, University of California-Davis, Davis, California, Brazil

The Zika virus (ZIKV) is spreading rapidly in Latin American and is likely to become an epidemic in the United States and other countries. It was already declared as a global public health emergence by the World Health Organization. After all, ZIKV is transmitted sexually and by infected mosquitoes and, more importantly, Zika infection in pregnancy causes severe fetal brain malformation, and other medical problems. It is commonly accepted that Aedes aegypti and Ae. albopictus are the main vector of ZIKV, but it is worth noting that up to the time of this writing not a single ZIKV-infected Aedes mosquito has been detected in the main outbreak areas, including Brazil, despite several attempts. Additionally, recent findings suggest that the common mosquito, Culex quinquefasciatus, is a potential ZIKV vector. Therefore, prophylactic measures to reduce virus dissemination must include prevention of mosquito bites by both Culex and Aedes mosquitoes. Here, we will discuss details (i.e., deets) on mosquito repellents for prevention of Zika infection. We will demystify the notion that the most expensive is the best and argue that to date DEET is still the gold standard of mosquito repellents. Additionally, we will discuss that some homemade repellent recipe may be misleading.

49

EFFECTS OF ALKALOIDS ON FEEDING BEHAVIOR AND METABOLISM OF RHODNIUS PROLIXUS, VECTOR OF CHAGAS DISEASE.

Ignacio J. MUÑOZ1; Pablo E. SCHILMAN1; Romina B. BARROZO1*

1Universidad de Buenos Aires, IBBEA, CONICET-UBA, Buenos Aires, Buenos Aires, Argentina

Through the sense of taste, an individual can evaluate what to eat and drink favoring consumption of nutritious and avoiding intake of harmful and toxic substances that can induce discomfort, illness or death. Aversive substances can lead an to avoid feeding, even if the alimentary source is not toxic per se. In different groups of insects, including triatomines (Reduviidae: Triatominae), it has been demonstrated that alkaloids stimulate taste receptors generating an aversive response. However, it is still not clear if the ingestion of alkaloids produces a physiological change in triatomines. In this work, we investigated the effect of different alkaloids with anti-appetitive potential in the feeding behavior of Rhodnius prolixus, and we analyzed its impact on their metabolism. For this purpose, we used an artificial feeder with different solutions at 37 ± 2 °C where the insects fed for 10 minutes. The effect on feeding behavior and metabolism was tested offering an appetitive solution (AS) of ATP in NaCl 0,15M alone or in combination with the potential anti-appetitive alkaloids: caffeine, quinine, and theophylline. We calculated the insect weight gain as feeding response and CO2 production as an indirect measurement of metabolism. CO2 production was measured by open flow respirometry for individual insects after being fed and three hours later. We found that R. prolixus detect alkaloids in a dose-dependent manner, decreasing feeding frequency and intake amount at higher concentrations. For all treatments, CO2 production slightly decreases after three hours of feeding. Also, CO2 production was about five times higher for insects that consumed caffeine and theophylline compared to insects that consumed AS or quinine, suggesting costs associated with mechanisms of detoxification. The understanding of this molecules’ mode of action would help to develop new tools designed to control and reduce host-vector interaction. Financial support by ANPCyT (PICT 2013-1253) and CONICET.

50

EVALUATION OF THE ACTIVITY OF THE ESSENTIAL OIL FROM AN ORNAMENTAL FLOWER AGAINST AEDES AEGYPTI: ELECTROPHYSIOLOGY, MOLECULAR DYNAMICS AND BEHAVIORAL ASSAYS.

Patrícia C. BEZERRA-SILVA1; Kamilla A. DUTRA1; Geanne K. N. SANTOS1; Rayane C. S. SILVA1; Jorge IULEK2; Paulo MILET-PINHEIRO3; Daniela M. A. F. NAVARRO3*

1UFPE, Recife, Pernambuco, Brazil 2UEPG, Ponta Grossa, Paraná, Brazil 3UFPE, Recife, Pernambuco, Brazil

Dengue fever has spread worldwide and affects millions of people every year in tropical and subtropical regions of Africa, Asia, Europe and America. Since there is no effective vaccine against the dengue virus, prevention of disease transmission depends entirely on regulating the vector (Aedes aegypti) or interrupting human-vector contact. The aim of this study was to assess the oviposition deterrent activity of essential oils of three cultivars of torch ginger (Etlingera elatior, Zingiberaceae) against the dengue mosquito. Analysis of the oils by gas chromatography (GC) —mass spectrometry revealed the presence of 43 constituents, of α-pinene, dodecanal and n-dodecanol were the major components in all cultivars. Solutions containing 100 ppm of the oils exhibited oviposition deterrent activities against gravid Ae. aegypti females. GC analysis with electroantennographic detection indicated that the oil constituents n-decanol, 2- undecanone, undecanal, dodecanal, trans-caryophyllene, (E)- -β- farnesene, α-humulene, n-dodecanol, isodaucene and dodecanoic acid were able to trigger antennal depolarization in Ae. aegypti females. Bioassays confirmed that solutions containing 50 ppm of n- dodecanol or dodecanal exhibited oviposition deterrent activities, while a solution containing the alcohol and aldehyde in admixture at concentrations representative of the oil presented an activity similar to that of the 100 ppm oil solution. Docking and molecular dynamics simulations verified that the interaction energies of the long-chain oil components and Ae. aegypti odorant binding protein 1 were quite favorable, indicating that the protein is a possible oviposition deterrent receptor in the antenna of Ae. aegypti.

51

HOW TO FIND HOST LARVAE IN A MILL: LONG-RANGE HABITAT AND HOST ATTRACTANTS AND CONTACT HOST KAIROMONES SHAPE THE FORAGING BEHAVIOR OF A LARVAL ECTOPARASITOID OF TRIBOLIUM.

Benjamin FÜRSTENAU1*; Monika HILKER1

1Freie Universität Berlin, Dahlem Centre of Plant Sciences, Institute of Biology, Applied Zoology/Animal Ecology, Berlin, Berlin, Germany

Here, we studied the chemically mediated host foraging behavior of the ectoparasitoid Holepyris sylvanidis (Hymenoptera, Bethylidae) which attacks larvae of the confused flour beetle Tribolium confusum (Coleoptera, Tenebrionidae). This beetle is one of the most important stored product pests in the food processing industry worldwide. While previous studies focused on long-range attraction of the parasitoid to its host by volatile cues, our recent studies addressed the relevance of contact kairomones for host location and host acceptance by the wasp. The previous olfactometer tests revealed that H. sylvanidis is highly attracted to host larval feces, in particular to two specifically host- associated components of the fecal odor, 1-pentadecene and (E)-2-nonenal. Volatile compounds ubiquitously present in the host´s habitat enhanced the parasitoid’s response to these host-specific cues. Thus, a blend of host-specific compounds and habitat odor serves as long-range attractant.We further investigated short-range attraction by H. sylvanidis and tested the hypotheses that female parasitoids i) utilize cuticular hydrocarbons of their host larvae for host recognition and ii) follow these hydrocarbons left by host larvae on the substrate. Indeed, the wasps recognized the host larvae by means of cuticular hydrocarbons and they were able to trail host cuticular extracts. GC- MS analysis of the cuticular hydrocarbon profile of T. confusum larvae revealed a blend of 23 long-chain linear and methyl-branched hydrocarbons (C25-C30). The time until a host larva was located significantly decreased when additional trails of host larval cuticular extracts were laid and also, when host feces were provided. Hence, fecal odor is crucial for olfactory long-range search by the parasitoid H. sylvanidis, but also supports the contact-mediated short-range host location.

52

INDEPENDENT AND JOINT ACTION OF THE REPELLENTS DEET E IR3535 ON TRIATOMA INFESTANS.

M.M. Noel REYNOSO1*; Javier A. CALCAGNO2; Eduardo N. ZERBA3; Raul A. ALZOGARAY3

1UNIDEF, CITEDEF, CONICET, CIPEIN, Villa Martelli, Buenos Aires, Argentina 2Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico (CEBBAD), Departamento de Ciencias Naturales y Antropológicas, CONICET, Buenos Aires, --, Argentina 3UNIDEF, CITEDEF, CONICET, CIPEIN. 3IA-UNSAM, Villa Martelli, Buenos Aires, Argentina

N,N-diethyl-3-methylbenzamide (DEET) and ethyl 3-[acetyl(butyl)amino]propanoate (IR3535) are synthetic insect repellents used worldwide to protect people from bites of blood-sucking . Triatoma infestans (Hemiptera: Reduviidae) is the main vector of Chagas disease in Argentina and neighboring countries. The objective of this work was to evaluate the repellence produced by these substances, applied individually and mixed, on fifth instar nymphs of T. infestans. The experimental arena consisted of a circle of filter paper (diam.: 11 cm). Half the circle was treated with a solution of repellent in acetone; the other half, with acetone alone. A nymph was gently placed on the middle of the arena, and during the next ten minutes its position was recorded using an image analyzer (Videomex V, Columbus). The following concentrations of pure DEET and IR3535 were tested: 0 (control), 0.38, 1.15, 3.5, 10.4 and 31.0 µg/cm2. Three mixtures of the two repellents were also evaluated (DEET:IR3535 1:1, 3:1 and 1:3). Four independent replicates of each assay were performed. Results were analyzed using one- way ANOVA, followed by post hoc comparisons when suitable. DEET and IR3535 showed the same Lowest Observed Effect Level (LOEL): 1.15 µg/cm2. In general, the mixtures showed a repellent effect slightly lower than their pure components, but the vast majority of these differences were not statistically significant. This is the first evaluation of the repellent effect of IR3535 on T. infestans. We will continue looking for interactions between repellents in Chagas disease vectors and other insects, focusing our effort on mixtures containing synthetic and natural compounds.

53

OLFACTORY AND GUSTATORY ADAPTATIONS IN ANTHROPOPHILIC INSECTS.

Coby SCHAL1*; Ayako WADA-KATSUMATA1

1North Carolina State University, Raleigh, North Carolina, United States

Insects that live in our homes (i.e., pests) evolved chemosensory adaptations to thrive and deal with their primary enemies – you and I!The insect cuticular lipid layer is a physical and chemical barrier that excludes pathogens and prevents desiccation. Insects maintain their antennal lipid layer and chemosensory acuity by two counteracting processes: grooming removes excess lipids while fresh lipids are deposited on the antennal surface. These two processes also contribute to fine-tuning of chemosensory acuity by physically removing environmental contaminants, including female contact sex pheromones, which are pervasive in cockroach aggregations. We demonstrate that (a) antennae accumulate more cuticular lipids in the scotophase than in the photophase, (b) when antennal grooming is prevented male antennae become contaminated with female pheromones and their courtship responses significantly decline, and (c) sexual recognition is disrupted as non-groomed males become “feminized” and elicit courtship responses in intact males. Antennal grooming prevents the accumulation of cuticular lipids that interfere with the interaction of semiochemicals with sensory neurons, and it thus prevents adaptation, desensitization and habituation of the male’s olfactory system after contact with female sex pheromone, preserving sexual recognition in aggregations.In response to strong selection with insecticidal baits containing glucose as a phagostimulant, populations of the German cockroach have rapidly evolved a novel heritable behavior, aversion of glucose, which is highly adaptive because cockroaches avoid toxic baits. In glucose- averse roaches bitter-responding gustatory receptor neurons acquired a new function – they detect glucose as a bitter compound. We recently discovered other adaptive sensory polymorphisms in the peripheral gustatory system, including fructose aversion. Thus, strong local anthropogenic selection pressures drive polymorphisms in peripheral gustatory function that contribute to the evolution of locally adaptive foraging and sexual behaviors.

54

OVIPOSITIONAL RESPONSES OF CULEX TARSALIS TO FISH-ASSOCIATED SEMIOCHEMICALS IN LABORATORY BIOASSAYS.

Adena M. WHY1*; William WALTON1

1University of California, Riverside, California, USA

The Western encephalitis mosquito, Culex tarsalis, has been shown to respond to the presence of fish-associated semiochemicals in oviposition sites by decreasing the number of egg rafts laid, in both laboratory and field bioassays. A decrease in oviposition rate on water that contains fish semiochemicals has been attributed to the presence of different classes of chemical compounds, which cause the mosquitoes to alter their oviposition behavior. Whether or not the compounds present act solely as attractants or deterrents has not been determined. To date we have identified three volatile compounds, and one non- volatile compound, that are associated with the Western mosquitofish, Gambusia affinis. Our results indicate that the volatile compounds may be acting as attractants while the non-volatile compound is functioning as an oviposition deterrent.

55

RESPONSE OF HEAD LICE PEDICULUS HUMANUS CAPITIS TO VOLATILES FROM HUMAN SCALP AND CHEMICAL IDENTIFICATION.

Federico G. GALASSI1*; Maria I.PICOLLO1; Ariel. TOLOZA1; Paola GONZALEZ- AUDINO1

1(CIPEIN) UNIDEF - CITEDEF - CONICET, VILLA MARTELLI, BUENOS AIRES, ARGENTINA

The head lice is an specific ectoparasite that mainly affects children of school age, in both developed and developing countries. It is well known that P. capitis prefer to infest some individulas above others. However, no studies have been performed towards the undestanding of the existence of susceptible and non-susceptible indviduals to head lice infestations. Susceptible individuals represent the main source of dispersion through head to head direct contact.In this work we adapted a T-shaped olfactometer to measure head lice walking activity in optimal conditions of light, temperatue and humidity according to our previous results. With this set up, we evaluated the response of headlice to volatiles from human scalp extracts obtained by rubbing filter paper on the heads surface. We found a significant attraction response for this volailes compared to controls.Volatile Organic Chemicals present in the human scalp extract were analysed by Solid phase microextraction -Gas Chromatography-Mass Spectrometry (SPME-CG-MS). The extracts both for bioassay and for chemical analyses were from the same volunteers.The results showed several alcohols, saturated and unsaturaded ketones, aldehydes and fatty and short chained acids.These results represent the basis for the study of chemical cues involved in human susceptibility to head lice.

56

SEMIOCHEMICALLY MEDIATED INTERACTIONS BETWEEN MOTHER AND OFFSPRING IN THE BED BUG, CIMEX LECTULARIUS.

Sydney CRAWLEY1; Kenneth F. HAYNES1*

1University of Kentucky, Lexington, Kentucky, United States

Adult insects are often responsible for the growth and development of offspring. For many insects, much of parent-offspring interaction is mediated by pheromones. Bed bugs, Cimex lectuarius release (E)-2-hexenal and (E)-2-octenal, and the signaling functions of these compounds are varied including alarm, aggregation and sex recognition. In very different contexts, we tested the hypothesis that female bed bugs mediate interactions with offspring using these (and other) pheromones. First, we tested the hypothesis that semiochemicals released by females mediate first instar feeding success. A behavioral assay indicated that first instar bed bugs held with groups containing adult females fed significantly more frequently than those without adults or with adult males. Also, older bed bug nymphs did not have the same effect on first instar feeding. Female bed bugs reached a blood source significantly faster than first instars, suggesting that there is opportunity to signal the presence of a host. Substrates that were walked across by females on their way to and from feeding (and not males or fifth instar nymphs), or extracts of those substrates improved feeding by first instars. We also investigated a novel behavior, female egg-marking, and the role of semiochemicals in bed bug oviposition. These results and the role of semiochemicals for the maintenance of mother offspring relationships in bed bugs will be discussed.

57

THE LUTZOMYIA LONGIPALPIS COMPLEX: A BRIEF NATURAL HISTORY OF AGGREGATION-SEX PHEROMONE COMMUNICATION.

Carolina SPIEGEL1; Denise DIAS2; Alejandra ARAKI2; James HAMILTON3; Reginaldo BRAZIL4; Theresa JONES5*

1Fluminense Federal University, Niteói, RJ, BRASIL 2Rio de Janeiro StateUniversity, , RJ, BRASIL 3Lancaster University, Lancaster, Lancaster, UNITED KINGDOM 4Oswaldo Cruz Institute/ FIOCRUZ,, Rio de Janeiro, RJ, BRASIL 5University of Melbourne, Melbourne, Victoria, AUSTRALIA

Species complexes are important models for understanding the role of chemical divergence in speciation. We review the history of aggregation-sex pheromones within the Lutzomyia longipalpis Lutz & Neiva species complex in an evolutionary context. This phlebotomine is the main vector of Leishmania infantum, agent of visceral leishmaniasis in the Americas. Aggregation-sex pheromones are produced by males when they display during lekking behavior and attracts the haematophagous female in synergy with host odors to a common site of copulation and feeding. Based on the analysis of the main terpene component of the gland extract, four distinct chemotypes can be recognized:(S)- 9-methylgermacrene-B,cembrene-1,(1S,3S,7R)-3-methyl-alpha-himachalene and cembrene-2.Unrecognized taxonomic substructures could exist within those chemotypes based on variation in the amount of specific terpenes and better knowledge of stereospecificity of cembrene molecules. Although those chemotypes exist in sympatry, the chemical hybrids (generated in laboratory experiments) are rare in nature. Field and laboratory studies show there is no significant cross attraction between different chemotypes. Insect sex pheromones have evolved and are maintained for a range of different function such as species recognition, mate recognition and mate assessment. Pheromone contribution to speciation in the L. longipalpis complex is clear in conjunction with courtship behaviours and courtship song acting as pre-zygotic barriers. Pheromone might play a role in mate recognition as pheromone production is age-related and contributes to mating success, nevertheless it is not known whether a decline in pheromone content is commensurate with senescence.The fact that males take multiple days to achieve maximum pheromone output suggests the cost of production and recent transcriptomics and proteomics studies clarified some of the biosynthetic pathway, but not the evolutionary pathway across the complex. Thus, we must assume that there are adaptive benefits of pheromone production including fitness benefits such as increased mating success and hence offspring production, contributing therefore to mate assessment.

58

TARSAL GLANDS AS A SOURCE OF ARRESTING PHEROMONES IN TRIATOMA INFESTANS.

Alicia LORENZO-FIGUEIRAS1*; Claudio LAZZARI2

1Laboratorio de Fisiología de Insectos, DBBE, FCEyN, Universidad de Buenos Aires, IBBEA, CONICET-UBA, Buenos Aires, Argentina., C.A.B.A., Buenos Aires, Argentina 2Institut de Recherche sur la Biologie de l’Insecte, UMR CNRS 7261, Université François Rabelais, Tours, France, Tours, Indre- Loira, France

The use of pheromones appears is the most important mode of intraspecific communication in many insects, including triatomine bugs, vectors of Chagas disease in Latin America. These insects spend daylight hours assembled inside shelters, in close contact with conspecifics. Chemical signals help keeping insects inside their refuges, which they leave at dusk to look for a potential host. There are evidences that the aggregation of bugs is mediated by thigmotaxis, volatile substances in their faeces, and by low-volatility non-polar substances that these insects left behind on walked substrates. The aim of the present work was identifying the source of the later signal, testing the hypothesis that their origin are exocrine glands located into the bugs legs, since in the pretarsi of adult T. infestans at least two types of glandular structures have been identified. So, we tested whether or not arresting on walked substrates is mediated by compounds secreted by these glands that can be detected by contact chemoreception. The bioassay consisted in presenting to fifth-instar nymphs of T. infestans (eight insects per assay) to binary choices in a circular arena. The tested stimuli were deposed in pieces of filter paper and consisted of dichlorometane (DCM) extract of tarsi, DCM extract of the rest of the leg and only solvent (DCM). The results evinced that bugs were significantly attracted and assembled around papers impregnated with the tarsi extract (p<0.05, one- sample t-test), but not on the DCM extracts of the rest of the leg or solvent-impregnated papers. The next step of our study will be performing the analysis of tarsal secretion to unravel their chemical nature.

59

THE FIRST RECEPTOR FOR COMPONENTS OF A CLASSICAL SEX PHEROMONE IN DIPTERA.

Martin N. ANDERSSON1*; Jacob A. CORCORAN1; Richard D. NEWCOMB2; Christer LÖFSTEDT2

1Dept. Biology, Lund University, Lund, Scania, Sweden 2The New Zealand Institute for Plant & Food Research, Auckland, N/A, New Zealand

Gall midges (Diptera: Cecidomyiidae) constitute an intriguing insect family in which the members have evolved intricate relationships with their hosts, including the capability to induce galls and ‘reprogramming’ of host plant physiology. Most cecidomyiids are specialists and many are serious pests. One of the worst is the Hessian fly (Mayetiola destructor Say), which is a severe pest of wheat. Olfaction is crucial for host localization and reproduction; the latter evidenced by a female-produced long-distance sex pheromone consisting of five to seven components. We previously annotated 122 odorant receptors (ORs) from the Hessian fly genome, and revealed major differences in OR gene expression levels between male and female antennae. Whereas 50 ORs display female- biased expression, 10 ORs, all confined to a recently expanded lineage, are highly overexpressed in males, suggesting that they may detect sex pheromone components. Using heterologous expression in HEK293 cells, we show that MdesOR115 responds primarily to (2S,8E,10E)-8,10-tridecadien-2-yl acetate, and to a lesser extent its Z,E- isomer, which are both required for sex pheromone attraction. In addition to pheromone receptors, members of the Sensory Neuron Membrane Protein (SNMP) family are also necessary for pheromone reception as demonstrated in Drosophila with the requirement for SNMP1 in the detection of the pheromone cis-vaccenyl acetate. Intriguingly the Hessian fly has six genes encoding SNMP1 orthologues. However, co-expressing each of the MdesSNMP1 genes with MdesOR115 in HEK293 cells did not affect the responses to the two active ligands. MdesOR115 is the first functionally characterized gall midge OR, and represents the first receptor identified in any dipteran that detects components of a classical long-distance sex pheromone.

60

USE OF AEDES AEGYPTI LARVAE ATTRACTANTS TO ENHANCE THE EFFECTIVENESS OF LARVICIDES.

Paula GONZALEZ1*; Laura HARBURGUER1; Paola GONZÁLEZ AUDINO1; Héctor MASUH1

1Centro de Investigaciones de Plagas e Insecticidas (CIPEIN), UNIDEF-CITEDEF- CONICET, Villa Martelli, Buenos Aires, Argentina

Aedes aegypti (L.) is an important dengue, Chikungunya, Zika and yellow fever vector. Immature stages of this species inhabit human-made containers placed in residential landscapes. Application of larvicides inside containers that cannot be eliminated is still considered a priority in control programs. Larvicidal efficacy is influenced by several factors, including the formulation used, the water quality and the susceptibility of larvae, among others. If an attractant can be incorporated into a slow release larvicide formulation it will be feasible to direct the larvae into the source of insecticide and thereby improving its efficacy. We studied the influence of 1-octen-3ol and 3- methylphenol on the rate of Ae. aegypti larvae mortality using the larvicides Bacillus thuringiensis var. israelensis (Bti), temephos and spinosad. These chemicals were combined with the larvicides mixed with agar during the bioassays. Mortality was registered every 10 min and a lethal time 50 (LT50) was calculated. The inclusion of the Ae. aegypti larvae attractants with the larvicides into a solid agar matrix improved their efficiency obtaining a strong and marked reduction in the LT50 compared with the use of larvicides alone. The results obtained in this work for Ae. aegypti could be extended to mosquitoes that live in large water bodies, such as Anopheles or Culex considering that larvae attractants for Culex pipiens quinquefasciatus and Anopheles gambiae have already been identified (Barber and Burnton 1983, Xia et al. 2008).

Barber and Burnton. 1983. J. Med. Entomol., 20: 641-643. Xia et al. 2008.Proc.Natl.Acad.Sci.USA.105:6433-6438.

61 CHEMICAL ECOLOGY OF PHYTOPHAGOUS FLIES

ANALYSIS OF CUTICULAR HYDROCARBON PROFILES IN BACTROCERA DORSALIS SPECIES COMPLEX (DIPTERA: TEPHRITIDAE).

Radka NAGY BŘÍZOVÁ1*; Lucie POMPEIANO VANÍČKOVÁ1; Antonio POMPEIANO2; Blanka KALINOVÁ3

1of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague,, Prague, Czech Republic, Czech Republic 22Global Change Research Centre, Czech Academy of Sciences, Brno, CZECH REPUBLIC, Brno, Czech Republic, Czech Republic 3of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague,, Prague, Czech Republic, Czech Republic

Fruit flies Bactrocera papayae Drew &Hancock, Bactrocera philippinensis Drew & Hancock, Bactrocera carambolae Drew & Hancock, and Bactrocera invadens Drew, Tsuruta &White are morphologically and genetically very similar to the oriental fruit fly Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) and all together are referred as Bactrocera dorsalis species complex. Recent coordinated international multidisciplinary research efforts yielded data, on which B. philippinensis has been synonymized with B. papayae, B. invadens with B. dorsalis, and B. philippinensis with B. dorsalis.B. carambolae has been determined as a distinct species as it exhibits a number of consistent differences from B. dorsalis (1).Cuticular hydrocarbons (CHCs) are components of insect epicuticle. Their primary function is protection against desiccation. Secondarily, CHCs are involved in intra- and inter-specific communication, such as species and gender recognition, fertility and mating status signaling, chemical mimicry and many others. Due to their species-specificity, the CHCs became a valuable tool in insect .To determine whether the CHCs compositions coincides with recent taxonomic adjustments we performed a comparative CHCs analysis of males and females of B. dorsalis, B. papayae, B. philippinensis, B. invadens, and B. carambolae.Flies were individually rinsed with hexane and obtained samples were analyzed using GCxGC-TOFMS. The data were statistically evaluated using principal component analysis (PCA). Data show clear differences in CHC profiles between sexes of all investigated taxonomic entities. Preliminary statistical analysis presumably supports the recent taxonomical changes. Data will be discussed with respect to both original species descriptions (2) and recent taxonomic changes (1, 3).References1. Schutze M.K., 30 co-authors, and Clarke A.R (2015) Systematic Entomology 40: 456-471.2. Drew R.A.I. (1989) Memoirs of the Queensland Museum 26: 1-521.3. Clarke A.R. and Schutze M.K, (2014) In: Trapping Tephritid Fruit Flies for Detection, Control, and Regulation pp. 611-632 Springer, Dordrecht, The Netherlands.

63

ATTRACTION OF MALE AND FEMALE ORIENTAL FRUIT FLY TO A NOVEL TERPENOID COMPOUND.

Alvin Kah-Wei HEE1*; Keng-Hong TAN2; Suk-Ling WEE3; Ritsuo NISHIDA4

1Universiti Putra Malaysia, Serdang, Selangor, MALAYSIA 2Tan Hak Heng Co., Penang, Penang, MALAYSIA 3Universiti Kebangsaan Malaysia, Bangi, Selangor, MALAYSIA 4Kyoto University, Kyoto, Kyoto, JAPAN

Oriental fruit flies, Bactrocera dorsalis Hendel is the most invasive and destructive pest of fruits that has been recorded in over 65 subtropical and tropical countries. Whilst direct damage to economically valuable fruits, such as mango, carambola and guava, has been mainly attributed to the ability of the gravid female flies to detect the fruits for oviposition, control of the species has been centred on the males due to their strong attraction to the potent male lure, methyl eugenol. Thus, application of a toxicant-baited lure in male annihilation technique coupled with other tactics such as sterile male releases have contributed significantly to the delimitation of the species spread or colonization. Hitherto, the control of females has been largely limited by application of protein baits that also attracts many other non-target arthropod species. Therefore, in addressing the progress of current fruit fly control methods, it is highly desirable and economical to have an ideal fruit fly lure attractive to both sexes - especially gravid females. In our search for new attractants, we have discovered a particular terpenoid compound that is attractive to both male and female B. dorsalis. Interestingly, this compound has been also observed to be significantly more attractive to gravid than virgin females.

64

CHEMICAL CUES USED FOR HOST INTERACTIONS AMONG VARIOUS FLIES.

Junwei (Jerry) ZHU1*

1USDA-ARS, AMRU, University of Nebraska, Lincoln, Nebraska, USA

Flies use chemical cues for their host and host environment location. In this presentation, I will report several case studies describing how these flies use various classes of chemical cues either associated with their hosts or host plants for locating food sources and oviposition sites. In addition, how these chemical cues can be used for developing novel pest management tools. Fly species included in this report are predacious flies, public health concerned flies and flies that attack livestock animals and human beings.

65

CHEMICAL ECOLOGY OF DROSOPHILA SUZUKII: ATTRACTION TO FRUIT VOLATILES.

Cesar RODRIGUEZ-SAONA1*; Aijun ZHANG2; Tracy LESKEY3; John ABRAHAM4

1Department of Entomology, Rutgers University, Chatsworth, New Jersey, USA 2USDA, ARS, Plant Sciences Institute, Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, Maryland, USA 3USDA-ARS Appalachian Fruit Research Station, Kearneysville, West Virginia, USA 4Department of Conservation Biology and Entomology, School of Biological Sciences, University of Cape Coast, Cape Cost, Cape Cost, Ghana

Spotted wing drosophila, Drosophila suzukii, is a new invasive insect pest in North America and Europe that attacks many soft, thin-skinned fruits including blueberries, cherries, raspberries, and strawberries. Unlike most other drosophila, D. suzukii oviposits and develops in ripening fruits, making it challenging to protect fruit crops against this pest. We are conducting studies to develop an attract-and-kill approach based on a combination of olfactory (fruit-based) and visual cues and a killing agent for managing D. suzukii in small fruit crops. Our objectives are to identify fruit-based attractants for D. suzukii, identify visual cues important in host location, and develop an attract-and-kill approach. Coupled gas chromatography electro-antennographic detection (GC-EAD) experiments revealed 11 antennally-active volatiles from raspberries. In choice tests, D. suzukii flies were attracted to a blend containing the 11 antennally-active raspberry compounds. In laboratory, semi-field, and field trials, spheres painted black or red had greater D. suzukii captures compared with other colors. Based on these results, we evaluated D. suzukii fly response to a number of insecticides formulated into red attracticidal spheres (originally designed for apple maggot) which included sugar as a feeding stimulant and wax. Based on 24 h mortality rates, spinosad, spinetoram, lambda- cyhalothrin, bifenthrin, acephate, permethrin, and dinotefuran resulted in mortality greater than 90%. In field trials, we compared attracticidal spheres formulated with spinetoram with weekly insecticide treatments, weekly insecticide treatments combined with attracticidal spheres, and an untreated control. There was a significant difference among treatments, with attracticidal spheres alone and weekly sprays reducing D. suzukii infestation by over 50% compared with the untreated control. When combined, attracticidal spheres and weekly insecticide applications reduced D. suzukii infestation by ~66%. In summary, these results indicate that attracticidal spheres offer promise in terms of a behaviorally-based management strategy to protect small fruit from D. suzukii and reduce insecticide applications.

66

DANGEROUS DUNG DETER DROSOPHILA.

Suzan MANSOURIAN1*; Marcus STENSMYR1

1Dept. Biology, Lund University, Lund, Scania, Sweden

Feces is an abundant, rich source of energy, utilized by a myriad of organisms, not least by members of the order Diptera, i.e. flies. How Drosophila melanogaster reacts to fecal matter remains unclear. Here we examined oviposition behavior towards a range of fecal samples from mammals native to the putative South Eastern African homeland of the fly. We show that D. melanogaster display a strong oviposition aversion towards feces from carnivorous mammals, but indifference or even attraction towards herbivore dung. We identify a set of four predictor volatiles, which with high reliability can be used to differentiate fecal from non-fecal matter, as well as separate carnivore from herbivore feces. Of these volatiles, phenol – indicative of carnivore feces – is repellent and detected by a single class of sensory neurons expressing Or46a. The Or46a expressing neurons are necessary and sufficient for oviposition site aversion. We further demonstrate that carnivore feces – unlike herbivore dung – contain a high rate of pathogenic bacteria taxa. These harmful bacteria synthesize phenol from L-tyrosine, an amino acid specifically enriched in high protein diets, such as consumed by carnivores. Finally we demonstrate that carnivore feces, and phenol, is also avoided by a ball rolling species of dung beetle, suggesting that phenol is a widespread avoidance signal.

67

EFFECT OF PLANT CHEMICALS ON THE SEXUAL BEHAVIOR OF A PHYTOPHAGOUS TEPHRITIDAE FLY.

M. Teresa VERA1*; Guillermo E. BACHMANN2; Patricia C. FERNANDEZ3; Blanka KALINOVÁ4; Francisco DEVESCOVI5; M. Laura JUÁREZ6; Radka BRIZOVA6; M. Josefina RUIZ6; Diego F. SEGURA7

1Cátedra Terapéutica Vegetal FAZ, UNT, San Miguel de Tucumán, Tucumán, ARGENTINA 2Instituto de Genética E.A. Favret, Instituto Nacional de Tecnología Agropecuaria (INTA) / CONICET, Hurlingham, Buenos Aires, Argentina 3Estación Experimental Agropecuaria Delta del Paraná, INTA, Buenos Aires, Buenos Aires, Argentina 4Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, Czech Republic 5Instituto de Genética E.A. Favret, Instituto Nacional de Tecnología Agropecuaria (INTA) / CONICET, Hurlingham, Buenos Aires, Argentina 6Facultad de Agronomía y Zootecnia (FAZ), Universidad Nacional de Tucumán / CONICET, Tucumán, Tucumán, Argentina 7Instituto de Genética E.A. Favret, Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Buenos Aires, Argentina

Tephritidae flies show a wide range of mating systems. In polyphagous species, female location is less predictable and males aggregate in groups (termed leks) to release a sex pheromone. Once the females are at the lek, chemical and physical signals are display and females choose a partner. Plant chemical compounds have been found to affect sexual communication in some of these species. Our objective was to evaluate the impact of fruit volatiles in the sexual communication of the South American fruit fly, Anastrepha fraterculus (Wiedemann), an economically important fruit pest. First, we exposed males to different fruit species and addressed if this modified their sexual performance. Then, we tested if the release rate of key components of male borne volatiles or the cuticle chemical profile wereaffected after exposure to guava volatiles. We found that guava fruit volatiles positively affected male sexual performance, whereas other fruit did not. In addition, guava volatiles exposure enhanced male calling behavior and the amount of (E,E -α-farnesene, anastrephin, epianastrephin and suspensolide collected in male borne volatiles. Exposure to an artificial blend, comprised of a subset of synthetic compounds present in guava fruit, also enhanced mating success. Yet, the chemical profiles of male volatiles and cuticle extracts did not differ qualitatively between exposed and non- exposed males. Finally, we addressed whether mating with an exposed male provided the female a direct benefit in terms of fitness. Females mated with males exposed to guava have higher fecundity than that mated with non-exposed males. Our results contribute to a better understanding of the reproductive system of A. fraterculus in a chemical context, in which the host plant seems to have a significant role. These and further studies may provide new tools for the management of this pest through the sterile insect technique by enhancing sexual performance of sterile males.

68

GUAVA VOLATILES ENHANCE THE SEXUAL BEHAVIOR OF SUDAMERICAN FRUIT FLY (DIPTERA: TEPHRITIDAE) MALES WITH NO EVIDENT CHANGES IN THEIR PHEROMONE AND CUTICLE CHEMICAL PROFILES.

Guillermo E. BACHMANN1*; Blanka KALINOVÁ2; Radka BŘÍZOVÁ2; Michal HOSKOVEC2; Patricia C. FERNÁNDEZ2; M. Teresa VERA3; Diego F. SEGURA4

1IGEAF-INTA / CONICET, Buenos Aires, Buenos Aires, ARGENTINA 2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, Czech Republic 3Cátedra Terapéutica Vegetal FAZ, UNT, San Miguel de Tucumán, Tucumán, ARGENTINA 4IGEAF-INTA / CONICET, Buenos Aires, Buenos Aires, ARGENTINA

The males of some tephritid fruit fly species obtain compounds from plants that affect the chemical communication during courtship. Previous studies showed that Anastrepha fraterculus Wiedemann (Diptera: Tephritidae) males who had access to guava fruit volatiles are preferred by females over other males. Here we evaluated whether female preference for guava exposed males results from a change in the chemical composition of the male cuticle after exposure to guava (perfume effect, sensu Shelly et al., 2007). Alternatively, we address if guava exposure resulted in the incorporation of precursors of sexual pheromones (Tan & Nishida, 1995). We used two dimensional gas chromatography coupled to mass spectrometry (GCxGCxTOF-MS), electroantennography (EAG) and gas chromatography coupled with an electroantennographic detector (GC-EAD) to compare both, the chemical profile of volatiles, and compounds retained on the cuticles of exposed and non-exposed males. There were no major differences in the chemical profile between males both, for volatiles and for compounds retained on the cuticle. GC-EAD with female antennae as biological detectors revealed 16 consistent areas of depolarizations corresponding to 16 specific compunds. Four of these compounds have never been reported as EAG-active for A. fraterculus. The results suggest that the discrimination of guava exposed and non- exposed males is not based on chemical signals. Future work should address other source of signals (i.e. acoustic and visual) during the sexual display.

References Shelly TE, Edu J, Pahio E & Nishimoto J. 2007. Scented males and choosy females: does male odor influence female mate choice in the Mediterranean fruit fly? J Chem Ecol. 33: 2308–2324. Tan KH & Nishida R. 1995. Incorporation of raspberry ketone in the rectal glands of males of the Queensland fruit fly, Bactrocera tryoni Froggatt (Diptera: Tephritidae). Appl Entomol Zool. 30: 494-497.

69

IDENTIFICATION OF ATTRACTANT FOR FRUIT FLY, SPOTTED WING DROSOPHILA.

Aijun ZHANG1*; Yan FENG1; Robert BRUTON1

1USDA-ARS, Beltsville, MD, USA

The spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), is a fruit-infesting fly native to Southeast Asia. Since its introduction in 2008, D. suzukii has become a devastating pest of soft-skinned fruit crops in the United States. Until today, no volatiles that can effectively attract this fly have been identified. By conducting laboratory and field tests, we have identified an attractive blend from fruit juice volatile aroma. A synthetic natural attractant based on the fruits would be useful for selectively detecting, monitoring, and managing this invasive species. In addition, the identified volatils are expected to lure SWD into traps and arrest them on insecticide strips or biocontrol dispensers. Classical biological control programs for SWD appear promising based on outcomes of similar programs for other invasive fruit flies; however, place and timing are critical for releasing. The identified attractants are useful to growers for SWD early infestation detection and population monitoring in support of SWD management programs.

70

MALE LURES AND THE SEXUAL BEHAVIOR OF TEPHRITID FRUIT FLIES.

Todd SHELLY1*

1United States Department of Agriculture, Waimanalo, Hawaii, USA

Males of certain tephritid fruit fly species are strongly attracted to naturally-borne plant products, or their synthetic analogues, and use of these lures has long been a part of surveillance and trapping programs against a handful of invasive species that attack fruits and vegetables, causing substantial economic closes and prompting trade restrictions. Only in the past few decades have studies been undertaken to understand the biological nature of this male attraction, and the object of this presentation is to summarize key findings of this research, highlighting in particular several species of Bactrocera fruit fly as well as Ceratitis capitata, the Mediterranean fruit fly. Discussion will focus on data that demonstrate enhanced male mating success following lure feeding, the mechanisms underlying elevated mating performance, and the effect of lure feeding on subsequent trap visitation/catch. Special emphasis will be given to work that examines possible female benefits derived from selecting lure-fed males as mates over control, non-lure-fed males. Finally, the potential use of “chemotherapy” to improve the Sterile Insect Technique against these pest species will be discussed.

71

PARALLEL EVOLUTION OF TOXIN RESISTANCE IN LEAF-MINER FLIES (DIPTERA: AGROMYZIDAE).

Georg PETSCHENKA1*; Vera WAGSCHAL2; Alexander DONATH3; Susanne DOBLER4

1Institute for Insect Biotechnology, Giessen, Hesse, Germany 2Molecular Evolutionary Biology, Hamburg, Hamburg, Germany 3Zoologisches Forschungsmuseum Alexander Koenig, Bonn, North Rhine-Westphalia, Germany 4Molecular Evolutionary Biology, Hamburg, Hamburg, Germany

Coping with natural toxins can lead to a striking level of molecular convergence of resistance adaptations in insects and vertebrates [1–3]. As predictability of molecular adaptations is one of the major questions in evolutionary genetics we have studied independent adaptation to toxic plant cardiac glycosides (cardenolides and bufadienolides) in five lineages of leaf miner flies (Diptera: Agromyzidae). While cardenolides usually block the ubiquitous animal sodium pump (Na,K-ATPase), many insects have convergently evolved target site insensitivity, which prevents inhibition based on specific amino acid substitutions in the cardenolide binding pocket of the Na,K- ATPase. As a result of potential molecular trade-offs we also observe convergent gene duplications that free the Na,K-ATPase from pleiotropic constraints while enabling the accumulation of substitutions conferring resistance to cardiac glycosides. We have studied both, amino acid substitutions and gene duplications, in a narrow taxonomic framework including four phylogenetic pairs of specialized leaf miner flies each composed of a cardiac glycoside adapted and a non-adapted species. Traditional research on cardenolide resistance has almost exclusively focused on insects feeding on Apocynaceae. Here, we also include flies feeding on cardiac glycoside producing Brassicaceae, Plantaginaceae, and Ranunculaceae. We found further instances of target site insensitivity, caused by toxins from non-apocynaceous families, and its correlation with gene duplications. Nevertheless we also found evidence for evolutionary plasticity in our narrow phylogenetic framework emphasizing that molecular adaptations are not entirely predictable.

1. Dobler, S., Dalla, S., Wagschal, V. & Agrawal, A. A. 2012 Proc. Natl. Acad. Sci. 109, 13040–13045. (doi:10.1073/pnas.1202111109)

2. Zhen, Y., Aardema, M. L., Medina, E. M., Schumer, M. & Andolfatto, P. 2012 Science 337, 1634–1637. (doi:10.1126/science.1226630)

3. Tarvin, R. D., Santos, J. C., O’Connell, L. A., Zakon, H. H. & Cannatella, D. C. 2016. Mol. Biol. Evol. (doi:10.1093/molbev/msv350 )

72

POTENTIAL ATTRACTANTS FOR THE FRUIT FLIES ANASTREPHA FRATERCULUS AND A. OBLIQUA (DIPTERA: TEPHRITIDAE).

Ruth R. DO NASCIMENTO1*; Adriana L. MENDONÇA1; Lucie VANICKOVÁ1; Nathaly C. DE AQUINO1; Jéssica DE LIMA SANTOS1; Luana L. FERREIRA1; Raphael F. TAVARES1; Carlos Eduardo DA SILVA1; Rafael Augusto N. TAVARES1; Regivaldo S. MELO1

1Laboratório de Ecologia Química, Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Alagoas, Maceió, Brazil

Anastrepha fraterculus and Anastrepha obliqua belong to a group of insects responsible for causing damages to crops of economic value. To diminish the growth of these insects, growers use a huge amount of pesticides, causing serious problems to the environment as well as to human and beneficial insects. To minimize these problems, fruit fly management strategies using food attractants were developed. However, a strategy based on the use of volatile substances released by tephritid host fruits is now being considered as one of the most promising technique to control fruit flies. This perspective has motivated researchers to find potential attractants for female tephritids. The present study was conducted to evaluate formulations as attractants for A. fraterculus and A. obliqua females based on the volatile compounds released by guavas and starfruits, using laboratory bioassays. The results showed that A. fraterculus females were more attracted to guava than to starfruit extract. On the other hand, A. obliqua females showed preference for starfruit extract. The number of A. fraterculus females attracted to guavas extract was higher than conspecific females attracted to the synthetic mixture of all tested compounds. This result was also observed for A. obliqua females which were more attracted to the formulation containing the starfruit extract rather than to the synthetic mixture formulation. Amongst the formulations containing the individual compounds, those loaded with 1 µg/µl and 0,1 µg/µl of α-copaeno attracted more females of A. fraterculus and A. obliqua than the synthetic mixture. Therefore, it is possible to conclude that the attraction of A. fraterculus and A. obliqua females to formulations containing extracts of their fruit hosts is due to the chemicals present in these extracts. The different manner that females of these two species respond to formulations is possibly because the lack of some compounds in the synthetic mixture.

73

TRACKING THE OBJECTS OF FLIES' AFFECTIONS ACROSS SPECIES AND CONTINENTS.

Shannon B OLSSON1*

1National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, Karnataka, India

One of the most important tasks for any organism is to identify objects in the world around them. All organisms must, for example, discriminate what to eat from what might eat them. Identifying complex objects in an even more complex world is a difficult task. Most insects, including phytophagous flies, are solitary. This means they must initially identify some objects, such as food or enemies, innately. Our group is interested in how phytophagous flies identify objects across different environments, and how they can detect new introduced objects in an environment, such as invasive species. Using field assays, multivariate analysis, and physiological analyses, we have found that cosmopolitan species of hoverflies use unique combinations of visual and olfactory cues to identify flowers in tropical South India, the alpine Himalayas, or subarctic Sweden. For the latter question, we have compared olfactory processing of host odors for different fruit-specific populations of Tephritid flies that have recently diverged in preference for various fruit within the past 300 years. Finally, we are quantitatively characterizing object identification itself using a novel -visual virtual reality arena. We hope to offer a comparative approach to understand how flies parse the environment to identify objects in nature.

74

THE RESPONSES OF WHITEFLIES TO GLUCOSINOLATES WITHIN A PHYLOGENETIC CONTEXT.

Osnat MALKA1*; Daniel G. VASSÃO2; Simon SPRINGER3; Michael REICHELT4; Susan SEAL5; John COLVIN5; Jonathan GERSHENZON6; Shai MORIN7

1The Hebrew University of Jerusalem,, Rehovot, ISRAEL, ISRAEL 2Max Planck Institute for Chemical Ecology, Jena, GERMANY, ,Jena, GERMANY, GERMANY 3University of Greenwich,, Kent, UNITED KINGDOM, UK 4Max Planck Institute for Chemical Ecology, Jena, GERMANY, ,Jena, GERMANY, GERMANY 5University of Greenwich, kent, UNITED KINGDOM, United Kingdom 6Max Planck Institute for Chemical Ecology, Jena, GERMANY, ,Jena, GERMANY, GERMANY 7The Hebrew University of Jerusalem,, Rehovot, Rehovot, ISRAEL

The ability to metabolize and detoxify plant defensive chemicals is considered one of the major responses that insect herbivores have evolved in order to colonise plants successfully. The vast majority of insect herbivores exhibit various degrees of host plant specialization reflected in various mechanisms to cope with plant secondary metabolites. Specialist insect herbivores encounter high levels of predictable toxicants and have often evolved efficient and specialized detoxification systems towards specific compound classes . Conversely, generalist insect herbivores must cope with a variety of toxic chemicals in their diet and are thus expected to have greater flexibility and capacity to detoxify variable plant chemicals than specialists. To elucidate the relationship between host plant adaptation and detoxification mechanisms of secondary metabolites by generalist and specialist whiteflies and within phylogentic context, four colonies representing different levels of specialization to plants from the Brassicaceae family were studied: Bemisia tabaci MEAM1 (generalist species) which has a good preformance on Brassicaceae plants, Bemisia tabaci MED Q2 (generalist species) which has a moderate preformance on Brassicaceae plants, B. tabaci MED Q Uganda (generalist species) that performs poorly on Brassicaceae plants and Aleyrodes proletella, a specialist on Brassicaceae plants. The four colonies were fed on Brassicaceae plants and their honeydew was analyzed for the occurrence and composition of glucosinolate-derived metabolites. We found that only the generalist Bemisia tabaci colonies (MEAM1 and Q2), utilize a desulfation mechanism for circumventing the glucosinolates-myrosinase defense system.In contrast, the mechanism was not detected in the Brassicacae specialist whitefly A. proletella or the B. tabaci MED Q Uganda species. These findings suggest the existence of different adaptation mechanisms to glucosinolates within a phylogentic context in whiteflies. The pros and cons will be presented and discussed.

75 CHEMICAL ECOLOGY OF VECTOR-HOST INTERACTIONS

AN INSIGHT INTO VARROA OLFACTION AND HOST FINDING MECHANISM

Nurit ELIASH1*; Nitin Kumar SINGH1; Inna STEIN1; Yosi KAMER1; Ilia ZEIDMAN1; Ada RAFAELI1; Victoria SOROKER1

1Volcani Center ARO, Beit Dagan, Israel, Israel

Little is known about the olfactory mechanism in non-insect Arthropods. We investigated the host chemosensing and selection of Varroa destructor Anderson and Truman, an ectoparasitic mite of honey bees (Apis mellifera Linnaeus) and a worldwide threat to apiculture. Varroa life cycle can be divided into two main phases: a reproductive phase in which the mite reproduces within the sealed brood cell and a phoretic one in which the Varroa parasitizes an adult bee. The mites, particularly phoretic, exhibit preference for nurse over forager bee, based on volatiles perceived by the pit organ located at the distal part of its foreleg (1). We implemented both chemo-ecological and molecular strategies to identify the components of the olfactory machinery behind the Varroa host selection. Using quantitative PCR we compared the expression of several potential transcripts between the mite’s forelegs from the two phases of Varroa life cycle. A pheromone receptor transcription factor homolog (PRTF) was one of the differentially expressed transcripts that showed higher expression in phoretic mites foreleg (2). Electrophysiological and behavioral studies revealed a reduction in the sensitivity of PRTF-like-silenced mites to bee headspace. This was consistent with a reduction in the mites’ ability to reach a host. The impact of this silencing on the expression of other Varroa genes and its consequences on mites' olfaction and host finding will be discussed.1. Eliash N. et al., PLoS One. 9, e106889 (2014).2. Singh N. K. et al., Insect Mol. Biol., in press, doi:10.1111/imb.12212.

77

ANTENNAL RESPONSES OF RHODNIUS PROLIXUS (REDUVIIDAE: TRIATOMINAE) TO VOLATILES PRODUCED IN VITRO BY BACTERIA ISOLATED FROM HUMAN SKIN FACES.

Melanie RAMÍREZ CASALLAS1*; Marcela TABARES1; Mario I. ORTÍZ YANINE1; David A. GUZMÁN1; Martha VIVES1; Jorge A. MOLINA ESCOBAR1

1Universidad de los Andes, Bogota D.C., Bogota D.C., Colombia

The host seeking behavior of hematophagous insects is highly associated with the volatile organic compounds (VOCs) released by the host. The attractive role of VOCs produced by bacteria from human skin faces has been previously shown in Rhodnius prolixus (Ortiz et al. 2011). The aim of this study was to assess the antennal responses of R. prolixus to VOCs produced in vitro by eight bacterial species in exponential and stationary growth phases and previously isolated from human skin faces. VOCs produced by cultured media without bacteria were used as control. By means of electroantennograms (EAGs), we evaluate the antennal responses during the photophase and scotophase of R. prolixus (n=4) to each stimuli treatment (k=16). One hundred twenty eight EAGs were recorded and analyzed. Statistical differences between voltage amplitudes in control and treatment responses were evaluated using sign tests. VOCs produced by three Staphylococcus species in stationary growth phases showed statistical differences in voltage amplitudes in comparison to controls during the photophase or scotophase of R. prolixus. During the exponential growth phases, no significant responses to VOCs were obtained in any of the insects tested in photophase. The electrophysiological evidence presented here reinforces the idea that VOCs produced in vitro by bacteria can be detected by R. prolixus and highlights the importance of the interaction Rhodnius prolixus-host skin microbiota in the transmission of Chagas disease.

Ortiz MI, Suarez-Rivillas A, Molina J. 2011. Behavioural responses to human skin extracts and antennal phenotypes of sylvatic first filial generation and long rearing laboratory colony Rhodnius prolixus. Memorias Instituto Oswaldo Cruz. 106:461-466. Acknowledgements: Colciencias project # 120456934423 and Convocatoria Nacional Doctorado No. 567-Colciencias, for financial support.

78

ALTERATION OF PLANT PHENOTYPES BY MICROBES: IMPLICATIONS FOR VECTOR-BORNE DISEASE TRANSMISSION AND MULTI-TROPHIC PLANT-INSECT INTERACTIONS.

Kerry MAUCK1*; Milica NENADIC1; Luca GRANDI1; Consuelo DE MORAES1; Mark MESCHER1

1ETH Zurich, Zürich, ZH, Switzerland

Pathogenic and beneficial microbes can indirectly modify interactions between plants and the broader insect community through effects on host phenotype. To understand these effects and their implications for microbial fitness, we explored the influence of virus infection on host chemistry and vector behavior in two pathosystems involving non- persistent viruses in the family Bromoviridae: Cucumber mosaic virus (CMV) and Alfalfa mosaic virus (AMV). Both viruses are acquired and inoculated by aphids during short probes of outer plant cells, and are best transmitted when aphids emigrate after pathogen acquisition. Consistent with these requirements for aphid behavior necessary for non- persistent virus transmission, both CMV and AMV induce changes in free amino acids and simple carbohydrates that stimulate aphid vector dispersal after probing. These changes to plant palatability and quality also influence the success of aphid parasitoids and the behavior and performance of non-vector herbivores - both of which will influence pathogen transmission through effects on vector behavior and host plant survival. CMV effects on plant palatability occur regardless of whether the host plant is in a sterile soil environment or a field environment that includes beneficial microbes. However, AMV effects on plant palatability and vector behavior only occur when the host plant has an association with Sinorhizobium meliloti, a co-evolved root symbiont that also has positive effects on virus titer. Our results suggest that virus-induced changes in host phenotype can vary depending on whether the host plant also has associations with other microbes. While selection may act on viruses to favor genotypes that alter host chemistry in ways that increase transmission, our results point out that virus evolution occurs in an environment that includes multiple organisms simultaneously influencing host plant phenotype.

79

APHID RESISTANCE IN BRAZILIAN HEXAPLOID WHEAT DOES NOT CORRELATE WITH LEVELS OF THE BENZOXAZINOID NATURAL PRODUCT DIMBOA.

Andre Lucio Franceschini SARRIA1*; Jorge Fernando PEREIRA2; Paulo Roberto Valle da Silva PEREIRA2; Gudbjorg Inga ARADOTTIR2; Janet MARTIN2; Lesley SMART2; John A. PICKETT2; Michael A. BIRKETT2

1Rothamsted Research, Harpenden, Hertfordshire, UK 2EMBRAPA TRIGO, Passo Fundo, Rio Grande do Sul, Brazil

Chemical control has been widely used for the control of aphid pest in Brazilian wheat (Triticum aestivum L.) production but due to development of insecticide resistance, and the prohibitive cost of insecticide use on family-run farms, sustainable and affordable solutions for wheat production are required.Plant secondary metabolism, accessed from biodiversity resources beyond current elite cultivars, offers a source of novel interventions for pest control, based on small lipophilic molecule (SLM) signalling, which can be delivered by the seed [refs].Here, we report on our initial experiments to deliver new aphid resistance in Brazilian wheat based on SLM signalling, using aphid performance (phenotyping) assays and reverse-phase HPLC analysis.In phenotype assays that measured the mean relative growth rate of the grain aphid, Sitobion avenae and Rhopalosiphum padi, eight selected Brazilian wheat genotypes were ranked in terms of successful aphid development (BR 32, BRS 327, BRS 328, BRS Guabiju, BRS Guamirim, BRS Parrudo, PF869120 and BRS Timbaúva) and one UK variety (Solstice).Reverse-phase HPLC analysis showed variation in the levels of the benzoxazinoid DIMBOA, varied from 5.38 to 32.56 Mmol.KgFW-1 in BRS Guabiju and Parrudo respectively. Although BRS Guabiju was also among the genotypes with the highest intrinsic rate of population increase (rm) and BRS Parrudo showing low percentage of rm (compared with Solstice) against both aphid species, levels of DIMBOA did not correlate with intrinsic rate of increase. There was no clear correlation between aphid performance and DIMBOA levels. These data suggest the presence of an alternative resistance mechanism to that of DIMBOA, which is currently being explored with the help of a novel artificial diet assay and bioassay-guided fractionation.

Refs 1. Pickett et al, 2014, Phil Trans Roy Soc B, 369, 20120281 2. Birkett and Pickett, 2014, Curr Opin Plant Biol 19, 59

80

CHANGES IN HUMAN SKIN ODORS ASSOCIATED WITH MALARIA INFECTIOUS STAGES.

Margaux MULATIER1*; Nina JOFFARD1; Serge YERBANGA2; Benoit LAPEYRE3; Roxane DELLE-VEDOVE4; Jean-Marie BESSIERE5; Thierry LEFEVRE6; Anna COHUET7; Laurent DORMONT7

1IRD, UMR MIVEGEC (UM-CNRS 5290 - IRD 224), Montpellier, Hérault, France 2IRSS, Bobo-Dioulasso, Bobo-Dioulasso, Burkina Faso 3CEFE, CNRS UMR 5175, Montpellier, Hérault, France 4Université Lille 1, Lille, Nord, France 5ENSCM, Montpellier, Hérault, France 6IRD, UMR MIVEGEC (UM-CNRS 5290 - IRD 224) + IRSS, Bobo-Dioulasso, Bobo- Dioulasso, Burkina Faso 7IRD, UMR MIVEGEC (UM-CNRS 5290 - IRD 224), Montpellier, Hérault, France

Vector-borne pathogens may alter traits of their hosts in ways that increase the frequency of host-parasite interactions and the probability of transmission. This phenomenon is called parasitic manipulation.Previous work has reported that people carrying infectious forms of Plasmodium falciparum were more attractive to the vector Anopheles gambiae than non-infectious people. It was hypothesized that it may be due to a modification in the host skin odor, however the mechanisms underlying this potential parasitic manipulation have not yet been elucidated.In this study, skin odors were captured from asymptomatic volunteers naturally infected by different stages of development of P. falciparum and from non-infected control individuals in Burkina Faso, an endemic area of malaria. Among the volatile organic compounds (VOCs) identified, we found different distributions in the odor profile according to the stages of infection. Unusual skin compounds, terpenoids, were present at higher relative quantities in people carrying gametocytes, the transmissible stage of the parasite, compared to non-infected people and to patients carrying the non-infectious asexual stages of parasites only. Electroantennography assays revealed that A. gambiae has the ability to detect those compounds at a concentration mimicking the biological one. Behavioral assays are in progress, with the aim of determining if the increased attractiveness of gametocytes carriers is linked to the presence of those compounds in the skin odor.These findings provide insights to understand mechanisms that mediate vector attractiveness of infectious people, and could have future implications for improving the understanding of malaria epidemiology.

81

EXPERIENCE-DEPENDENT BEHAVIORAL PLASTICITY: WHAT CAN KISSING BUGS LEARN ABOUT THEIR HOSTS?

Sebastian MINOLI1*

1University of Buenos Aires, Buenos Aires, Buenos Aires, ARGENTINA

Although innate responses assure a quick and conservative way of surviving under stable conditions, cognitive abilities allow animals to better adapt to changing or unpredictable environments. For an haematophagous insect, innate responses to food-related cues common to most of their hosts (like CO2 or heat, among other) are necessary for attaining a quick and unequivocal food reward. However, after several experiences, bugs can improve their feeding abilities by starting to select or to avoid particular hosts or situations that previously rendered positive or negative experiences, respectively. Recent and actual studies carried out in our laboratory show that kissing bugs (Heteroptera: Reduviidae: Triatominae) possess cognitive capacities relevant for their lives in numerous contexts. These processes are expressed as changes in behaviors such as: 1) the escape response to the alarm pheromone, 2) the attraction to the aggregation pheromone, 3) the preference for different surfaces or textures, 4) the feeding behavior mediated by gustatory the sense, 5) the attraction to odors released by their hosts, among others. In all cases, we applied associative and non-associative experimental protocols that provoked changes in the behavior of triatomines, evincing the relevance of learning and memory in the life of these haematophagous insects, vectors of the Chagas Disease in Latin America. With this presentation we would also like to focus the attention of researchers in the experience-dependent plasticity of behavior exhibited by most of biological models.

82

INFLUENCE OF AEDES AEGYPTI LARVAE EXTRACTS IN THE BEHAVIOR OF INTRA-SPECIFIC OVIPOSITION.

Gabriel Bezerra FAIERSTEIN1; Eloína Maria Mendonça de SANTOS1; Rosângela Maria Rodrigues BARBOSA1*; Walter S. LEAL2

1Centro de Pesquisas Aggeu Magalhães-Fiocruz-PE, RECIFE, PERNAMBUCO, BRASIL 2University of California-Davis, Davis, California, USA

Aedes aegypti is an urban species that is challenging health programs, it is difficult to control and responsible for the transmission of arboviruses as DENV, YFV, CHIKV, ZIKV. The selection of breeding sites of many mosquito species is strongly influenced by chemical stimuli. In order to improve the attractiveness of oviposition traps, a large number of chemical compounds, with effect on the oviposition behavior, it has been identified. The study aimed to evaluate the effect of different solutions in the choice of oviposition site by pregnant female A. aegypti. The choice tests were conducted cage chamber with 30 females / test with twelve repetitions. They were used as a site, two or three glass container (200 ml) with filter paper, each containing 150 ml of the solution to be tested: distilled water (control), water feed, larval extract (L4), ether and hexane. Larval extract was prepared using 50 larvae L4, macerated. For water feed used 0.025 g of the diluted/ 150 ml feed. Bioassays were also performed using larvae treated with 1,25mL of ether or hexane. For analysis used the nonparametric analysis of variance. The results showed that the containers tests with larvae extract (L4) versus control (distilled water) and food extract, collected significantly more eggs, with 71.60% - 66.04% oviposition respectively, OAI varied from +0.43 to +0.61 (p <0.05). In tests with ether solvent and hexane observed a large number of test containers in position with 71.98% and 61.19 oviposition respectively, OAI varied from +0.22 to +0.43 (p <0.05) compared to control. The improvement of these extracts may lead to the development of baits for use in oviposition traps.

83

NON-HOST SEMIOCHEMICALS DELIVERED TO SUSCEPTIBLE DOGS USING SLOW-RELEASE FORMULATIONS REPEL RHIPICEPHALUS SANGUINEUS SENSU LATO TICKS.

Jaires G. de O. FILHO1; Lorena Lopes FERREIRA1; Andre Lucio F. SARRIA2; John A. PICKETT2; Michael A. BIRKETT2; Gabriel Moura MASCARIN3; Adalberto A. P. de LEÓN4; Lígia M. F. BORGES5*

1Universidade Federal de Goiás, Goiânia, Goiás, Brazil 2ROTHAMSTED RESEARCH, Harpenden, Hertfordshire, United Kingdom 3EMBRAPA Arroz e Feijão, Santo Antônio de Goiás, Goiás, Brazil 4USDA, ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, Kerrville, Texas, USA 5UNIVERSIDADE FEDERAL DE GOIÁS, Goiânia, Goiás, Brazil

Domestic dog breeds are hosts for the tick Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to this tick infestations than English Cocker Spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as tick repellents. We report the use of a slow release formulation of these compounds to reduce the burden of R. sanguineus s. l. on dogs. Twelve English Cocker Spaniel, kept in kennels, were randomly assigned to two groups (control and treated) with six dogs each. Three dogs of each group were housed per stall, and rotated between the stalls every seven days. Slow-release formulations of the compounds were attached pairwise to collars placed on the dogs in the treated group; the control group received clean formulations. Five environmental infestations were performed with 2,000 larvae, 100 nymphs, 30 males and 30 females. The first infestation was made 10 days before the second one, with subsequent infestations being done every seven days. Collars were replaced by new ones after 15 days and removed after the fifth infestation. The dogs were inspected twice a day for 45 days. The tick average in the treated group was significantly lower than in the control. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. In the present study we used a 1:1 release rate of the repellents. Recently we verified that the ratio of 2-hexanone and benzaldehyde released naturally from beagles is 2:1. The present study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs. We can hypothesize that the use of formulations containing the natural ratio released by resistant dogs can improve the control.

84

TASTE AVERSION FOR SALTS AND ALKALOIDS: CAN KISSING BUGS DISCRIMINATE THEM?

Santiago MASAGUÉ1*

1Laboratorio de Fisiología de Insectos, DBBE, FCEyN, Universidad de Buenos Aires, IBBEA, CONICET-UBA, Buenos Aires, Argentina, C.A.B.A., Buenos Aires, Argentina

The taste system of animals can help them in making appropriate feeding decisions, favoring the ingestion of nutritious foods and avoiding noxious ones. Although the kissing bug Rhodnius prolixus attains high epidemiological importance as vector of the Chagas disease, its taste sense is particularly disregarded. The existence of phagostimulants such as ATP or low doses of salts has been already described. However, the existence of aversive gustatory compounds is by far less studied in haematophagous insects. In this work, we show in 2-choice experiments that bugs avoid substrates with high-salt concentrations (sodium and potassium salts) or with alkaloids (caffeine and quinine, commonly named as bitter compounds). By applying an aversive operant conditioning we analyzed whether insects perceive salts and alkaloids as different aversive compounds or if instead they are perceived as similar but with different degree of aversiveness. First, we established that R. prolixus can learn to occupy zones of an arena loaded with aversive stimuli if the control side is punished with a mechanical disturbance. Second, we found that bugs are not capable to distinguish sodium from potassium salts. Likewise, bugs cannot discriminate salts from alkaloids. Our results suggest the existence of a common treatment of aversive information (high-salt or alkaloids) with independence of the chemical identity of the stimuli. This kind of studies serves to gain more insight into the taste sense of a blood-sucking model but also to know about their learning abilities.

85

THE ROLE OF TRYPANOSOMA RANGELI TEJERA INFECTION OF RHODINIUS PROLIXUS STAHL ON THE MATING BEHAVIOR AND METASTERNAL GLAND PROTEASE ACTIVITY.

Rosane LOPES1; Gulherme MACHADO1; Carolina SPIEGEl1; Simone OLIVEIRA2; Andre SANTOS2; Denise FEDER3; Suzete GOMES3*

1Universidade Federal Fluminense, Niterói, RJ, BRASIL 2Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, BRASIL 3Universidade Federal Fluminense, Niterói, RJ, BRASIL

Rhodinius prolixus is a triatomine vector of Trypanosoma cruzi Chagas, etiologic agent of Chagas disease and invertebrate host of Trypnosoma rangeli. These insects have one pair of exocrine glands, the metasternal glands (MGs) located in the ventral metathorax, involved in sexual communication. T. rangeli is a protozoan that invades all R. prolixus tissues and its parasitism is appointed as populational control. However, there are a few reports on the effects of this parasitism on the reproductive behavior. In this context we investigated whether T. rangeli infection can affect the mating behavior of R. prolixus and the involvement of proteases activities from MGs in this process. The copulation behavior analysis was done with infected female and control males compared with control females and control males in 3 periods: pre-mating; copulation and guard behavior. MGs were extracted from females at 7 days after blood infected meal and an enzymatic profile were determined using peptidic fluorogenic substrate for aspartic, cysteine and trypsin peptidases. The pre-mating period and copulation has changed significantly in the infected group compared to the control group. Several factors can modify the copulation behavior as changes in the chemical volatiles of the metasternal glands. It has been demonstrated that Tenebrio molitor Linnaeus, females decrease the production of pheromones when infected with Hymenolepis diminuta Rudolphi. Assessing the proteolytic activities we observed none aspartic and cysteine peptidase activities differences, but a significant alteration of trypsine-like serino protease activity in infected MGs compared to control. Since serino proteases are involved in several insects’ physiological processes, such as biologically active peptides production, it should have also an effect on sex pheromone biosynthesis. More studies of sexual behavior and volatiles analysis should be made to know if T. rangeli affects the chemical volatile profile of the MGs and consequently the reproductive behavior of R. prolixus.

86

THE LABORATORY-FIELD EXPERIMENTAL INTERFACE: TESTING AND MEASURING ATTRACTION TO ODORANTS IN THE LABORATORY AIMING AT DEVELOPING AN ODOR-LURE, A STUDY ON KISSING BUGS.

Fabio GUIDOBALDI1; Celina BRATOVICH2; Irving MAY CONCHA3; Mailen GARCÍA4; Nora BURRONI4; Pablo GUERENSTEIN5*

1CICyTTP-CONICET, Diamante, Entre Ríos, Argentina 2CICyTTP-CONICET & Fac. de Ingeniería, UNER, Diamante/Oro Verde, Entre Ríos, Argentina 3Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción (CICYTTP-CONICET), Diamante, Entre Ríos, Argentina 4CENTRO DE INVESTIGACIONES CIENTÍFICAS Y TRANSFERENCIA DE TECNOLOGÍA A LA PRODUCCIÓN (CICYTTP-CONICET) | Cidade: Dia, Diamante, Entre Rios, Argentina 5CICyTTP-CONICET & Fac. de Ingeniería, UNER, Diamante/Oro Verde, Entre Rios, Argentina

Candidate odor attractants are often tested in the laboratory to develop odor lures to attract insects in the field. However, sometimes, good attractants developed in the laboratory are not efficient in the field. This could be due to many reasons, including the use of a non-ideal laboratory experimental design. Different experimental designs are used to estimate attraction in insects. Moreover, for a certain experimental design, different variables are quantified. Using triatomines and a dual-choice trap-olfactometer, we asked which behavioral variables are best to predict capture in the laboratory (ultimately, we aim at asking which tests and variables are best to predict capture in the field). The olfactometers included an insect release-zone and, on the opposite side, two capture-tubes (emanating control and hostodor) connected to the arena from below. The insects could let themselves drop into the capture-tubes (triatomines let themselves drop when sensing hostodor from below). Using a video camera, we recorded the behavior of individual bugs during the experiments. The video recordings were analyzed to measure variables that are used to estimate attraction in the literature. Some of the variables measured were: first choice, and position at the end of the (5-to-30 min) experiment. Two stimuli were tested: a mouse and a synthetic hostodor blend, and we analyzed the degree of discrimination between the stimuli according to the different variables mentioned comparing to the variable “capture”. Whereas “capture” indicated that the synthetic mixture is a worse attractant than mouse, the other variables suggested little or no difference in the responses to the two stimuli. Therefore, using those other variables the response reached a maximum with a non-ideal stimulus, thus making difficult to improve the synthetic stimulus. Our results suggest that certain behavioral variables used in the literature are not adequate to extrapolate results on attraction to laboratory capture.

87

VECTOR-HOST INTERACTIONS: NEW OPPORTUNITIES FOR PROTECTION OF PLANTS AND VERTEBRATE ANIMALS AGAINST ARTHROPOD VECTORED PATHOGENS BY EXPLOITED STRESS RELATED SIGNALLING.

Professor John A PICKETT1*; Dr. Mike A BIRKETT1; Professor Zeyaur R KHAN2; Dr Charles A O MIDEGA2

1Rothamsted Research, Harpenden, Hertfordshire, UK 2icipe, Nairobi, Kenya, Africa

When hosts, whether plant, animal or from other kingdoms, become exploited, Small Lipophilic Molecules (SLMs) are released and can act as volatile semiochemicals that are detected by incoming, and particularly congeneric, species and interfere with the subsequent attack. Also, for organisms antagonistic to these species, such as parasitoid wasps, the same SLMs can serve to promote foraging for and attack on the species attacking the host. In plant protection these stress related SLMs repel pests, and increase foraging by parasitoids which adds further control of the pests by, for example, introducing conservation biological control. Delivery can be by slow release formulations but is particularly effective when companion plants are deployed in the stimulo-deterrent diversionary or push-pull system. The successful demonstration, initially in East Africa, has confirmed the potential value of these type of semiochemicals as targets for new GM based crop protection. For vertebrate hosts similar opportunities are being exploited and attempts again made to understand the underlying genetics which is already demonstrated as being based on hereditary principles in human hosts. The push-pull system for biting flies is being exploited in the field and the semiochemical tools are in place for use against arthropod pests attacking fish in marine ecosystems. The induction and priming of stress related SLM release from intact plants by other stress related SLMs shows tremendous promise for development of sentinel plants that either simply offer early warning of attack or can switch on defence genes in the main crop with the potential for exploiting analogous systems in vertebrates. These types of elicitors that don’t require hosts tissue damage for activity can greatly facilitate identification of genes involved in biosynthesis and regulation of expression, opening up exploitation by new molecular genetic techniques.

88 CHEMICAL INTERACTIONS MEDIATED BY MICROORGANISMS

ANTIFUNGAL COMPOUNDS PRODUCED BY PAENIBACILLUS POLYMYXA ALLI-03-01 ISOLATED FROM THE STINGLESS BEE MELIPONA SCUTELLARIS.

Carla MENEGATTI1*; Fábio S. do NASCIMENTO1; Cameron R. CURRIE2; Jon CLARDY3; Mônica T. PUPO4

1School of Pharmaceutical Sciences of Ribeirão Preto – University of São Paulo (FCFRP-USP), Ribeirão Preto, São Paulo, Brasil 2Department of Bacteriology – University of Wisconsin, Madison, Wisconsin, USA 3Department of Biological Chemistry and Molecular Pharmacology – Harvard Medical School (HMS), Boston, Massachusetts, USA 4School of Pharmaceutical Sciences of Ribeirão Preto – University of São Paulo (FCFRP-USP), Ribeirão Preto, São Paulo, Brasil

Insects establish various relationships with microorganisms [1] and some novel and bioactive natural products have been identified from insect microbial symbionts. Based on this ecological evidence, this work aimed to study the natural products biosynthesized by symbiotic microorganisms associated with the stingless bee Melipona scutellaris. Bees and their brood comb were collected and microorganisms were isolated following published methodology [2]. All microorganisms were assayed against two entomopathogenic fungi - Beauveria bassiana and Metarhizium anisopliae. One selected bacterium strain coded as ALLI-03-01 was isolated from larvae food inside brood comb and was active against both entomopathogenic fungi. This strain was identified as Paenibacillus polymyxa based on 16S rDNA sequencing, a known bacterium for its high ability to produce secondary metabolities [3]. P. polymyxa ALLI-03-01 was cultured in liquid medium and 3-phenyllactic-acid was isolated from the ethyl acetate extract. This compound showed satisfactory fungal inhibition against the entomopathogenic fungus B. bassiana. Due to this activity, 3-phenyllactic acid might play a fungal control inside bee colony allowing healthy growth of bee larvae. A series of fusaricidins was also isolated from methanol extract of the same strain cultured in ISP-2 solid medium. The compounds were identified by NMR and mainly MALDI-TOF-MS MS/MS. The fusaricidins are cyclic lipopeptides containing 15-guanidino-3-hydroxypentadecanoic acid as fatty acid component, recognized by their high antimicrobial activity, especially antifungal properties [4]. There is a lack of studies on the microbiota associated with bee colonies. Our results show that P. polymyxa ALLI-03-01, isolated form the larvae food, produces antifungal compounds that may play an ecological function in the protection of M. scutellaris colony against pathogens. [1] Moran, N. A. 2006. Curr. Biol. 16: 866-871. [2] Poulsen, M. et al. 2010. Plos One. 5:1-14. [3] Lal, S. et al. 2009. Indian J. Microbiol. 49:2-10. [4] Li,S. et al. 2013. Appl. Microbiol. Biotechnol. 97:9479-9489.

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BIO- AND CHEMODIVERSITY OF THE BACTERIAL CUTICULAR MICROBIOTA OF ANTS IN FRENCH GUIANA.

Caroline BIRER1; Lucie ZINGER2; Gregory GENTA-JOUVE3; Alexandre MACIUK4; Yannick ESTEVEZ5; Bruno FIGADERE6; Niklas TYSLKIND7; Christophe DUPLAIS7*

1Ecology of French Guianan Forests (EcoFoG), UMR8172 CNRS-CIRAD- INRAAgroParisTech- Université des Antilles-Université de Guyane, Kourou, French Guiana, France 2Evolution and Biological Diversity (EDB), UMR5174 Université Paul Sabatier- CNRSENFA, Toulouse, France, France 3Organic, Medicinal, Extractive Chemistry and Experimental Toxicologie (COMETE) UMR8638, CNRS-Université Paris Descartes, Paris, France, France 4Biomolecules : Conception, Isolation and Synthesis (BioCIS), UMR8076 Université Paris Sud-CNRS, Chatenay-Malabry, France, France 5Ecology of French Guianan Forests (EcoFoG), UMR8172 CNRS-CIRAD- INRAAgroParisTech- Université des Antilles-Université de Guyane, Kourou, French Guiana, France 6Biomolecules : Conception, Isolation and Synthesis (BioCIS), UMR8076 Université Paris Sud-CNRS, Chatenay-Malabry, France, France 7Ecology of French Guianan Forests (EcoFoG), UMR8172 CNRS-CIRAD- INRAAgroParisTech- Université des Antilles-Université de Guyane, Kourou, French Guiana, France

Symbiotic interactions with microorganisms have played a major role in the evolutionary history of species (1). In tropical forests, ants (Formicinae) constitute a relevant model for studying such processes. Their high species diversity, their social behavior, and their versatility in nesting modes make them excellent candidates for comparative studies to better understand the roles of these traits in determining their symbiotic microbiota. Recent work has shown the role that gut bacteria have played in the adaptation of ants to lownitrogen diets, thus enabling certain species to colonize and persist in arboreal habitats (2). Others work demonstrate that cuticular bacteria associated with attine ants (fungus-growing ants) biosynthesize antimicrobial metabolites to protect the fungus cultivar from various pathogens (3). However, diversity, host-specificity and function of ants-associated cuticular bacteria are unknown. Our aim is to correlate metabolomics and metabarcoding analysis to better understand which communities is producing a specific molecules’ cocktail and how this mixture is an advantageous trait in host species adaptation and diversification in Amazonian forests. We have recently optimized a protocol to extract and amplify bacterial DNA from a single ant’s cuticle, suitable for metabarcoding studies (MiSeq technology), and we have performed in parallel a metabolomics study ants’ cuticle (ESI-MS). Additionally 46 actinobacteria were isolated in order to evaluate the antimicrobial activity of their co-culture extracts. 1. M. McFall-Ngaia, et al. Animals in a bacterial world, a new imperative for the life sciences.Proc. Natl. Acad. Sci. USA, 2013, 110, 3229-3236.

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2. J. A. Russell, et al. Bacterial gut symbionts are tightly linked with the evolution ofherbivory in ants. Proc. Natl. Acad. Sci. USA, 2009, 106, 21236-21241. 3. C. S. Sit, et al. Variable genetic architectures produce virtually identical molecules inbacterial symbionts of fungus-growing ants. Proc. Natl. Acad. Sci. USA, 2015, 112, 13150-13154.

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FRAGRANCE AND MICROBIAL DIVERSITY IN CECIDOMYIIDAE (DIPTERA)-INDUCED HAPLOPAPPUS FOLIOSUS (ASTERACEAE) APICAL GALLS.

Cristian A. Villagra1*; Valéria Cid Maia2; Jan Bergmann3; Vinko Zadjelovic4; Esteban Severino4; Cristina Dorador4

1Instituto de Entomología, Universidad Metropolitana de Ciencias de la Educación, Santiago, RM, Chile 2National Museum, Entomology Dep., Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brasil 3Instituto de Química, Pontifica Univesidad Católica de Valparaiso, Curauma, Valparaiso, Chile 4Centro de Biotecnología y Bioingeniería (CeBiB), Universidad de Antofagasta., Antofagasta, Antofagasta, Chile

Gall-inducing arthropods modify plant structures. These chances can be mediated by the help of symbiotic microorganisms colonizing plant tissue together with cecidogenic arthropod. These symbionts may help bypassing plant resistance by altering host’s secondary metabolic products among other factors. In this work we explore these kind of changes in Haplopappus foliosus (Asteraceae) a native shrub from the Mediterranean matorral of central Chile. We discovered that these plants are attacked by a cecidogenic gall midget (Diptera) capable of inducing galls in apical terminal branches of this plant. We studied if the occurrence of these deformations may also involve changes in plant’s fragrance and microbial-associated biodiversity. Based on headspace scent collection of normal vs galled apical branches and GCMS analysis we discovered that fragrance composition of infected vs healthy (non-galled) branches differed with statistical significance. These differences were focused on β-myrcene concentration, which was higher on galled apical branches than healthy ones. By the contrary, p-cymene was more abundant in healthy branches than galled ones. These compounds have been found in essential oil volatile fraction of several plant species. β-myrcene has been suggested to have antibacterial property while p-cymene it is been characterized by its insecticidal and antifungal properties. Complementarily, based on the dissection of healthy and galled branches and the cultivation of their microorganisms with different methods we discovered 72% from total microorganisms biodiversity was extracted from gall- deformed tissues while only 28% belonged from apical branches from healthy H. foliosus. (DMG=10,2 y DMG=1,9 respectively). From total microorganism found 52% corresponded to fungi. We discuss our results under the frame of the potential host- microbial metabolic changes related to the development of apical galls and plant resistance traits. Funding: FONDECYT Iniciación Nº 11100109 and CONICYT inserción Nº 79100013 and Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia”.

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FUNGAL STRAINS ASSOCIATED TO THE AMBROSIA BEETLE MEGAPLATYPUS MUTATUS AND THEIR VOLATILE EMISSIONS.

Mariel SLODOWICZ1*; Esteban CERIANI2; Cecilia CARMARAN2; Paola GONZALEZ AUDINO2

1(CIPEIN) UNIDEF - CITEDEF - CONICET, VILLA MARTELLI, BUENOS AIRES, ARGENTINA 2FCEN, Buenos Aires, Buenos Aires, ARGENTINA

Megaplatypus mutatus is an ambrosia beetle native to South America, that represents the main forest pest in Argentina of Poplar plantations and is an emergent foreign pest in Europe, representing a potential risk to forest and fruit plantations globally due to its low specificity.In the fungal community associated, nineteen taxa are present, being the most relevant Fusarium solani species complex, three species of Raffaelea and Graphium basitruncatum. Gas chromatography-Mass spectrometry combined with Solid Phase Microextraction was used to identify the volatile organic compounds profiles from several fungal species associated to the forest pest. Compounds identified in the equilibrium vapor phase that came exclusively from fungal organisms where 3-methyl butanol, 2-methyl butanol, 2-phenyl ethanol and 2-undecanone were the most frequently volatiles found in the microbial samples. Analysis of volatile organic compounds of fungal strains is a primary tool for development of strategies for forest pest’s management with low environmental impact. Ceriani Namurake et al, Forestry, 2016.

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GLYCEOLLIN INDUCTION AFTER PHAKOPSORA PACHYRHIZI INFECTION AND ITS INTERACTION WITH MEVALONATE PATHWAY IN SOYBEAN.

Talitta R. PARMEZAN1; Jefferson T. FUJISAO1*; Alessandra YOKOYAMA1; Moisés AQUINO1; Estela O. NUNES1; Ricardo VILELA ABDELNOOR1; Clara B. HOFFMANN-CAMPO1; Francismar C. MARCELINO-GUIMARAES1

1Universide Estadual de Londrina, Londrina, Paraná, Brazil

Phytoalexins are a group of inducible low molecular weight secondary metabolites with antimicrobial activity and thus having plant defencing properties. In soybeans, glyceollin induction in response to Asian soybean rust (ASR), Phakpsora pachyrhizi infection has been reported, related to the traditional pathway, i.e., involving the precursors daidzein, and the action of the enzyme 4-dimethylallyltransferase (G4DT - EC2.5.1.36) (AKASHI et al., Plant Physiology 149: 683–693, 2009). Experiments were carried out to evaluate glyceollin contents in soybean, P. pachyrhizi infected leaves and the potential contribution of cytosolic pathway of Mevalonate in glyceollin production, by DMAPP precursor generation. Glyceollin levels on soybean leaves were quantified at 12, 24, 48, 72, 96 and 192 hours after infection (hai) with ASR by UPLC-MS. In parallel, the expression profile of glyceollin synthase enzymes (EC 2.5.1.36), isopentenyl-diphosphate Δ-isomerase (5.3.3.2) and geranyl diphosphate synthase (2.5.1.1) were evaluated. The analysis were carried out using soybean genotypes containing different ASR resistance genes (Rpp2, Rpp4 and Rpp5) and the susceptible genotype [Williams 82 (W82)]. The infection resulted in glyceollin accumulation mainly at the final time analysed, being higher in 192 hai, in W82, Rpp2 and Rpp5 genotypes. In contrast, the mRNA levels of G4DT were induced in all genotypes, at least in one of initial time point and from 96 hai, confirming the involvement of this phytoalexin in soybean resistance response to ASR. Additionally, the expression levels of genes encoding the enzymes 5.3.3.2 and 2.5.1.1, in the bifurcation of the Mevalonate pathway, acting before and after DMAPP production, respectively, were not correlated to glyceollin content in W82, Rpp4 and Rpp5 genotypes. In Rpp2, the induction of the mRNA corresponding to the enzyme 5.3.3.2, in parallel with the reduction of the levels of 2.5.1.1, would indicate a possible accumulation of DMAPP by Mevalonate pathway to be converted to glyceollin.

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INFLUENCE OF CULTURE CONDITIONS ON ANTIBIOTIC PRODUCTION BY KRASILNIKOVIA SP. T082, A RARE ACTINOBACTERIUM.

Rita de Cássia PESSOTTI1*; Matthew F. TRAXLER2; Jon CLARDY3; Roberto KOLTER4; Mônica T. PUPO4

1School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirão Preto, Sao Paulo, Brazil 2Department of Plant & Microbial Biology, University of California, Berkeley, California, USA 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA 4Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

Microorganisms are an important source of bioactive compounds, yet the discovery of new ones has diminished over the years. Therefore, different culture approaches should be tested in an attempt to diversify the biosynthesis of compounds, such as varying culture conditions (1). Previous studies highlighted Krasilnikovia sp. T082 as a promising strain for chemical studies. Genome mining demonstrated that this strain harbors in its genome several biosynthetic gene clusters related to secondary metabolism that were not previously described. This strain was cultivated on TSA and ISP2 solid media for seven days at 30 °C. Colonies were overlaid with the multi-resistant actinobacterium Amycolatopsis sp. AA4 and incubated overnight. Bioactivity was analyzed by visualization of inhibition halos. Liquid culture was also performed on TSB and ISP2 media for ten days at 30 °C, 200 rpm. Cultures were extracted by liquid-liquid partition with ethyl acetate. Extracts were tested against the same indicator strain. Bioactivity was detected only when Krasilnikovia sp. T082 was cultivated in liquid ISP2 medium. Bioactivity-guided fractionation by solid-phase-extraction and HPLC, and the combination of genome mining, NMR and MS analysis led to the isolation and elucidation of three analog compounds identified as SW-163D (2), RK-1355A (3) and the newly discovered Retimycin B. These analogs belong to a chemically interesting and bioactive class of NRPS DNA-bisintercalators that possess rare moieties for natural products, such as the non-proteinogenic amino acid norcoronamic acid and sulfoxide group. These results demonstrate how changing culture conditions can influence the expression of secondary metabolism in bacteria, and, therefore, the relevance of varying it for bioactive natural products research. References (1) Bertrand, S. et al. Biotechnol Adv. 32: 1180-204, 2014. (2) Nakaya, M. et al. Biosci Biotechnol Biochem. 71: 2969-76, 2007. (3) Lim, C.L. et al. J Antibiot (Tokyo). 67: 323-9, 2014.

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LIPOPEPTIDES FROM MARINE CYANOBACTERIA INDUCING BEHAVIORS ALONG A TROPHIC CASCADE.

Louis BORNANCIN1; Isabelle BONNARD1; Suzanne MILLS2; Bernard BANAIGS3*

1CRIOBE, USR 3278 CNRS-EPHE-UPVD, University of Perpignan Via Domitia, Perpignan, Languedoc-Roussillon, France 2CRIOBE, USR 3278 CNRS-EPHE-UPVD,, Papetoai, Moorea, French Polynesia 3CRIOBE, USR 3278 CNRS-EPHE-UPVD, University of Perpignan Via Domitia, Perpignan, Languedoc-Roussillon, France

In the lagoon of Moorea in French Polynesia, we have identified a relatively simple tropical marine ecosystem consisting of two filamentous cyanobacteria (Lyngbya majuscula and Anabaena cf. torulosa), two herbivorous mollusks (Stylocheilus striatus and Bulla orientalis), an omnivorous nudibranch (Gymnodoris ceylonica) and a carnivorous crab (Thalamita coerulipes). L. majuscula and A. torulosa, that can proliferate across a wide sandy area and even on corals, are prolific producers of secondary metabolites, mainly cyclic lipopeptides, which may either be toxic or act as feeding deterrents to potential consumers. However, the compounds do not prevent feeding by the sea hare S. striatus, which is known to sequester and transform some secondary metabolites produced by L. majuscula. But do mollusks have the ability to detect these chemical cues? Can they use the chemical information in their decision making? In the study of this model ecosystem, we combine cyanobacterial metabolome profiling and behavioral bioassays in order to study the cascading effects of chemical mediators in multi-trophic relations; expression by primary producers, sequestration and biotransformation of secondary metabolites acquired from dietary sources, and chemical recognition mechanisms in inter-specific relationships. Using extensive 1D and 2D NMR and mass spectrometry we characterized new acyclic lipopeptides derived from the known cyclic laxaphycins, five in the cyanobacteria and four in the mollusks. Although a biotransformation of lipopetides from A. torulosa occurs in sea hare, peptides from L. majuscula are found intact. The sea hare S. striatus, considered as a L. majuscula specialist, was found feeding on A. torulosa and was less susceptible to predation by the nudibranch G. ceylonica when it fed on A. torulosa than on L. majuscula. Finally we show that the two opistobranchs were influenced by the lipopeptidic blend in feeding choice, and in foraging behavior; they were able to extract information from cyanobacterial chemical defenses.

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MOLECULAR AND GENETIC EXCHANGES IN THE FUNGUS-GROWING ANT SYSTEM.

Jon CLARDY1*

1Harvard Medical School, Boston, Massachusetts, United States

Four types of animals – humans, ants, termites and beetles - have derived agricultural systems, and this talk will focus on the chemical ecology that regulates the multilateral symbioses of fungus-growing ants. Small molecules regulate many aspects of the behavior of the ant farmers, their fungal crop, the specialized fungal pathogens, and the symbiotic bacteria that provide chemical defenses. Since the ant system evolved from a single origin in the Amazon 50-60 million years ago to include over 200 species, it has created a large set of diverse molecules that is beginning to be systematically explored at the molecular and genetic level. This talk will discuss some early but important examples of both the molecular diversity and the genetic strategies that created it.

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NATURAL PRODUCTS FROM ENTOMOPATHOGENIC BACTERIA - FROM CHEMICAL ECOLOGY TO SYNTHETIC BIOLOGY.

Helge B. BODE1*

1Merck-endoweg Chair for Molecular Biotechnology, Goethe University Frankfurt, Frankfurt, Frankfurt, Germany

Most antibiotics and several other therapeutics used in the clinic are derived from natural products produced by bacteria and fungi. Despite the importance of such compounds their natural function is often unknown but clearly they are not made originally to cure diseases.Using entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus we are trying to answer the following questions: What is the natural function of these natural products? How have these compounds been optimized for what mode of action? How is their biosynthesis regulated? These typical chemical ecology questions can be addressed since we can study the function of the bacterial natural products in the bacteria alone, together with their nematode host or the insect prey that is infected and killed by the nematodes carrying the bacteria in their gut and we can maintain all levels of this complex life cycle in the lab. Once the basic mechanisms have been identified they can be applied to manipulate the regulatory mechanisms for the production of specific natural products. Moreover, the detailed analysis of several bacterial genomes with their encoded biosynthetic capacity allows the identification of rules for synthetic biology enabling the modification of biosynthesis pathways and even the de novo design of “non-natural” natural products.

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PRODUCTION OF INDOLE-3-ACETIC ACID BY ATTA SEXDENS RUBROPILOSA ASSOCIATED PSEUDONOCARDIA SP.

Eduardo A. SILVA-JUNIOR1*; Emily MEVERS2; Camila R. PALUDO2; Antonio C. RUZZINI2; Weilan G. P. MELO2; Fabio S. NASCIMENTO3; Cameron R. CURRIE4; Jon CLARDY4; Mônica T. PUPO4

1School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirão Preto, Sao Paulo, Brazil 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA 3FACULTY OF PHILOSOPHY, SCIENCES AND LITERATURE OF RIBEIRÃO PRETO - UNIVERSITY OF SÃO PAULO, Ribeirão Preto, São Paulo, BRAZIL 4Department of Bacteriology – University of Wisconsin, Madison, Wisconsin, USA

A number of fungus-farming ants in the tribe Attini engage in a symbiosis with antibiotic-producing bacteria to protect their colonies against pathogenic microbes. The relationship between these bacteria and leaf-cutter ants is unclear. Ants in the genus Atta lack visible exoskeletal colonies of bacteria. As part of our efforts to study the Actinobacterial symbionts of fungus-farming ants, we investigated the possible association between Pseudonocardia spp. and the leaf-cutter ants Atta sexdens rubropilosa. Ants and fungal gardens were collected from three laboratory colonies of Atta sexdens rubropilosa. Actinobacteria were isolated from both ants and fungal garden material using a chitin-based selective medium. Among these bacteria, eight isolates belonged to the genus Pseudonocardia, the preeminent microbial associate of lower Attine ants. Each of the isolated Pseudonocardia sp. inhibited Escovopsis growth and bioassay-guided isolation revealed that the active compound is indole-3-acetic acid (IAA). This compound selectively inhibited the growth of the fungal garden pathogen Escovopsis, but not the fungal cultivar. Further chemical analyses by LC-MS revealed that all eight Pseudonocardia isolates can produce IAA. Despite it’s initial characterization as a plant auxin, IAA is a component of the antimicrobial metapleural glandular secretions of Atta sexdens. The results suggest that Pseudonocardia spp. can contribute to the chemical defenses used to protect Atta sexdens rubropilosa colonies.

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PLAGUE INSECTS’ GUT BACTERIA AS A RICH SOURCE FOR BACTERIAL POLYHYDROXYALKANOATE PRODUCERS.

Bruno PERLATTI1; Maria Fátima das Graças Fernandes DA SILVA1; João Batista FERNANDES1; Moacir Rossi FORIM1*

1Universidade Federal de São Carlos - UFSCAR, São Carlos, São Paulo, Brasil

Polyhydroxyalkanoates (PHA) are a class of polyester produced from bacteria that might pose an interesting alternative for production of polymers without using petroleum-based monomers. The major problems restraining the widespread application of PHA are the production in an economically viable scale, from which the major problems can be related to cost of substrate, and moderate bacterial productivity. Strategies to overcome these obstacles are the utilization of highly productive strains and cheap sources of carbon substrates. As such, this work had as aim the screening of a collection of bacteria obtained from the gut of a pest insect using fluorescent dye method, and evaluation of the production of PHA from the most promising strains using conventional substrates such as glucose, and acetate, as well as industrial by-products such as glycerol and light and heavy fractions from biomass pyrolysis. Overall 73 strains from 17 genera were screened using Nile Red, with 78% of the isolates showing PHA production to some extent. Further experiments with most promising strains revealed that Aurantimonas sp. and Delftia sp. were able to accumulate up to 50 and 95% of PHA, respectively. While the former produced mainly polyhydroxybutyrate (PHB) homopolymer, the latter produced poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PBHV) with up to 11% of valerate. Production of PHA from pyrolysis fractions were low probably due to absence of nutrient limitation studies, but apparently did not inhibit bacterial development.

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THE ROLES OF STINGLESS BEES AND BACILLUS SPP. INTERACTIONS.

Camila R. PALUDO1*; Eduardo A. SILVA-JUNIOR1; Antonio C. RUZZINI2; Gleb PISHCHANY3; Cameron R. CURRIE4; Fábio S. NASCIMENTO5; Roberto G. KOLTER5; Jon CLARDY5; Mônica T. PUPO5

1School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirão Preto, Sao Paulo, Brazil 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA 3Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA 4Department of Bacteriology – University of Wisconsin, Madison, Wisconsin, USA 5Faculty of Philosophy, Sciences and Literature of Ribeirão Preto - University of São Paulo, Ribeirão Preto, São Paulo, BRAZIL

Bacillus spp. engage in symbiotic relationships with social bees (1). In particular, the abdominal colonization of stingless bees by Bacillus is thought to be an ancient association, supported by the nucleotide identity of bacterial DNA extracted from abdominal tissue of a bee specimen preserved in amber dating back to 25 to 40 million years in age (2). Studying insect-associated bacteria is an opportunity to obtain antimicrobial compounds and to understand the ecological role involving these organisms. In this study, we have isolated Bacillus sp. SDLI1 from the gut of the stingless bee Scaptotrigona depilis in larval phase. The whole-genome sequence of this microorganism revealed that it encodes putative biosynthetic gene clusters for production of the antimicrobial compounds fengycin, bacillomycin, bacillaene, macrolactin, surfactin, bacilysin, bacillibactin, and difficidin (3). Bacillus sp. SDLI1 is capable to inhibit the entomopathogens Beauveria bassiana and Metarhizium anisopliae and in a bioassay-guide fractionation using Candida albicans ATCC MYA-2876, we have isolated and identified the antifungal compound anteiso-C17 Bacillomycin D. These evidences suggested that Bacillus sp. SDLI1 could maintain a symbiotic relationship with S. depilis, furnishing antimicrobial compounds to protect the stingless bee’s colony against invaders. The gut colonization of larvae indicates that this bacterium is particularly important in this period of insect development. 1. Gilliam M. FEMS Microbiol Lett, 155:1-10, 1997. 2. Cano RT, Borucki MK. Science, 268:1060-1064, 1995. 3. Paludo CR, Ruzzini AC, Silva-Junior EA, Pishchany G, Currie CR, Nascimento FS, Kolter RG, Clardy J, Pupo MT. Genome Announc, 4:e00174-16, 2016.

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THE A-FACTOR GOES VOLATILE? NEW BUTYROLACTONES FROM MARINE SALINISPORA BACTERIA.

Stefan SCHULZ1*

1Institute of Organic Chemistry, Technische Universität Braunschweig, Braunschweig, Niedersachsen, Germany

The A-factor and the related virginiae-butanolides are water soluble signals used by Streptomyces (actinomycetes) bacteria [1]. These lactones are active in low concentrations, inducing e. g. sporulation and biosynthesis of certain antibiotics. Usually they occur in species specific mixtures, activating specific genes. Receptor genes are also reported outside of Streptomyces, indicating a broader communicative role of these compounds. These signaling compounds contain a butyrolactone ring, and one hydroxyl and a keto group or two hydroxyl groups. In culture, they stay in the water phase and are not detectable in the headspace of Streptomyces cultures.To detect potential signaling compounds, volatiles of marine Salinispora bacteria, another actinomycetes genus, were analyzed by GC/MS. A group of unknown compounds was detected, present in about 80 % of the 30 strains investigated. A genomic analysis revealed that in all of these strains a gene cluster potentially responsible for butyrolactone formation occurred. Analysis of the mass spectra revealed that these compounds might be related to the A-factor, but were less polar. Their structures were elucidated by mass spectra interpretation and synthesis. Contrary to other butyrolactones, the new lactones are volatile. Further research will be focusing on the function of these compounds on Salinispora and other bacteria. More than 200 compounds are produced by the Salinispora strains investigated, including well- known insect pheromones, e. g. spiroacetals. [1] S. Schulz, S. Hötling, Nat. Prod. Rep. 32, 1042 (2015). doi: 10.1039/c5np00006h.

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THE COMBINATION OF PLANT GROWTH PROMOTING RHIZOBACTERIA AND METHYL JASMONATE PRODUCE POSITIVE AND SYNERGETIC EFFECTS ON PHYTOCHEMICAL AND MORPHOLOGICAL CHANGES IN MENTHA X PIPERITA L.

Lorena del Rosario CAPPELLARI1; Julieta CHIAPPERO1; Walter GIORDANO1; Erika BANCHIO1*

1UNIVERSIDAD NACIONAL DE RIO CUARTO, RIO CUARTO, CORDOBA, ARGENTINA

Peppermint is one of the most important crops cultivated worldwide for fresh or dried leaves, and essential oils (EOs). The EOs are produced inepidermal structuresknown as glandular hairs.Numerous species of bacteria associated with the plant rhizosphere, have shown beneficial effects on plant growth, yield and quality of crops. Such bacteria are collectively “plant growth- -promoting rhizobacteria” (PGPR).M. piperitashootswere inoculated with PGPR strains Bacillus subtilisGB03, Pseudomonas fluorescensWCS417r or P. putidaSJ04. The inoculated plants were sprayed with methyl jasmonate (MeJA) at different concentration. At 7 and 14 days after the treatmentwith MeJA shoot biomass, EOs yield, major essential-oil components, total phenolics content, and peltate glandular trichome density were evaluated.Treated plants (inoculated with PGPR or MeJAsprayedin different concentrations) showed a similar increases in all parameters evaluated except MeJA treated plants exhibit a slight decrese in shoot biomas.Total EOs content in inoculated plantswas 2-3 folds higher than the control.Exogenous application of MeJA increase the total phenolics content.The combined (PGPR + MeJA) treatmentexhibit an increases in total EO yield and marked quantitative changes in monoterpene content; total phenolic and trichome density much higher than that observed in the individual treatments. Aproximately was2-fold higher in the different parameters evaluated. The exogenous application of MeJA in combination with rhizobacteria causes an increase on secondary metabolites content and the number of peltate trichomes in M. piperitahigher than individual treatments. As might be suggested that both rhizobacteria and the methylated derivative of JA trigger the activation of defense responses in the plant, involving stimulation of the biosynthesis of secondary metabolites. Improved knowledge of factors that affect biosynthesis of secondary metabolites will lead to strategies for improved cultivation and productivity of aromatic plants and other agricultural crops without the use of chemical fertilizers.

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VOC BIOMARKER AND INSECT ANTENNA-BASED BIOSENSOR FOR DETECTION OF FUNGAL CONTAMINATION IN GRAIN.

Vincas BŪDA1*; Laima BLAŽYTĖ-ČEREŠKIENĖ1; Violeta APŠEGAITĖ1; Rita BUTKIENĖ1; Irena NEDVECKYTĖ1; Dalė PEČIULYTĖ1

1Nature Research Centre, Vilnius, Lithuania, Lithuania

Fungi invading stored grain influence quality of food. Volatile organic compounds (VOCs) among metabolites produced by fungi can be characteristic biomarkers for detection of fungal presence as alternative to mycotoxin detection. The objective of this study was to reveal insects’ ability to recognise fungus-infected substrate and to find out the most suitable insect antenna-based biosensor for detection of biomarker characteristic for fungal contamination in grain.To estimate insect ability to recognise fungus-infected substrate the two-choice oviposition behaviour of mated Plodia interpunctella females was tested. To reveal the substrate changes during fungal growth on grain, weight changes of the grain sample, moisture, and CO2, as well as volatile secondary metabolite formation were investigated. The results of our study demonstrated that pest of stored products P. interpunctella (Lepidoptera: Pyralidae) discriminated between uninfected and Aspergillus flavus infected wheat grain preferring for oviposition uninfected grain versus fungus-infected grain. Dynamics of insect oviposition behaviour clearly correlated with fermentation processes in infected grain, i.e. moisture content, CO2 and 3-methyl-1- butanol emission changes. Grain volatile organic compounds’ changes and appearance of 3-methyl-1-butanol might be an early indicator for P. interpunctella of fungi presence in the substrate. EAG-dose responses showed high sensitivity of the insect antennal receptors to the compound.Three insect species were revealed which antennae showed explicit responses to fungal volatile metabolite 3-methyl-1-butanol: P. interpunctella, Drosophilamelanogaster and Apis mellifera. The compound could serve as early biomarker for detection grain contamination by fungi. Thus, we suggest to use the insect antenna as biosensor for detection spoilage in stored grain.

105

VOLATILES OF THE HUMAN SKIN MICROBIOME ACT AS QUORUM QUENCHER.

Marie Chantal LEMFACK1; Srinivasa Rao RAVELLA2; Stefan SCHULZ2; Birgit PIECHULLA3*

1University of Rostock, Institute for Biological Sciences, Rostock, Mecklenburg- Vorpommern, Germany 2Technical University of Braunschweig, Institute for Organic Chemistry, Braunschweig, Lower Saxony, Germany 3University of Rostock, Institute for Biological Sciences, Rostock, Mecklenburg- Vorpommern, Germany

The human skin covers the body and serves as a barrier to protect the body against excessive loss of water, harmful substances and pathogens. It is the part of the body, which is in contact with the external environment and therefore colonized by diverse species of microorganisms, the skin microbiome. This microbiome is dominated by actinobacteria (51.8%, largely Corynebacterium sp.) followed by firmicute (24.4%, largely Staphylococcus sp.) (Grice et al., 2009). These bacteria are often beneficial for the host, e.g. they synthesize compounds with antimicrobial properties. Presently not much is known about volatile compounds released from bacteria of the human microbiome and their functions as well as relevance within this community. Therefore we investigated the volatile profiles of several Corynebacterium and Staphylococcus strains. The volatile organic compounds (VOCs) appear species-specific or are common in several strains. Interestingly, the volatile profile of Staphylococcus schleiferi revealed new amino/imino ketones, which are new in nature and have never been reported to be emitted by any organism. In order to study their biological effects, the new compounds were chemically synthesized and applied to Gram-positive and Gram-negative bacteria. It turned out that these volatiles affect the growth of Gram-positive bacteria and quorum sensing controlled markers in Gram-negative bacteria. These results will provide new aspects regarding VOCs-based communication between skin microorganism communities to better understand the balance between host and microorganisms as well as its importance in healthy and disease conditions.

106

YEAST VIS-À-VIS CHEMICAL ECOLOGY OF GREEN LACEWINGS (NEUROPTERA: CHRYSOPIDAE).

Jeffrey R. ALDRICH1*; Elda VITANOVIC2; Frank G. ZALOM3

1Department of Entomology & Nematology, UC Davis, California, USA 2Institute for Adriatic Crops & Karst Reclamation, Put Duilova 11, 21000 Split, Croatia 3Department of Entomology and Nematology, University of California, Davis, CA, Davis, California, USA

Green lacewings (Chrysopidae; ~1200 species), especially Chrysopa and Chrysoperla species whose larvae are predators of aphids and other small soft-bodied pests, are the most agriculturally important family in the order Neuroptera (Aldrich and Zhang 2016). Adults of Chrysopa are also predacious, with robust and asymmetrical mandibles modified for chewing, and are the only lacewings known to produce aggregation pheromones. Recent evidence suggests that Chrysopa adult males must feed on aphid oviparae and/or certain plants to produce their pheromones (Aldrich et al. 2016). However, in non-carnivorous green lacewings such as Chrysoperla, the adult mandibles are symmetrical and relatively poorly developed, whereas their tracheae are much larger than in Chrysopa, an adaptation supporting intestinal symbiotic yeasts (Gibson and Hunter 2005); Chrysopa species do not harbor yeast in the crop (Albuquerque et al. 2012). The University of California at Davis is home to one of the largest yeast collections in the world; the Phaff Yeast Culture Collection, with ~800 species and 6,000 yeast strains. One of us (EV), while screening yeasts for attraction of the olive fruit fly, incidentally discovered that certain yeasts are significantly attractive to green lacewings. The current research to be described is focused on the field-testing and volatile chemical results of the lacewing-attractive yeasts from the UC Davis Yeast Culture Collection.

Albuquerque GS et al. (2012) In: Panizzi AR, Parra JRP (eds) Insect Bioecology and Nutrition for Integrated Pest Management. CRC Press Boca Raton, FL, pp 593-631

Aldrich JR et al. (2016) PeerJ http://dx.doi.org/10.7717/peerj.1564:1-17

Aldrich JR & Zhang Q-H (2016) Chemical ecology of Neuroptera. Annu Rev Entomol 61:197–218.

Gibson CM, Hunter MS (2005) Biol Control 32:57-64.

107 GENERAL CHEMICAL ECOLOGY

A MULTIDISCIPLINARY APPROACH TOWARDS UNDERSTANDING TRITERPENOID METABOLISM AND SIGNALING IN PLANTS.

Bastien CHRIST1; Jing-Ke WENG1*

1Whitehead Institute for Biomedical Research, Cambridge, Cambridge, USA

Plant specialized metabolism remains largely unexplored due to its richness and complexity. The vast network of enzymes and metabolites not only provides opportunities for revealing fundamental principles of complex biological systems, but also presents a natural source for catalysts and natural products of pharmaceutical and industrial significance. My research focuses on non-steroidal triterpenoids, a mostly uncharacterized class of isoprenoids enriched in the plant kingdom. Recent studies suggest that several groups of non-steroidal triterpenoids may also serve as signaling molecules modulating plant growth and development, and inter-species communication. However, how structurally diverse non-steroidal triterpenoids are biosynthesized and exert hormonal functions in vivo remain a quandary. We recently developed a new workflow implementing state-of-the-art metabolomics, genome editing, and metabolic engineering in yeast, which greatly facilitates identification and characterization of the biochemistry of largely unexplored plant specialized triterpene metabolism. Ultimately, we seek to understand the role and action mechanisms of plant non-steroidal triterpenoids in plants’ chemical communication with their biotic environments.

109

ALDEHYDE OXIDASE FOUND IN HALYOMORPHA HALYS (STAL) (HETEROPTERA: PENTATOMIDAE) AND DEGRADATION OF DEFENSIVE ALDEHYDES.

Koji NOGE1*; Shigeru TAMOGAMI2

1Akita Prefectural University, Akita City, Akita Prefecture, JAPAN 2Akita Prefectural University, Akita City, Akita, JAPAN

Most terrestrial heteropterans protect themselves by producing pungent secretions containing aliphatic aldehydes, such as (E)-2-hexenal, (E)-2-octenal and 4-oxo-(E)-2- hexenal (OHE). These aldehydes function either repellent, deterrent or toxin against insect predators. (E)-2-Hexenal and (E)-2-octenal repell ants and mantids at ecologically relevant doses. In contrast, OHE functions as deterrent and toxin but without repellent properties against predators. Exposure to OHE in a jar for 2 h impaired the locomotive ability of crickets, an experimental model (EC50=1.2 mg/L), resulting in permanent paralysis and death. OHE also affected other insects, such as cockroach and dragonfly, but OHE-producing heteropterans, for example, Halyomorphahalys, show better resistance to this toxic compound (EC50>50 mg/L). Although this exposure experiments were performed under laboratory conditions, the results may indicate that OHE- producing heteropterans possess some barrier against toxic aldehydes to avoid autotoxicity caused by released their own defensive secretions. Thus we planned to investigate the enzyme(s) in H. halys that might be involved in detoxification of aldehydes. First, we detected aldehyde oxidase activities from the antenna, head, secretory gland and gut of H. halys on a polyacrylamide gel by activity staining method using nitro blue tetrazolium. The enzymes found in the antenna, head and gut seemed to be the same by native polyacrylamide gel electrophoresis, but the enzymes in secretory gland was different from the others. Second, we purified the aldehyde oxidase from the head through four steps and examined its enzymatic properties. The purified enzyme was determined to be a 256 kDa homotetramer composed of 65 kDa subunits. The enzyme oxidized not only aliphatic aldehydes (hexanal, decanal, (E)-2-hexenal and (E)-2- decenal), but also aromatic aldehydes (benzaldehyde and phenylacetaldehyde). The broad substrate specificity may indicate that this enzyme play a role in degradation of various aldehydes.

110

AN UNUSUAL PATTERN OF SEX PHEROMONE PRODUCTION AND CALLING BEHAVIOR IN HELICOVERPA ARMIGERA (LEPIDOPTERA: NOCTUIDAE).

Hernane Dias ARAÚJO1*; Janice de Souza LOPES1; Eraldo LIMA1

1UNIVERSIDADE FEDERAL DE VIÇOSA, Viçosa, Minas Gerais, Brazil

Helicoverpa armigera is one of the most important pests in the Old World. Recently, it has been documented in Brazil and is already distributed all over the country. The process of adaptation to this new habitat can affect the moth reproductive behavior. Here, we assessed the rhythm of the calling behavior and of the sex pheromone production in a Brazilian H. armigera population from Luís Eduardo Magalhães, State. The mean onset time of calling declined and the mean time spent calling increased after the first day of calling, but remained constant thereafter. The mean number of calling bouts was not affected. The calling behavior occurred only in the last quarter of the 10h scotophase, with the highest frequency reached in its last hour. Females of 1 and 3 days old produced the highest amounts of (Z)-11-hexadecenal, the major pheromone component, at the last 4 hours of the scotophase, while 5 days old females had the pheromone highest amounts in the last 2 hours. However, there was no difference in the amount of pheromone produced regarding the female age. The calling behavior was synchronized with the pheromone production, as expected. But this so late calling pattern and pheromone production is an unusual behavior, being reported for the first time in H. armigera. Few other moths present this late calling rhythm. Other H. armigera populations around the world call more frequently in the second half of scotophase, but it is common that the activity begins in the first half of the scotophase. The invasive nature of this insect in Brazil opens a new range for research of the reproductive behavior of H. armigera. Its close relationship with the native species, Helicoverpa zea, is something that should receive attention in the coming years.

111

ANTENNAL ELECTROPHYSIOLOGICAL RESPONSES OF WORKERS TO QUEEN CUTICULAR HYDROCARBONS IN A HIGHLY EUSOCIAL BEE, TETRAGONISCA ANGUSTULA (APIDAE: MELIPONINI).

Arodí FAVARIS1; Denise ALVES1; José Maurício BENTO1*

1University of São Paulo, Piracicaba, São Paulo, Brazil

Chemical communication is the most efficient mode of spreading reliable information between insects of the same (or different) species. The perception of chemical compounds occurs via sensory organs (sensilla), and can be exploited by gas chromatographic-electroantennographic detection (GC-EAD), an efficient method for chemical ecology studies. However, many interesting behavioural ecology questions, using CG-EAD are still unexplored in highly eusocial insects, including the stingless bees. In order to gain more insights into this issue, the aim of this study was evaluate if the antennae of Tetragonisca angustula workers react to queen cuticular compounds. This is an excellent model species, since workers just activate their ovaries and produce male-destined eggs in queenless conditions. To achieve this goal, we used antennae from workers, which were sampled at brood comb area, and the cuticular extract of one fertile mother-queen. Workers’ antennae were excised and prepared on electro conductive gel electrode coupled with signal amplifier. The workers’ antennae reacted to pentacosane, heptacosane and four unidentified compounds. These results showed that GC-EAD provides a variety of novel research approaches using T. angustula as a study model.

112

BEHAVIORAL RESPONSES OF BT -RESISTANT AND -SUSCEPTIBLE SPODOPTERA FRUGIPERDA MALES TO FEMALE SEX PHEROMONE.

Nataly de La PAVA1; Jeremy MCNEIL2; Eliseu J. G. PEREIRA2; Eraldo LIMA2*

1UNIVERSIDADE FEDERAL DE VIÇOSA, Viçosa, Minas Gerais, Brazil 2UNIVERSITY OF WESTERN ONTARIO, LONDON, ONTARIO, CANADA

Spodoptera frugiperda is the most important economic pest of maize crop in tropical areas of the American continent, and the use genetically modified corn producing Bacillus thuringiensis (Bt) toxins is the main means of pest control. However, despite the use of resistance management strategies (refuge/high dose) resistant lines of the fall armyworm are now present in the field. While it has been well established that there may be costs associated with the development of resistance (e.g. increased development time, reduced fecundity), little attention has been given to the potential effects of resistance on pheromone mediated mating. We evaluated the response of virgin males from Bt resistant and susceptible lines to pheromone gland extracts (0.15 female-equivalent/μl released by an ultrasonic sprayer) in a wind tunnel. Both resistant and susceptible males showed a higher response to the pheromone extracted from the glands of females from their own line, and resistant males showed a significantly decreased response to gland extracts from susceptible females. These findings suggest that assortative mating could occur when resistant and susceptible genotypes of the fall armyworm are both present in the field and, if this is the case, would limit the efficacy of using non-Bt corn as a refuge crop for resistance management. Acknowledgments to CNPq, INCT -Semioquímicos na Agricultura and FAPEMIG - CNPq Grant 407504/2013-3.

113

BIOSYNTHESIS, CHEMICAL SYNTHESIS AND OCCURRENCE OF HEMOLYMPH TOXINS IN CHRYSOMELINA.

Tobias BECKER1; Wilhelm BOLAND1*; Antje BURSE1; Kerstin PLOSS1; Gerhard PAULS2

1MPI Chemical Ecology, Jena, Thüringen, Germany 2MPI Chemical Ecology, Jena, Thüringen, Germany

Leaf beetles (Chrysomelina), legumes and fungi produce 3-nitropropanoate (3-NPA) derived compounds for their chemical defense. 3-NPA is an irreversible inhibitor of succinate dehydrogenase. Thus, this compound is a general poison, showing high toxicity especially against nerve cells. These compounds protect all life stages of the insects. Esters of this toxin are highly concentrated in the hemolymph as well as in the adult secretion. Although 3-NPA derivatives are well characterized in terms of toxicity, little is known about their biosynthesis in insects and plants. In addition, no biological function of a major hemolymph metabolite lacking a 3-NPA ester is described. To study biosynthetic aspects as well as the occurrence and biological importance of these compounds within many leaf beetle species, we developed novel synthetic protocols for isoxazolin-5-one glucosides. Intermediates of the biosynthetic pathway have been synthesized and injected or fed to the larvae. Applying these methods, a novel and unexpected biosynthetic pathway, derived from propanoate, for the formation of isoxazolin-5-one and 3-nitropropanoic acid derived glucosides was revealed in Chrysomelina. Quantitative data have been acquired in many different species of Chrysomelina larvae to determine the amounts of these compounds in the larval hemolymph as well as in whole larva extracts. These findings indicate that isoxazolin-5- one glucosides are chemomarkers in this subfamily.1-3 References 1. G. Pauls, T. Becker, P. Rahfeld, R. R. Gretscher, C. Paetz, J. M. Pasteels, S. H. von Reuss, A. Burse and W. Boland, J. Chem. Ecol., 2016. 2. T. Becker, P. Kartikeya, C. Paetz, S. H. von Reuss and W. Boland, Org. Biomol. Chem., 2015, 13, 4025. 3. T. Becker, H. Görls, G. Pauls, R. Wedekind, M. Kai, S. H. von Reuss and W. Boland, J. Org. Chem., 2013, 78, 12779.

114

CHARACTERIZATION OF THE PHEROMONE COMMUNICATION CHANNEL IN MAMESTRA CONFIGURATA.

Edyta SIEMINSKA1; Martin A. ERLANDSON1*; Dwayne D. HEGEDUS1; John R. GRAY1

1Agriculture and Agrifood Canada/ University of Saskatchewan, Saskatoon, Saskatchewan, CANADA

Mamestra configurata (bertha armyworm) is a major insect pest in Western Canada, but despite its economic importance, little is known about its genetic diversity across its geographical range. To characterize the genetics and biology of the M. configurata pheromone communication channel, we established colonies from three distinct geographic locations in Western Canada in addition to a pre-existing colony. Total RNA samples from head and antennae for each M. configurata strain were sequenced using pair-end Illumina and the resulting data subjected to de novo transcriptome assembly and bioinformatics analysis. The reference transcriptome was used to identify and characterize 80 proteins involved in the M. configurata pheromone communication, including 15 olfactory receptors and 3 pheromone binding proteins. Furthermore, individual libraries from the four M. configurata colonies were compared to the reference transcriptome to assess genetic differences among genes involved in pheromone communication. Real-time droplet PCR confirmed the theoretical expression profiles and determined sex-biased expression of relevant genes. Detailed chemical analysis of female pheromone gland extracts performed with gas chromatography-coupled mass spectroscopy was used to investigate the chemical composition of the female pheromone gland and differences between M. configurata strains. Wind tunnel and gas chromatography-electroantennographic detection assays indicate possible differences in response of males to; a) commercially-available pheromone blends, b) strain-specific pheromone gland extracts, and c) the effect of the presence of host plant volatiles on male response to the female pheromone gland extracts. This project combines the power of next generation sequencing with established physiological and behavioural bioassays to further our understanding of the lepidopteran pheromone communication channel. The knowledge gained during the course of this research will contribute to development of a more responsive and accurate pheromone trap monitoring system.

115

CHEMICAL ECOLOGY OF THE BEARDED PALM WEEVIL RHINOSTOMUS BARBIROSTRIS (COLEOPTERA: CURCULIONIDAE).

Analú C. REIS1; Palmira L. S. NETA1; Jéssica P. JORDÃO1; Luana N. SANTOS1; Ivon P. LÔBO1; José Inácio L. MOURA1; Paulo H. G. ZARBIN2; Carla F. FÁVARO3*

1State University of Santa Cruz, DCET, Ilhéus, BA, BRAZIL 2Federal University of Paraná, Depart. of Chemistry, Curitiba, PR, BRAZIL 3State University of Santa Cruz, DCET, Ilhéus, BA, BRAZIL

The beetle Rhinostomus barbirostris Fabricius 1775 (Coleoptera: Curculionidae) is a pest that attacks coconut trees, oil palm and other palms. The larval stage form galleries in the stem which reduce or interrupt the sap flow, causing breakage of the leaves and weakening of the plant. The use of pesticides to control R. barbirostris is ineffective by failing to achieve the insects. So far, the only method of control to this weevil is visual inspection and removal the highly infected plant. The need for effective and environmentally safe control methods of R. barbirostris lead the semiochemicals to be a very promising technique. Aiming the identification of the attractiveness compounds produced by R. barbirostris, the mating behavior was observed and the presence of an aggregation and a contact pheromone was suggested. To confirm the production of the pheromones, the volatiles released by males and females adults were collected by aeration and extracts from insects cuticle were made. The extracts were analyzed by GC- FID and GC-MS, and olfactometer bioassays are underway to investigate the bioactivity of the natural compounds.

116

CONTACT CUES DETERMINE OVIPOSITION OF THE SPECIALIST WILLOW SAWFLY NEMATUS OLIGOSPILUS.

Celina Laura BRACCINI1; Maria Victoria COLL ARAOZ2; Romina BARROSO3; ZAVALA Jorge Alberto3; FERNANDEZ, Patricia Carina4*

1INTA, Hurlingham, Buenos Aires, Argentina 2PROIMI- CONICET, San Miguel de Tucuman, Tucuman, Argentina 3FCEN-CONICET, CABA, CABA, Argentina 4INTA - CONICET, Campana, Buenos Aires, Argentina

Nematus oligospilus is a highly specialized sawfly feeding on selected willows (Salix spp). In the southern hemisphere, females are parthenogenetic. Adults do not seek for food, conspecifics or mating partners. As soon as they hatch, females look for oviposition sites. This specialization for egg laying suggests a sensory system highly tuned for recognition of suitable oviposition sites. Here we studied the oviposition behavior of N. oligospilus on two willow genotypes, a preferred (S. nigra) and a non-preferred one (S viminalis). First, we performed an ethogram during the recognition of the leaf surface before oviposition on both S nigra and S viminalis genotypes. Second, in order to estimate the importance of leaf cuticular waxes for species recognition, we offered willow leaves as oviposition sites with their cuticular waxes intact or removed by means of gum Arabic treatment. In addition, we analyzed the polar and non-polar components of the cuticular waxes by chromatography following dichloromethane and methanol extractions of the leaves. Finally, we performed a morphological study by means of SEM of the antenna and ovipositor of sawflies, in order to look for chemosensory structures. Sawflies spent ca. 40 % of their time on the leaf surface of the preferred genotype while antennating, fanning, tapping with the abdomen end or ovipositing. Conversely, sawflies spent only 20% of their time on the less preferred S. viminalis leaves, where they remained mainly quiet or antennating. According to chemical cues, both polar and non- polar extracts showed qualitative differences among willow genotypes, suggesting that oviposition cues can be polar, non polar or either kind. Putative chemoreceptor sensilla were found in both, the cerci of the ovipositor and the antenna. Our results suggest the presence of contact cues on the leaf surface of preferred genotypes, which seem to increase locomotor activity and trigger egg laying in sawflies.

117

CHANGES IN PHEROMONE EMISSION BY A SEED-FEEDING HEMIPTERAN CORRESPOND TO HOST PLANT QUALITY.

Mariana A. STANTON1; Jens PREβLER2; Christian PAETZ3; Wilhelm BOLAND3; Aleš SVATOŠ3; Ian T. BALDWIN3*

1Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, São Paulo, São Paulo, Brazil 2Department of Bioorganic Chemistry. Max Planck Institute for Chemical Ecology, Jena, Thuringia, Germany 3Research group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology, Jena, Thüringen, Germany

As consumers of plant tissues with transient apparency, florivores and seed-feeders must frequently migrate between host plants to synchronize colonization with plant phenology. Furthermore, the defensive chemistry and persistence of plant tissues determines their suitability and apparency - the likelihood of being discovered - to insect herbivores. Here we show how a hemipteran seed-predator, Corimelaena extensa (Hemiptera: Thyreocoridae), which forms feeding aggregations on the annual Nicotiana attenuata (Solanaceae) and also on the closely related perennial N. obtusifolia. We identified a novel pheromone released by C. extensa males – (5Z,8Z)-tetradeca-5,8-dienal – and performed field assays with the synthetic pheromone, showing that it stimulates formation of feeding aggregations on the post-fire annual N.attenuata. C. extensa pheromone emission was 40-fold higher when feeding on N. attenuata compared to the perennial N. obtusifolia, as was adult fecundity and seed capsule content of the putative biosynthetic precursor, linoleic acid. Additional field studies using transgenic plants, showed that C. extensa is unaffected by N. attenuata jasmonate-induced defenses, and avoids nicotine, to which it is demonstrably sensitive. We conclude that higher pheromone emission increases the apparency and colonization of the ephemeral, high- quality host, N. attenuata and that C. extensa avoids plant defenses by stealthy feeding. This plant-specific variation in insect signaling could facilitate host-finding by generalist seed-feeders migrating between plant patches as is the case of many insects that attack seed crops.

118

CLIMATE CHANGE: ATMOSPHERIC CO2 CONCENTRATION AFFECT APHID ALARM SIGNALING.

Antoine BOULLIS1*; Frédéric FRANCIS1; Georges LOGNAY1; Stéphanie HEUSKIN1; François J. VERHEGGEN1

1Evolutionary and functional entomology / Gembloux Agro-bio Tech / University of Liège, Gembloux, Province of Namur, Belgium

Temperature and atmospheric greenhouse gas concentrations are rising and should not stabilize in the foreseeable future. Assessment of these changes on species interactions is one ecological challenge. Like many insect species, aphids communicate with each other thanks to semiochemicals, and because of their economical importance in agriculture, the understanding of their adaptation to a changing climate remains a major issue. Here, we test the hypothesis that changes in atmospheric carbon dioxide (CO2) concentration affect alarm signaling in aphids, and specifically (E)-β-farnesene (Eβf - main compound of alarm pheromone) (1) production, (2) emission, (3) perception and (4) induced behavioral response. Indeed, it is important to consider the effect of elevated CO2 concentration on all the steps that a semiochemical has to pass by, from its biosynthesis and release by the emitter individual, until its perception and behavioral response expressed by the receiver. Using pea aphids (Acyrthosiphon pisum) reared under ambient or elevated CO2 concentrations (respectively a[CO2] and e[CO2]), we first quantified the whole body content of Eβf, and found no significant differences. Secondly, using dynamic headspace volatile collection, we quantified the amount of Eβf released by pea aphids under predation. Pea aphids reared under e[CO2] were found to release lower amount of Eβf. Third, we found no significant differences in terms of neuronal responses to Eβf when using electroantennography. Finally, the escape behavior expressed by an aphid colony when exposed to natural doses of Eβf greatly differed between aphid colonies reared under ambient or elevated [CO2].We conclude that the chemical communication of aphids reared under CO2 concentrations that mimic those predicted for the coming century will be affected. The potential impact of [CO2] and other abiotic parameters associated with climate change should be evaluated on each trophic level.

119

COULD PROSTERNAL INDENTATIONS BE ASSOCIATED WITH PRODUCTION OF VOLATILE PHEROMONES IN THE CERAMBYCID BEETLES AGLAOSCHEMA CONCOLOR AND ORTHOSTOMA ABDOMINALE?.

Weliton SILVA1*; Jocelyn MILLAR2; Lawrence HANKS3; José Maurício BENTO4

1University of São Paulo, Piracicaba, São Paulo, Brazil 2University of California, Riverside, California, USA 3University of Illinois at Urbana-Champaign, Urbana, Illinois, Brazil 4University of São Paulo, Piracicaba, São Paulo, Brazil

Previous research has found a strong correlation between the presence of sex-specific prothoracic gland pores and the production of aggregation sex pheromones by male beetles of species in the family Cerambycidae. Here we report a remarkable sexual dimorphism in the indentations on the prosterna of Aglaoschema concolor (Gounelle) and Orthostoma abdominale (Gyllenhal), South American cerambycids in the subfamily Cerambycinae, tribe Compsocerini. Prosternal indentations were deeper in males than in females. We hypothesized that these indentations could house pores involved in production and release of pheromones by males of these species. The GC-MS and GC- EAD analyses of extracts of the headspace volatiles from male A. concolor and O. abdominale revealed the presence of sex specific compounds that stimulated responses from the antennae of both sexes in electroantennogram assays. Moreover, the indentations on the prosternum of male A. concolor and O. abdominale are similar in structure to those reported from males of other cerambycine species that are known to produce aggegation sex pheromones. It therefore seems likely that these indentations have pores with the same function in males of A. concolor and O. abdominale.

120

CROTALARIA SPECTABILIS SEEDS EXTRACT AND ASSOCIATED ADJUVANTS: NEMATICIDE EFFECT ON THE PRATYLENCHUS BRACHYURUS JUVENILES.

Tamires SCUPINARI1; José P. da GRAÇA1; Ivani de O. N. LOPES1; Waldir P. DIAS1; Clara B. HOFFMANN-CAMPO1; Estela O. NUNES1*

1Universidade Estadual de Londrina - UEL, Londrina, Paraná, Brasil

Crotalaria spectabilis is used in the crop rotation system due to its antagonist effect on entomopathogenic nematodes. Alternative approaches, like adoption of cultivars with moderate to high nematode resistance and chemical treatments, also face challenges of their own. Therefore, the diversification of nematode control strategies is imperative in a competitive agricultural system. This work studies the nematicide effect of C. spectabilis seed extract against infective juveniles (J2) from P. brachyurus, in association with the adjuvants Tween 20 (1.5%) and Dimethylsulfoxide [DMSO (0.05%)]. The experiment consisted in exposing J2 samples to ethanolic extract of C. spectabilis seeds at five different concentrations, in association or not with the adjuvants and three controls: distilled water and distilled water plus adjuvants. The samples were evaluated after two moments: 24h-exposure and 24h-recovery, as described below. Initially, the samples were incubated during 24h, at °C and darkness. After the quantification of immobile individuals in Peter’s Chamber, the samples were filtered and kept in distilled water, under same incubation conditions for the 24h-recovery test. The immobility rates after the 24h-exposure were 100% on adjuvant-free extracts, minimum of 90% on extracts plus adjuvants, against an average of 10% in the controls. After 24h-recovery, these rates dropped to a maximum of 53% (adjuvant-free extracts), 35% (extracts plus DMSO) and 9% (average of Blank and DMSO controls). However, J2 samples from Tween 20 control (extract-free) presented mortality rate of 91%, indicating toxicity at tested concentration; so further studies establishing the lethal dose to P. brachyurus should be performed. Extract plus DMSO treatment showed average reduction of 7.6% in mortality when compared to adjuvant-free extract; probably due to permeation effect (stabilizing cuticle components and activating the immune system). Our results suggest that the 24h- exposure criteria is only useful to evaluate nematostatic action, but is not be reliable to determine nematicide effect.

121

EPIDERMAL GLANDS IN THE FOOD ROLLING BEETLE, DELTOCHILUM FURCATUM (COLEOPTERA: SCARABAEINAE).

Manuel IX-BALAM1*; Karen SALAZAR-NIÑO1; José SERRÃO1; Julio LOUZADA2; Jeremy N. McNEIL3; Eraldo LIMA3

1UNIVERSIDADE FEDERAL DE VIÇOSA, Viçosa, Minas Gerais, Brazil 2Universidade Federal de Lavras, Lavras, Minas Gerais, Brazil 3UNIVERSITY OF WESTERN ONTARIO, LONDON, ONTARIO, CANADA

Coprophagous beetles use dung and/or carrion but these nutrient rich resources are ephemeral both in time and space. Consequently this results in intense intra and interspecific competition, with Dipterans being the most important interspecific competitors for food. Laboratory experiments demonstrated that the blow-fly Lucilia cuprina (Diptera: Calliphoridae) laid significantly fewer eggs on food balls rolled by Deltochilum furcatum (Coleoptera: Scarabaeinae) than on those not rolled by beetles. During ball rolling the thorax, abdomen and pygidium come into frequent contact with the food source. As these regions are known to possess many exocrine integumentary glands it is possible that they secrete chemicals that affect fly oviposition behaviour. Scanning electron microscopy analysis of the thorax, abdomen and pygidium of both D. furcatum females and males found numerous circular pores on the body suface, below which there were unicellular glands consisting of a secretory cell and a duct. The latter had three distinct regions: a basal receptor canal, an apical receptor canal and a conducting canal. These content of these glands will be determined and assayed to determine which ones, if any, produce allomones that deter oviposition by L. cuprina.

122

EFFECT OF LEAF DAMAGE ON FLORAL HEADSPACE AND VISITATION RATES OF A TROPICAL SHRUB.

Luiz H G REZENDE1*; Gustavo Q. ROMERO1; Martín PAREJA1

1Universidade Estadual de Campinas – UNICAMP, Campinas, São Paulo, BRAZIL

Leaf damage can affect floral attractiveness through changes in floral volatile organic compounds (VOCs) or through a modified emission of leaf VOCs that change the background upon which the floral display is presented. Loss of leaf area can also stimulate the production of defensive compounds whose biosynthesis compromises the synthesis of attractive compounds such as those that make up the floral scent. These changes in scent environment can attract or repel pollinators sensitive to alterations in the volatile profile. We test whether leaf damage affects floral visitation rates and VOC profiles of Trichogoniopsis adenantha (DC) (Asteraceae), a native evergreen shrub in Southeastern Brazil. This is a chemically conspicuous plant, since it has glandular trichomes all over the leaves and branches. Damage can easily break these trichomes, releasing VOCs that can change the chemical background upon which inflorescences are presented. In the field we caused different levels of mechanical leaf damage on reproductive branches and assessed whether visitation to the inflorescences was altered immediately and 24 hours later. In order to assess changes in the scent around reproductive branches, damage treatments were applied to greenhouse-grown plants and leaf and floral VOCs were collected using both dynamic headspace sampling and solid phase microextraction. The experiment is currently underway and the most common floral visitors in all treatments so far were Diptera and Lepidoptera. We have tentatively identified 27 VOCs, all monoterpenes and sesquiterpenes, though we have not yet detected any specific floral VOCs. The community of floral visitors can respond differently to such compounds, enhancing or compromising their interaction with the plant. Studies on plant signalling in natural tropical environments are rare, but essential for understanding the ecology of VOCs in highly biodiverse ecosystems.

123

EFFECT OF SINGLE AND MULTIPLE HERBIVORY IN ATTRACTION OF THE GENERALIST PREDATOR ORIUS INSIDIOSUS (SAY, 1832) TO ROSE PLANTS.

Ana Luiza V. SOUSA1*; Maria Fernanda G. V. PEÑAFLOR1; Guilherme G. SILVA1; Brígida SOUZA1

1Federal University of Lavras, Lavras, Minas Gerais, BRAZIL

Previous studies have shown that rose plants infested with a single herbivore species emit a volatile blend that attracts predatory insects. However, these plants are often attacked by more than one arthropod species under natural conditions, leading to changes on plant volatile composition compared to single herbivore attack, which can consequently alter predator’s behavior. In this context, this study investigated the olfactory response of the minute pirate bug, Orius insidiosus (Say, 1832), to volatiles emitted by roses under single or double infestation by the spider mite Tetranychus urticae Koch, 1836 and the thrips Frankliniella insularis (Franklin, 1908). Minute pirate bug females were individually assessed in a glass Y-tube olfactometer for a maximum of five minutes. The predator O. insidiosus did not discriminate between non-infested rose odors and clean air, but preferred volatiles emitted by singly-infested plants (T. urticae or F. insularis) or doubly- infested (T. urticae + F. insularis) over non-infested plants. When these attractive treatments were compared among them, the predator did not discriminate between volatiles emitted by T. urticae and F. insularis-infested roses as well as singly-infested (T. urticae or F. insularis) against doubly-infested plants. Therefore, our results show that O. insidiosus is equally attracted to volatiles emitted by singly-infested roses regardless of the prey species, and that double infestation with mites and thrips in roses exert a neutral effect on predator attractiveness relative to singly-infested plants. As O. insidiosus is a generalist predator, the observed behavior can be an adaptive strategy to efficiently locate their prey. Next steps of this study consist of: (i) chemical characterization of rose volatile profile to examine whether infestation with the different species, as well as double infestation, modifies plant induced response; and (ii) evaluation of quality of both preys to the predator.

124

EVOLUTION OF PHEROMONE RECEPTORS IN A PRIMITIVE MOTH, ERIOCRANIA SEMIPURPURELLA (ERIOCRANIIDAE: LEPIDOPTERA).

Jothi Kumar YUVARAJ1; Martin N. ANDERSSON1; Jacob CORCORAN1; Dan-Dan ZHANG1; Olle ANDERBRANT1; Christer LÖFSTEDT1*

1Dept. Biology, Lund University, Lund, Scania, Sweden

Female moths produce a species-specific sex pheromone to attract males for mating. Advanced moths have specialized pheromone receptors (PRs), a subset of odorant receptors (ORs), to detect the sex pheromone components. Although it is known that pheromone detection evolves with respect to variations in pheromone production, it is still not known at which evolutionary point the moths evolved their specific PRs. Pheromone compounds of the primitive moth Eriocrania semipurpurella (Eriocraniidae: Lepidoptera) are similar to general plant volatile compounds. Therefore, we hypothesized that their PRs might have evolved from ORs for general plant volatiles. We sequenced RNA samples from the antennae of E. semipurpurella and found a number of PR candidates, based on their expression level, alignment with other moth PRs and the presence of the PR motif that is commonly found in moth PRs. We have also identified the odorant receptor co-receptor (ORCO), which is essential for the function of ORs. The identified ORCO and PR candidates were cloned and transfected into HEK293 cells. HEK cells stably expressing the ORCO and PRs were functionally tested for response to known pheromones of E. semipurpurella and plant volatiles. Among five PR candidates, EsemOR3 and EsemOR5 responded to the pheromone compounds. In addition, EsemOR3 and EsemOR5 also showed responses to some of the plant volatiles, however these responses were not as strong as to pheromones. We speculate that during birth- death evolution the plant volatile-detecting ORs obtained the ability to detect the pheromones but still retained their ability to detect plant volatiles. At the same time, phylogenetic analyses showed that the non-functional PR candidates root the PR clade and the functionally active PRs are outside the PR clade. Thus, the phylogenetic analyses and the functional studies suggest that PRs may have evolved from ORs that are tuned to structurally similar plant volatiles.

125

FRIENDS AND FOES ATTRACTED BY THE CONSPICUOUS CHEMICAL FLORAL ADVERTISEMENT OF THE BOCAIUVA PALM (ACROCOMIA TOTAI; ARECACEAE).

Artur Campos Dália MAIA1*; Letícia Koutchin dos REIS2; Eduardo Gomes GONÇALVES2; Geanne Karla Novais SANTOS3; Luis Alberto Núñez AVELLANEDA4; Flavio ARISTONE5; Daniela Maria do Amaral Ferraz NAVARRO6

1Universidade Federal de Pernambuco, Recife, Pernambuco, BRAZIL 2Universidade Católica Dom Bosco, Campo Grande, do Sul, BRAZIL 3Universidade Federal de Pernambuco, Recife, Pernambuco, BRAZIL 4Universidad de La Salle, Bogotá, ---, COLOMBIA 5Universidade Federal do Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, BRAZIL 6Universidade Federal de Pernambuco, Recife, Pernambuco, BRAZIL

The bocaiuva palm (Acrocomia totai; Arecaceae) is a ubiquitous element in the vast pasture lands that dominate the landscape in the transition between the Cerrado and Pantanal ecoregions of Central Brazil. Remarkably high fruit and seed sets observed in populations of this species are testimony to an unwavering association with specialized pollinators (Andranthobius sp., Curculionidae), driven to night-blooming thermogenic inflorescences by intense floral scent emissions. Curiously enough, the very same volatile organic compounds (VOCs) that ensure the reproductive success of the bocaiuva palm might be involved in the attraction of florivorous cyclocephaline scarabs (Cyclocephala spp.; Melolonthidae), which often appear in large numbers and severely diminish fruit yield. We analyzed inflorescence headspace samples of A. totai (n = 15) and identified a methoxylated benzenoid (4-methylanisole) and two methoxylated pyrazines (2-methoxy- 3-isopropylpyrazine and 2-methoxy-3-sec-butylpyrazine) as their major constituents. Field-trapping bioassays evidenced that these three VOCs were attractive to Andranthobius sp. (up to > 300 captures per trap), either individually or in mixed blends. 4-methylanisole, either pure or in mixed blends with the pyrazines, was additionally attractive to both male and female Cyclocephala forsteri (up to >10 captures per trap). Effective pollinators and injurious florivores associated to the bocaiuva palm evolved towards the recognition of similar sets of chemical cues to locate an abundant source of nutritious resources.

126

GENISTEIN (4’, 5, 7-TRIHYDROXYISOFLAVONE): A CONSTITUTIVE NON INDUCIBLE SOYBEAN SEEDS RESPONSE AT HIGH SOWING DENSITY AND CONSTITUTIVE INDUCIBLE AT LOW SOWING DENSITY.

Lucía G. BARRIGA1*; Hugo D. CHLUDIL1; Jorge A. ZAVALA1

1Facultad de Agronomía, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina

Soybean (Glycine max L.) is one of the most important crops in Argentina and its cultivated area has continuously expanded among the past thirty years. Seed-sucking insects such as stinkbugs (Nezara viridula L., Piezodorus guildinii Westwood) negatively impact on soybean yield. Due to plants wide diversity of secondary metabolites reported as defensive compounds (antiherbivore response effect), understanding how these defenses increases in soybean seeds should help us reducing stinkbug attack. Genistein is one of the most relevant direct chemical responses of soybean. This secondary metabolite derivate from phenylalanine and is constitutively synthesize in soybean plants. The inducible response of soybean seeds after stinkbugs attack has been confirmed, but the effect of sowing density over this response have never been evaluated. The aim of this work was to study the effect of soybean plants intraspecific competition over the inducible concentration of seeds isoflavonoids, as a response to Piezodorus guildinii Westwood attack. Don Mario 4670 cultivar was sowed at three densities: 20, 40, and 60 cm between plants. After 24 and 72 hours of P. guildinii Westwood herbivore, attacked and non attack (control) pods were harvested and conserved under -80 °C. Isoflavonoid identity and quantification was studied trough a methanol extraction followed by HPLC- UV analysis. Our results showed high constitutive levels of genistein under high sowing density and a non inducible response after stinkbug attack. In contrast, low constitutive levels of genistein were found under low sowing density (high availability of resources), with a strong induction after stinkbug attack.

127

HEAD LICE-HOST INTERACTION: HUMAN SCALP EXTRACTS MODIFY THE BEHAVIOUR OF PEDICULUS HUMANUS CAPITIS.

Paola GONZALEZ AUDINO1*; Federico GALASSI1; Isabel ORTEGA INSAURRALDE1; Ariel TOLOZA1; Maria PICOLLO2

1(CIPEIN) UNIDEF - CITEDEF - CONICET, VILLA MARTELLI, BUENOS AIRES, ARGENTINA 2(CIPEIN) UNIDEF - CITEDEF - CONICET, BUENOS AIRES, BUENOS AIRES, ARGENTINA

P. h. capitis is a cosmopolitan ectoparasite that affects the head of their human hosts. It is present in children of school age, in both developed and developing countries. It is been already stablished that P.h. capitis prefer to infest some individulas above others. However, no studies have gained insight into the reasons behind the existence of susceptible and non-susceptible individuals. The main mechanism by which louse transmision occurs is the direct host to host contact.Behavioural assays were performed in order to study environmental factors that affect locomotor activity and to evaluate if human scalp extracts modify the behaviour of head lice. Extracts were collected by rubbing filter paper onto scalp surface. Data were processed with video tracking system EthoVision XT 10.1. VOCs present in the human scalp extracts were collected and analysed by Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry (SPME-CG-MS).In a circular arena, headlice increase their locomotor activiy at 30°C and dark conditions and are arrested by the extracts collected from human scalp.In olfactometer, head lice were significantly attracted to volatiles from the human source. The chemical composition of the extracts showed the presence of hydrocarbons, alcohols, diols, saturated and unsaturated ketones, aldehydes and fatty and short chain acids. The role of these compounds alone and in mixtures is currently under study. These results represent the basis for the study of chemical cues involved in human susceptibility to head lice.Ortega Insaurralde et al., J. Med. Entomol. 2015.

128

HOST LOCATION CUES OF SPINTHERUS DUBIUS (NEES) (HYMENOPTERA: PTEROMALIDAE), THE CLOVER WEEVIL PARASITOID.

Nicoletta FARAONE1*; Olle ANDERBRANT1; Glenn SVENSSON1; Maj RUNDLÖF1; Mattias LARSSON2; Göran BIRGERSSON2; Åsa LANKINEN2

1Department of Biology, Lund University, Lund, N/A, SWEDEN 2Department of Plant Protection, Swedish University of Agricultural Sciences, Alnarp, N/A, SWEDEN

Insect herbivores and their natural enemies use chemical cues for locating food and oviposition sites. In the case of parasitoids, chemical signals from the insect host or their combination with the plant itself may contribute to localizing the host1. The most abundant parasitoid in clover fields is Spintherus dubius, a small wasp responsible for the death of young weevil larvae belonging to the genus Apion2. The chemical cues involved in this process which allow S. dubius to locate their host are still unknown and we aim to clarify the role of the volatiles that mediate the host recognition. From preliminary behavioural studies carried out using a Y-tube olfactometer, female parasitoids responded positively to the volatiles released from the feces of adult A. trifolii fed on clover, Trifolium pratense, which seemed to be attractive versus clean/uninfested clover flowers and weevil larvae. Female parasitoids may use volatiles released from the feces as cues for locating their hosts. It is well documented that natural enemies can be attracted to chemical cues emitted from the larva of their insect host or from the host plant the larva feeds on3. However, to our knowledge, attraction by a parasitoid to volatiles from feces of the adult host has not been documented before. The next step is to identify the volatiles in feces from adult weevils, and check the parasitoid’s behavioural response to these compounds. These findings open a new scenario of multitrophic interactions and they need to be deepen for elucidating the chemical nature of compounds involved in host recognition.

References: 1Wäschke N, Hardge K, Hancock C, Hilker M, Obermaier E, and T Meiners (2014) PLosOne: e85152. doi:10.1371/journal.pone.0085152

2Lundin (2013) Doctoral thesis. Swedish University of Agricultural Sciences, Uppsala. Acta Universitatis agriculturae Sueciae 20013:39

3Cournoyer M and G Boivin (2004) Entomol. Exp. et Appl. 112: 117-124

129

INJURIES OF HELICOVERPA ARMIGERA AND ITS IMPACT ON ISOFLAVONES CONTENT IN SOYBEAN GENOTYPES.

Barbara F. DIAS1; Paulo A.S. GHIZONI1; Jefferson TAKEO1; Patrícia BRAGA2*; Tamires SCUPINARI3; Diego GAZOLA3; Tatiane E. UEDA3; José P. GRAÇA3; Maria C. N. OLIVEIRA3; Estela O. NUNES3; Clara B. HOFFMANN-CAMPO3

1Universidade Norte do Paraná, Londrina, Paraná, Brazil 2Universidade Tecnológica Federal do Paraná, Dois Vizinhos, Paraná, Brazil 3UNIVERSIDADE ESTADUAL DE LONDRINA - UEL, Londrina, Paraná, Brazil

Isoflavones are secondary metabolites largely presented by leguminous plants and in general can affect feeding behaviour of insects. The production of these compounds by host plants usually increases after pest injuries. This study aimed to quantify isoflavones in different soybean genotypes with or without Helicoverpa armigera injuries. Six selected soybean genotypes (Anta, Apollo, BRS 257, BRS 391, and TMG 132) were compared to PI 227687 in the experiments. At V3 stage, soybean leaves were offered to H. armigera (Hübner, 1808) caterpillars (third to fourth instars) for a 24 h trial. A methanolic extract was prepared using injured and non-injured leaves, collected at V3 stage. The obtained extracts were submitted to chromatographic analysis by using HPLC- PDA equipment to identify and quantify secondary compounds produced by soybean leaves. Daidzin, malonyl daidzin, genistin, malonyl genistin, malonyl glycitin (isoflavones glycosides), daidzein, genistein, glycitein (aglycones), and rutin (flavonol glycoside) were detected in the chromatograms. Isoflavone quantitative differences were observed among genotypes. Considering cultivar average, regardless damage, Anta and Apollo exhibited higher amounts of glycosides daidzin, malonyl daidzin and the aglycones daidzein and genistein. BRS 257 and PI 227687 genotypes showed lower levels of these isoflavones. Apollo and TMG 132 showed larger amount of genistein, either in plants with and without injuries caused by H. armigera. Regarding responses of genotypes to injuries, non-injured TMG 132 showed a significantly lower amount of genistein when compared to injured plants. On the other hand, genistein increase due to H. armigera herbivory was not observed on PI 227687, which is reportedly one of the most resistant genotypes to leaf-feeder insects. Consequently, further feeding experiments must be carry out to correlate metabolite induction, timing and severity of injury.

130

IDENTIFICATION OF 2,5-DIKETOPIPERAZINES IN XYLELLA FASTIDIOSA AND CULTURE MEDIUM PW.

Michelli SILVA1*; Moacir ANDRADE2; Alessandra SOUZA3; Norberto LOPES4; Maria Fátima SILVA4

1Universidade Federal de São Carlos - UFSCAR, São Carlos, São Paulo, BRASIL 2Universidade Federal de Sergipe, Itabaiana, Sergipe, BRAZIL 3Instituto Agronômico de Campinas, Cordeirópolis, São Paulo, BRAZIL 4School of Pharmaceutical Sciences of Ribeirão Preto – University of São Paulo (FCFRP-USP), Ribeirão Preto, São Paulo, BRASIL

Xylella fastidiosa, bacteria Gram-negative, xylem-limited bacterium is known pathogen of a number of economically important plants, including citrus, grapevine and coffee. In Brazil, X. fastidiosa causes citrus variegated chlorosis (CVC) disease, leads to losses of millions of dollars to the citrus industry. X. fastidiosa produces 2,5-diketopiperazines (DKPs) described in the literature as substances involved in the cell-to-cell communication process in many microorganisms, becoming interesting targets for the control of this phytopathogen. However, the origin of diketopiperazines has been questioned, because many cyclic dipeptides has been found in fermentation broths and yeast cultures. 1 Thereby, this work has as aim to compare the chromatographic profile of the bacterial and culture medium PW extracts and accomplish the relative quantification of DKPs cyclo (Pro-Tyr), cyclo (Pro-Leu/Ile) and cyclo (Pro-Phe) by LC-MS/MS using the tecnic Multiple Reaction Monitoring (MRM). With the results from the quantification, elevated levels of the three DKPs was shown in the bacterial extract of X. fastidiosa regarding medium PW extract, wich indicates that the DKPs were produced by X. fastidiosa, or could be the result of some bilateral reaction of bacteria metabolism. In the extract obtained of bacterial cells of the biofilm was confirmed the presence of cyclo (Phe-Tyr), cyclo (Pro-Phe) and cyclo (Pro-Leu). Lastly, extracts obtained of several ingredients of the culture mediums submitted and no to sterilization process were analysed by UPLC-MS and GC-MS. From this analyses, were identified in the extracts of phytone peptone, trypticase peptone (ingredients for the PW) submitted to sterilization: cyclo (Pro-Val), cyclo (Pro-Leu/Ile), cyclo (Pro-Phe) and cyclo (Pro-Tyr), besides the cyclo (Pro-Ser) in phytone peptone. On the other case weren’t identified any DKP. This data indicates that the temperature has an important part in the DKPs formation from the culture mediums. 1- PRASAD, C. Peptides, 1995, 16, 151.

131

INTERACTION DIVERSITY AMONG PIPERACEAE SPECIES AND THEIR ASSOCIATED FAUNA.

Massuo J. KATO1*; Lydia F. YAMAGUCHI1; Marcilio M. MORAES1; Mariana A. STANTON1; Patricia A. MACHADO2; André R. NASCIMENTO2; Simeão S. MORAES2; André V. L. FREITAS2

1Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, São Paulo, São Paulo, Brazil 2University of Campinas, Campinas, SP, Brasil

The high species diversity of some families of the magnoliid clade is intriguing. While some families such as Myristicaceae (e.g. nutmeg – Myristica fragrans) contain nearly 440 species (20 genera), Piperaceae (e.g. – Piper nigrum) has about 3600 species (5 genera), with 2000 and 1600 Piper and Peperomia, respectively. The diversity of plant-insect interactions observed to Piper species led us to select these species as model plants to study tropical ecology and to test the hypothesis of secondary chemistry as possible factor involved in relationship specificity. Therefore, major aspects under study included the study of the components of multi-trophic interaction of plant- herbivores-parasitoids in South America by means of morphology and molecular taxonomy. The communities of Lepidoptera included the specialist Geometridae (e.g. Eois olivacea, E. tegularia and E. loprosa) eating preferentially on P. malacophyllum, P. gaudichaudianum, P. amplum and P. crassinervium and Hesperiidae (Quadrus cerealis and Q. u-lucida) are specific to various species including P. regnelli. Additional species included the polyphagous Nymphalidae (Memphis sp) and few weevils (Piridinetus zinckenii and Naupactus bipes – Coleoptera). Microgastrinae species comprise the majority of parasitoids associated with Eois, while three species of Diptera dominated the interactions with Quadrus. The fate of major secondary compounds during digestion of leaves by herbivores suggests various metabolic adaptation to cope with the large array of secondary compounds in Piper species.

PRP-USP, FAPESP, CAPES, CNPq.

132

INVESTIGATING THE PHEROMONE COMPOSITION OF CYDIA ARAUCARIAE (LEPIDOPTERA, TORTRICIDAE): A SEED BORER OF THE ENDANGERED TREE SPECIES ARAUCARIA ANGUSTIFOLIA.

Camila B. C. MARTINS1; Marcílio J. THOMAZINI2; Paulo H. G. ZARBIN2*

1Universidade Federal do Paraná, Cutitiba, Paraná, BRASIL 2Embrapa Florestas, Colombo, Paraná, Brazil

Every year, thousands of Araucaria trees seeds are used by Cydia araucariae larvae as food source impeding their development into new trees. Thus, these seeds are sold in markets in the south of Brazil (April-July) for human consumption. Diminishing the seed attack by these insects is the first step into an improved seed production collaborating with reforestation programs and expanding its commercialization. Because there are no efficient methods to deter the seeds’ attack, producers wait for the development of alternative methods to monitor and control this pest, such as with pheromone traps. Infested seeds were collected in Araucaria plantations or purchased by collectors, and maintained under laboratory controlled conditions (20 ± 2°C, 50 ± 10°C RH and 12L:12D). Pupae were sexed and individualized in small plastic containers until adults’ emergence. Couples were rapidly observed to determine the period of sexual activity. Virgin female pheromone glands were extracted during the period of sexual activity with tweezers using hexane as solvent. Extract analyses are underway on GC, GC-MS and GC-EAD. Because pheromone components of Cydia spp. are shared by various species, some reference compounds, such as acetates and alcohols were also tested against males’ antenna in GC-EAD tests. Kovats indexes values (KI) were calculated for all antennal responses. Six consistent male antennal responses were observed against female extracts. Comparing their KI values with reference compounds’ KI values, five of them matched for E8,E10-12Ac, E8-12Ac, 12Ac, 14Ac and 16Ald. Thus, all of them were active on male’s antenna. We concluded that couples are sexually active on photophase. Female gland extracts were active on EAG tests against males’ antenna which were sensitive to five female extract compounds. Two of these compounds matched with acetates and an aldehyde already described as pheromone components of other Cydia species.

133

INVESTIGATING VOLATILE EMISSION PATTERNS TO ELUCIDATE THEIR ROLE IN ECOLOGICAL INTERACTIONS.

Andrea CLAVIJO MCCORMICK1*; G. Andreas BOECKLER2; Jonathan GERSHENZON2; Sybille B. UNSICKER2

1Massey University, Palmerston North, Manawatu, New Zealand 2Max Planck Institute for Chemical Ecology, Jena, Thuringia, Thuringia

Herbivore-induced plant volatiles attract natural enemies mediating indirect plant defense. The dynamics of plant volatile emission are strongly influenced by biotic and abiotic factors as shown by studies on cultivated annuals. However, less is known about volatile emission dynamics on woody perennial species. In this study we investigated the effect of factors such as time of herbivory, herbivore identity, developmental stage, feeding amount and photoperiod on the volatile emission of black poplar (Populus nigra) in order to make inferences about the ecological roles of individual compounds. We found that emission dynamics were similar for compounds within the same chemical class, but individual compounds reacted differently to biotic and abiotic factors. Some compounds were released immediately upon herbivore attack or shortly afterwards, irrespective of light conditions, and stop being emitted shortly after herbivore removal. These compounds, which include minor nitrogen-containing compounds, green leaf volatiles, and some aromatics could be reliable indicators of herbivore presence for natural enemies. The amount of plant biomass removed had a significant effect on the emission of the majority of compounds investigated, indicating that most volatiles are good cues for herbivore density irrespective of their chemical class. Only a few compounds differed between the induced volatile blends of different caterpillar species (Lymantria dispar and Laothoe populi) in the same developmental stage. Nevertheless, the developmental stage of the herbivore had a strong impact on volatile emission with early instar L. dispar inducing significantly more nitrogen-containing volatiles and terpenoids than late instars. The emission patterns of minor nitrogen-containing volatiles (aldoximes) suggest that they are reliable cues for natural enemies, providing information about herbivore presence, identity, developmental stage and density. This work demonstrates that understanding volatile emission patterns is useful to elucidate their roles in ecological interactions and may contribute to optimize pest management strategies based on plant semiochemicals.

134

J.H. FABRE (1823-1915) AND T. EISNER (1929-2011) - TWO EXCEPTIONAL CHARACTERS IN CHEMICAL ECOLOGY OF THE 19TH AND 20TH CENTURY.

Prof. Hans. E. HUMMEL1*; Simone S. LANGNER1

1Justus-Liebig-University Giessen, Giessen, Hesse, Germany

Through ergography, two centres of gravity may be identified which substantially shaped the subject of chemical ecology to this very day: one (Fabre) active during the transition to the 20th, the other (Eisner) to the 21st century. If, in the words of Victor Hugo, Fabre was the Homer of insects, Eisner by comparison would be their Voltaire or Goethe. Both scientists were "baroque men" gifted equally with an enormous productivity and with an immense love for arthropods, to whom they dedicated the majority of their lifetime. Two ergograms depict the immense richness of their experience: Fabre with an oeuvre of 64 titles (43 various science books, 10 normal research articles and 11 as his famous contiguous book series, "Souvenirs Entomologiques", packed with experimental observations on behaviour and instinct of arthropods. Eisner, in contrast, published some 580 research articles and 11 books, thousands of photographs, films, audio and video sequences, commentaries, and political statements. The wealth and originality of their field and laboratory observations is impressive beyond the ordinary. For Fabre, the LIVE insect and its behaviour was important, as it was for Eisner. Both were equally gifted communicating their research findings in lucid, appealing style. But there were major differences: The young Fabre was a high school teacher and never left his home country. The second period of his life he spent in seclusion. Eisner, in contrast, was constantly on the move and can be considered a world citizen. He travelled Europe, South and North America, and Australia, which allowed him worldwide comparison of arthropods and their ecology. Eisner received recognition as distinguished Cornell professor, taught dozens of graduate students and postdocs and stood always in the limelight. Fabre was recognized and honoured only very late in life when he, at age 91, was the 1914 Nobel laureate for literature.

135

LUCIFERIN SULFATE IS BIOSYNTHESIZED BY A SPECIALIZED LUCIFERIN SULFOTRANSFERASE IN FIREFLIES.

Tim FALLON1; Fu-Shuang LI1; Maria ALLENDE2; Jing-Ke Weng3*

1Whitehead Institute/MIT, Cambridge, MA, USA 2Williams College, Williamstown, MA, USA 3Whitehead Institute/MIT, Cambridge, MA, USA

Firefly luciferin is a specialized metabolite restricted to fireflies (family Lampyridae) and other select families of beetles (order Coleoptera). Firefly luciferin undergoes luciferase- catalyzed oxidation to produce light, thereby enabling the luminous mating signals essential for reproductive success in most bioluminescent beetles. Although firefly luciferin and luciferase have become widely used biotechnological tools, questions remain regarding the physiology and biochemistry of firefly bioluminescence. Here we report sulfoluciferin to be an in vivo derivative of firefly luciferin in fireflies, and report the cloning of luciferin sulfotransferase (LST) from the North American firefly Photinus pyralis. LST catalyzes the production of sulfoluciferin from firefly luciferin and the sulfo donor PAPS. Sulfoluciferin is present in several surveyed firefly genera as well as in the bioluminescent Elaterid beetle Pyrophorus luminosus at a trace level. We propose that sulfoluciferin could serve as a luciferin storage molecule in fireflies, and that LST may find use as a new tool to modulate existing biotechnological applications of the firefly bioluminescent system.

136

LUPANINE DETERMINES THE LEVEL OF ACCEPTABILITY AND PALATABILITY OF LUPINE LUPINUS SPP. VARIETIES TO THE PEA APHID ACYRTHOSIPHON PISUM HARRIS.

Beata GABRYŚ1*; Bozena KORDAN2; Katarzyna DANCEWICZ2; Anna WRÓBLEWSKA-KURDYK2

1University of Zielona Góra, Zielona Góra, Lubuskie, Poland 2University of Warmia and Mazury, Olsztyn, Warmia and Mazury, Poland

In Poland, the cultivated varieties of lupines Lupinus (Fabaceae: Genistae) belong to three indigenous species: Lupinus albus L., L. luteus L., and L. angustifolius L. The wild species, Lupinus polyphyllus Lindl., is an alien anthropophyte in the Polish flora, but it is well established and even considered an invasive species as it is all over Europe. The pea aphid Acyrthosiphon pisum Harris (Homoptera: Aphididae) is a well known and widely distributed oligophagous pest species of leguminous plants. Due to the rising economic importance of the sweet low-alkaloid varieties of lupines, A. pisum may become a threat to this crop. The aim of this work was to establish the level of acceptability of all species of lupines that occur in Poland to the pea aphid infestation, and to determine to what extent the variation in acceptability is related to their alkaloid profiles. Altogether, the alkaloid profile and the pea aphid probing behaviour were studied using five varieties of L. angustifolius, four varieties of L. luteus, two varieties of L. albus, and one wild species L. polyphyllus. The pea aphid probing behaviour was monitored using the electrical penetration graph (EPG) technique.Eighteen alkaloids were identified: one piperidine alkaloid, one indole, ten quinolizidine alkaloids, and six esters. All lupine varieties with high alkaloid content were rejected by the pea aphid. At the same time, all lupine varieties that contained lupanine, its derivatives and especially their esters appeared to be unacceptable to the pea aphid, independent of the total concentration of any specific lupanine alkaloid. On these varieties, aphid probes were usually epidermal. The phloem phase occurred rarely, and when it did, it was short and consisted mainly of watery salivation. In contrast, sparteine and its derivatives did not seem to affect aphid probing significantly.

137

METHOD FOR EXTRACTION OF VOLATILE ORGANIC COMPOUNDS IN NESTS OF THE BEE CENTRIS ANALIS.KEY WORDS: HS-SPME-GC-MS, NESTING, VOLATILE ORGANIC COMPOUNDS, CENTRIS ANALIS.

Paulo Herbesson Pereira de SOUSA1; Cládia Inês da SILVA2*; Breno Magalhães FREITAS3; Guilherme Julião ZOCOLO4; Tigressa Helena Soares RODRIGUES4

1Universidade Federal do Ceará, Fortaleza, Ceará, Brasil 2USP -University of São Paulo, Ribeirão Preto, São Paulo, Brazil 3Universidade Federal do Ceará, Fortaleza, ceará, Brasil 4Embrapa Agroindústria Tropical, FORTALEZA, CEARÁ, Brasil

The presence of solitary bees such as Centris analis in agricultural settings is facilitated by the introduction of inhabited nests, but there are limited studies on the factors that influence their choice for nesting sites. In this study we aimed to test three types of SPME fibers in developing a method to extract volatile organic compounds present in nest constituents such as food, brood and floral oils to assess their importance in attracting Centris analis females.Samples, a known mass of each nest constituent, were accommodated in glass vials with medal lid and added 3 g of NaCl and 8 mL of deionized water. The extraction and characterization was carried out using a Headspace - Solid Phase Microextration (HS-SPME) combined with Gas Chromatography - Mass Spectrometry, and the three types of SPME fibers: polydimethylsiloxane (PDMS), divinylbezene/ carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) and carboxen/ polydimethylsiloxane (CAR/PDMS). Each type of fiber was subjected to three periods of extraction (15, 30 and 45 min) and analyzed in a chromatography Agilent GC-MS equipped with a quadrupole detector (MSD 5977A), bearing a HP-5MS (30 m x 0.25 mm x 0.25 µm) column and Helium 1 mL.min-1 with gas carrier. The temperatures of the injector and detector were 240°C and 150°C, respectively. The furnace temperature was maintained at 40° C during four minutes and then increased to 80°C with an increment of 2.5°C min-1, then 5°C min-1 to 110°C and 10°C min-1 to 220°C while maintaining the final temperature for 13 min. In general, the CAR/PDMS was the one with the greatest amount of extracted compounds, followed by DVB/CAR/PDMS and PDMS to a lesser extent. Therefore, we concluded that all fibers produced good results and can be used to extract volatile organic compounds from bee nests.

138

METHOXYLATED PYRAZINES AND THE ATTRACTION OF FLORIVOROUS CYCLOCEPHALINE SCARABS (MELOLONTHIDAE, DYNASTINAE) ASSOCIATED TO SOUTH AMERICAN PALMS (ARECAEAE).

Artur Campos Dália MAIA1*; Geanne Karla Novais SANTOS1; Eduardo Gomes GONÇALVES2; Luis Alberto Nuñez AVELLANEDA3; Daniela Maria do Amaral Ferraz NAVARRO3

1Universidade Federal de Pernambuco, Recife, Pernambuco, BRAZIL 2Universidade Católica Dom Bosco, Campo Grande, Mato Grosso do Sul, BRAZIL 3Universidad de La Salle, Bogotá, ---, COLOMBIA

In tropical South America, several species of night-blooming palms (Arecaceae) visited by cyclocephaline scarabs (Melolonthidae, Dynastinae) exhibit unusual methoxylated pyrazines as major floral scent constituents. Assuming the likely involvement of these compounds in the olfatory-oriented behavior of the beetles, attractiveness field biotests were conducted in forest fragments in the municipalities of Hato Corozal (Colombia; April - May 2015; n = 4) and Igarassu (Brazil; April 2014; n = 2). Flight-interception traps individually baited with 100 μL of a) 2-Isopropyl-3-methoxypyrazine and b) 2-(sec- butyl)-3-methoxypyrazine yielded anywhere from 1.500 captures per installed unit in a 2h interval after dusk. Cyclocephala amazona and C. distincta, known florivores associated to Acrocomia, Astrocaryum, Attalea, Bactris, Cryosophila and Phytelephas, were the sole identified species in Hato Corozal and Igarassu, respectively. The exceptional selective attractiveness of methoxylated pyrazines make them an interesting propect as a tool for controlled management of florivorous cyclocephaline scarabs.

139

OLFACTORY-MEDIATED REPELLENCY IS OBSERVED IN BLATTELLA GERMANICA EXPOSED TO PYRETHROIDS.

Emiliano BONÉ1; Paola GONZÁLEZ AUDINO2; Valeria SFARA3*

1Intituto de Investigación e Ingeniería Ambiental Universidad Nacional de San Martín, San Martin, Buenos Aires, ARGENTINA 2Centro de Investigaciones de Plagas e Insecticidas (CIPEIN), UNIDEF-CITEDEF- CONICET, Villa Martelli, Buenos Aires, ARGENTINA 3Instituto de Investigación e Ingeniería Ambiental Universidad Nacional de San Martín, San Martín, Buenos Aires, ARGENTINA

Pyrethroids are synthetic insecticides used worldwide. In addition to their insecticidal effect, they have also repellent effect. This repellency is associated with an increase in the locomotor activity of insects caused by the interaction of the pyrethroid with a central nervous system target. As a consequence, insects avoid the source of the pyrethroid, with a kinetic response. This phenomenon is called excito-repellency (1).On the other hand, an insect repellent is defined as a chemical that produces oriented movements against the source of repellency. In this case, the repellent interacts with olfactory receptors. In this work we studied whether the pyretroid d-allethrin elicits an oriented repellency response mediated by olfaction in Blattella germanica.The following experimental series were performed: insects exposed to a film of d-allethrin; insects exposed to d-allethrin vapors at room temperature; insects exposed to d- One male of B. germanica was placed in the center of the arena and its spatial distribution was determined. Additionally, the electrical activity of the cockroaches’ antennae was recorded using electroantennography, to ensure that the insecticide was detected by olfactory receptors. In the behavioral experiments repellency was observed in a dose- dependent manner. When insects were exposed only to d-allethrin vapors at room temperature, repellency was not observed, unless the vapour fraction of the insecticide was increased by heating. In the electroantenographic assays a significant electrical response was recorded when the antennae were exposed to d-allethrin. This indicates that the insecticide is detected by olfactory sensilla, and repellency is the behavioral associated response to that stimulus. We conclude that d-allethrin produces olfactory- dependent repellency in B. germanica.

(1) Alipour H, Abaie MR, Ladonni H, Kadivar AA. A Comparative Study on Excito- Repellency Effects of Permethrin, Deltamethrin and Etofenprox Treated Bed Nets against Anopheles stephensi Liston, 1901 (Diptera: Culicidae). J Health Sci Surveillance Sys. 2013;1 (2):94-97.

140

OLFATOMETRY OPTIMIZATION FOR TUTA ABSOLUTA LARVAE.

Anna Paula BURGUEÑO1; María Laura UMPIÉRREZ1; Andrés GONZÁLEZ1; Carmen ROSSINI1*

1Laboratorio de Ecología Química, Universidad de la República, Montevideo, Montevideo, URUGUAY

In the laboratory, insect responses to odors are usually studied in olfactometers. These must be adapted to the specific problem under investigation in order to obtain representative data of natural behaviors1,2. Some lepidopteran caterpillars disperse and search for new host plants to feed on, sometimes causing crop damage. To control these pests, essential oils (EOs) provide a potential resource to develop environmentally friendly means of control3. While some EOs can be used as contact control agents for the tomato pest Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)3, their potential role as repellents also deserves investigation. The aim of this study was to develop an appropriate olfactometer method to study the behavioral response of T. absoluta larvae to potential volatile repellents (for which there are not previous reports). We evaluated 7 different Y-olfactometer settings by offering tomato leaves as a positive stimulus. These settings differed in factors such as vertical/horizontal orientation, open/closed system, larval stage (L2-L4), airflow, amount of stimulus and light. The percentage of responsive larvae, their first and last arm choices, as well as the number of entries and time spent on each arm, were the variables recorded. None of the larval stages responded in the horizontal olfactometer. L2 were the most mobile and responsive stage, especially towards light, while L4 (near to pupate, and probably less motivated to feed), showed weaker responses. Overall, the best setup for obtaining larval responses was the vertical open Y olfactometer (12 cm stem, 4.5 cm arms, 90o between arms) with an air flow of 600 mL/min and lit from above (3000 lux).

1) Physiological Entomology 2004, 29, p.45-55. (2) Journal of visualized experiments : JoVE 2013, 72. (3) Phytochemistry Reviews 2012, 11, p.339-350.

141

OPTIMISING THE MALE-PRODUCED SEX PHEROMONE OF GALLERIAMELLONELLA (PYRALIDAE): ELECTROPHYSIOLOGICAL AND BEHAVIOURAL ANALYSES OF STEREOISOMERS OF 5,11- DIMETHYLPENTACOSANE.

Glenn P. SVENSSON1*; Zainab AZHAR1; Fredrik ANDERSSON2; Erik HEDENSTROM2; Kenji MORI3; Olle ANDERBRANT4

1Dept. Biology, Lund University, Lund, Scania, SWEDEN 2Dept. Chemical Engineering, Mid Sweden University, Sundsvall, Vasternorrland, SWEDEN 3University of Tokyo, Tokyo, Tokyo, JAPAN 4Dept. Biology, Lund University, Lund, Scania, SWEDEN

Background: The greater wax moth, Galleriamellonella (L.), is a serious and global pest on honeybees, Apismellifera L. This species represents a rare case of sex-role reversal in moths, where males release a long-range sex pheromone in combination with ultrasound to attract conspecific females. Nonanal and undecanal were previously identified from wing gland extracts of male G. mellonella, but the blend of aldehydes failed to elicit attraction of females over large distances (1). We have recently identified a third male- produced pheromone component, the long-chain hydrocarbon 5,11-dimethylpentacosane, and shown that it acts as a behavioural synergist with the aldehydes (2).Aim: Analyse the bioactivity of the four possible stereoisomers of 5,11-dimethylpentacosane (3) to optimise the sex pheromone of G. mellonella.Methods: Coupled gas chromatography and electroantennographic detection (GC-EAD) was used to study the physiological response in antennae of female wax moths to individual isomers of 5,11-dimethylpentacosane. Also, flight tunnel assays with female moths were performed using blends including one of the isomers in combination with the aldehydes as stimulus.Findings: The 5R,11R isomer elicited the strongest antennal response in the GC-EAD analyses, and the blend including this isomer triggered strongest attraction in the flight tunnel tests. Still, the blend of nonanal, undecanal and 5R,11R-dimethylpentacosane is suboptimal because it is not as attractive to female moths as is the male extract.Conclusion: Our data showed increased response to the 5R,11R isomer in female G. mellonella. In spite of these promising results, further optimisation of the pheromone is needed to develop an efficient long-range attractant for monitoring of this pest.

References: 1. Flint HM, Merkle JR. (1983). J. Econ. Entomol. 76:467-472.

2. Svensson GP, Gunduz EA, Sjoberg N, Hedenstrom E, Lassance JM, Wang HL, Lofstedt C, Anderbrant O. (2014). J. Chem. Ecol. 40:387-395.

3. Mori K, Akasaka K. (2015). Tetrahedron 71:4102-4115

142

PREFERENCE-PERFORMANCE CORRELATION IN A MONOPHAGOUS SAWFLY.

Paula ALTESOR1; Andrés GONZÁLEZ1*

1Facultad de Agronomía, Universidad de la República, Montevideo, Montevideo, URUGUAY

The relationship between female oviposition choice and offspring performance is a central issue in insect-plant interactions. A recent meta-analysis found that the linkage between preference and performance in herbivorous insects varies with diet breath: they correlate well in oligophagous insects, but not in monophagous and polyphagous species. However, insects with restricted diets, especially monophagous, are expected to discriminate plants in relation to offspring performance. In this study, we explored the preference-performance linkage in a monophagous Neotropical sawfly, Tequus schrottkyi (Konow). Females were offered two congener plants, the cultivated Solanum tuberosum and the wild S. commersonii, on which the larvae are usually found. Performance was evaluated from newly-emerged larvae until adult emergence, using individual larvae raised on single-germplasm potted plants. Female oviposition preference was studied in two-choice bioassays in which individual females were offered whole plants. Comparative plant chemistry was performed on both plants, including protein and non- structural carbohydrate contents, as well as glycoalkaloid (GA) concentrations - the main secondary metabolites in Solanum plants. The results showed that female T. schrottkyi oviposited exclusively on the wild species S. commersonii, and that their offspring developed better on the preferred host plant. No differences were found in performance during the larval stage, but the pre-pupal and pupal stages and the emergence of adults were better on S. commersonii. Performance differences were not likely due to differential nutritional value, since primary metabolites measured were not different. Likewise, poorer performance cannot be attributed to GA concentrations, since more GAs were found in S. commersonii. A differential effect of GAs is possible, because GAs differ mainly in their glycosidic moieties in both Solanum species, and the larvae prefer S. commersonii based on its GAs. Regardless the role of GAs, our study shows that female preference and offspring performance are linked in the monophagous sawfly T. schrottkyi.

143

PHYTOCHEMISTRY DIVERSITY AT THE INTERFACE OF TRI-TROPHIC INTERACTIONS PIPER-LEPIDOPTERA-PARASITOIDS.

Lydia Fumiko YAMAGUCHI1; Mariana A. STANTON1; Patrícia Avelino MACHADO2; André Nascimento RANGEL2; Simeão S. MORAES2; André V. L. FREITAS2; Massuo Jorge Kato3*

1Instituto de Química, USP, São Paulo, São Paulo, BRASIL 2Departamento de Zoologia, Unicamp, Campinas, São Paulo, BRASIL 3Instituto de Química, USP, São Paulo, São Paulo, BRASIL

The chemical communication is the basis of multi-trophic interactions among organism. The knowledge on ecological consequences of the chemical signaling has advanced significantly but it is still scarce when the biodiversity as a whole is considered. Thus, the study of model systems such as Piperaceae represents a unique opportunity to understand the complexity of the three-trophic interactions plant–herbivores-parasitoids in a large- scale approach in the tropics. Thus, the determination of phytochemical diversity and the role of compounds in these interactions is one of the focus of our current project. An extensive survey on the components in these three trophic levels Piper-Lepidoptera- parasitoids in different sites of Atlantic Forests has revealed high level of specificity between a Geometridae genus and Piper species but a complex web of interactions.

PRP-USP, FAPESP, CAPES, CNPq.

144

REPELLENT AND ANTIFEEDANT PROPERTIES OF CRUDE EXTRACTS AGAINST EPICAUTA (COLEOPTERA: MELOIDAE).

Fernández Estrella NATALÍN1*; Sequin Christian JAVIER1; Campos Soldini Maria PAULA1; Wagner Leandro SEBASTIAN1; Safenraiter Melania EDITH1

1UADER-FCyT, Diamante, Entre Ríos, Argentina

Epicauta is one of the most diverse genera of Meloidae and it is considered an important horticultural pest in the world. Adults of species from South America have been reported as damaging several species of different plants families like Amaranthaceae, Asteraceae, Brassicaceae, Fabaceae, Mimosaceae, Portulacaceae, Solanaceae and Zygophillaceae. The chemical control has given rise to several problems including the pollution of the environment by insecticide residues, the selection of resistant pest populations, the negative side effects on beneficial parasites and predators and the deleterious effects on human health. In this context the main objective are study the repellent and antifeedant properties of ethanolic extracts of several plants with the purpose of searching for environmentally-friendly compounds controls for Epicauta. The plants used are Amaranthus quitensis; Artemisia dracunculus; Brassica oleracea V. Capitata; B. oleracea V. Italica; Beta vulgaris V. conditiva; Borago officinalis; Capsicum annum V. Bell; Clematis bonariensis; Dysphnia ambrosioides; Lavandula sp.; Origanum vulgare; Passiflora caerulea; Rosmarinus officinalis; Spinacia oleracea; Tagetes patula; and Thymus vulgaris. For feeding choice assay an antifeedant index (AI) was calculated as [1- (T/C)] x 100 where T and C represent consumption on treated and untreated food, respectively. The repellence tests were conducted using filter paper separated in halves. Repellence percentages were determined for each extract. The ethanolic extracts from A. dracunculus and T. vulgaris showed more than 100% of repellent activity and ethanolic extracts from T. patula, T. vulgaris, R. officinalis and Artemisia dracunculus showed more than 90% inhibition of the feeding at 100 µg/cm2 against Epicauta. For the application of these extracts in the Epicauta control, we propose categorize each extract as an insect control product taking into account the antifeedant activity and repellent. With this aim in mind, we will establish a rank of the most active extracts, using their activity on Epicauta “in situ”.

145

RESPONSE OF GRAVID FEMALES OF CODLING MOTH (CYDIA POMONELLA) TO VOLATILES OF FOUR VARIETIES OF QUINCE (CYDONIA OBLONGA) IN A Y-TUBE OLFACTOMETER.

Ariel DIAZ1; Flavia JOFRE BARUD1; Sandra LOPEZ1; Gabriela FERESIN1; Alejandro TAPIA1; Liza LÓPEZ1*

1Instituto de Biotecnología-FI, Universidad Nacional de San Juan., San Juan, San Juan, ARGENTINA

Knowledge of the interactions between codling moth, Cydia pomonella L., and its main hosts (apples and pears) is reasonably advanced from the perspective of chemical ecology. However, for quince, Cydonia oblonga Miller, such studies are absent. Since varieties of fruit crop species show different patterns of susceptibility to pest attack, we studied the response of gravid females of codling moth against four varieties of quince. Mated females were offered a dual choice between fruit volatiles and control (clean air) in a Y-tube olfactometer under laboratory conditions (24 ± 2◦C, 50 ± 5% RH). Assays were performed in the scotophase and twenty females were tested for each fruit variety. Time elapsed until the election and, first and final choices of the females were registered in a 10 min period. In addition, the time (s) spent in each arm of the device was recorded. Females showed preference for fruit volatiles (75 to 81% went to the fruit arm). At the first choice, only Smyrna and Mendoza-147 varieties displayed significant elections (p=0.04). Females spent more time in the arm with fruit volatiles than in the control but, only Smyrna and Champion varieties showed significant differences (p=0.007 and p=0.026, respectively). After 10 min period, the four varieties were preferred over the control (p<0.05). The lowest average time to initiate an election was registered against Smyrna and the greatest was for Champion varieties. According to our preliminary studies, the volatile profile for the variety Smyrna has the highest relative content in pear ester, (E,Z)-2,4-decadienoate, the kairomone of adult codling moth. The marked response to the variety Smyrna by females could be related with its high content in pear ester. More studies are necessary to evaluate this hypothesis.

Acknowledgements: To CICITCA-UNSJ, ANPCyT, DSVAA from San Juan Government and EEA-INTA, San Juan, ARGENTINA.

146

SECONDARY METABOLITES EXTRACTED FROM SPODOPTERA FRUGIPERDA ADULT BODIES AS INDICATORS OF SOYBEAN FEEDING AT LARVAL STAGE.

Tatiana E. UEDA1*; José P. da GRAÇA1; Jefferson T. FUJISAO1; Samuel MARTINELLI2; Renato A. de CARVALHO3; Maurício U. VENTURA4; Clara B. HOFFMANN-CAMPO4

1Universidade Estadual de Londrina, Londrina, Paraná, BRASIL 2Monsanto Company, St. Louis, Missouri, USA 3Monsanto do Brasil Ltda, São Paulo, São Paulo, BRASIL 4Universidade Estadual de Londrina, Londrina, Paraná, BRASIL

The understanding about feeding host patterns is a prerequisite to population dynamics studies and that knowledge is necessary for designing appropriate pest management programs. Likewise, it figures as an important strategy for monitoring the succession of crops, especially when technologies can generate selection pressure, as Bt crops is being used. Experiments were carried out to identify secondary metabolites available in the most cultivated soybean in Brazil and to assess the possibility of Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) to sequester and accumulate such compounds as defensive strategy. To test the hypothesis, eleven selected genotypes (V. Max RR, BMX Apollo RR, BMX Potência RR, BRS 284, BRS 360 RR, NA 5909 RR, ANTA 82 RR, TMG 115 RR, M9144 RR, 98Y30 and TMG 132 RR) were sowed at greenhouse conditions. S. frugiperda were maintained under controlled environmental conditions feeding on the same cultivars from hatching to adulthood. To identify and quantify phenolic compounds in soybean cultivars, leaves were collected at the V6 stage, and MeOH-extracted, according to the Embrapa Soybean Chemical Ecology Laboratory methodology. The resulting extracts were submitted to HPLC-PDA analysis. Adult body extracts were analysed by LC-MS for identification of secondary metabolites sequestered by the insects. The aglycones genistein (C15H10O5) and daidzein (C15H10O4) were observed in the leaves extracts; genistein was detected in higher concentration (16.86 mcg.g-1) than daidzein (1.01 mcg.g-1). In insect body analysis, genistein was detected at 269.16 m/z and daidzein at 253.17 m/z at concentration of 8.6 ppt and 4.4 ppt, respectively. Although, both isoflavones were detected in adult body extracts, the largest amount detected qualifies genistein as a better feeding indicator of soybean feeder S. frugiperda caterpillars.

147

SOYBEAN ISOFLAVONOIDS AS CHRYSODEIXIS INCLUDENS FEEDING INDICATOR.

Tatiana E. UEDA1*; José P. da GRAÇA1; Jefferson T. FUJISAO1; Samuel MARTINELLI2; Renato A. de CARVALHO3; Estela O. NUNES4; Maurício U. VENTURA4; Clara B. HOFFMANN-CAMPO4

1UNIVERSIDADE ESTADUAL DE LONDRINA | Cidade: Londrina, Londrina, Paraná, BRASIL 2Monsanto Company, St. Louis, Missouri, USA 3MONSANTO DO BRASIL LTDA, São Paulo, São Paulo, BRASIL 4Empresa Brasileira de Pesquisa Agropecuária, Embrapa Soja, Londrina, Paraná, BRASIL

Some herbivorous insects that feed on important economic crops are highly polyphagous. In general, for protection against natural enemies, these insects sequester plant-derived secondary compounds and use them as an important defense strategy. This study was carried out to assess the possibility of the soybean looper, Chrysodeixis includens (Walker, 1858) (Lepidoptera: Noctuidae) sequester and accumulate secondary metabolites available in the most cultivated soybean in Brazil. To test this hypothesis, eleven commercial soybean cultivars (V. Max RR, BMX Apollo RR, BMX Potência RR, BRS 284, BRS 360 RR, NA 5909 RR, ANTA 82 RR, TMG 115 RR, M9144 RR, 98Y30 and TMG 132 RR) were cultivated under greenhouse conditions. Leaves of such cultivars were offered to insects throughout the larval development. Insects were maintained on under controlled environmental conditions, from hatching to adulthood. In order to identify and quantify phenolic compounds, leaves at the V6 stage was submitted to methanolic extraction and analyzed by HPLC. Adult C. includens body extracts were analyzed by LC-MS for identification of secondary metabolites sequestered by the insects. Both extracts were hydrolyzed before performing the analysis. The aglycones genistein (269.16 m/z) and daidzein (253.17 m/z) were detected in adult body extracts, as well as on soybean leaves extracts of all eleven cultivars; the general average concentration of genistein in soybean leaves was fifteen times higher in comparison to daidzein. However, from insects fed on most the cultivars, the moth sequestered daidzein (6.81 ppt) in a very similar proportion as genistein (5.22 ppt). Thus, considering the concentration of isoflavones identified in C. includens body extracts, it is possible to conclude that both aglycones can be an appropriated feeding indicator to define whether soybean plants were the natural host of C. includens.

148

SEARCH FOR BUMBLEBEE FATTY ACYL REDUCTASES, WHICH UNDERLAY SPECIES-SPECIFIC COMPOSITION OF MALE MARKING PHEROMONE.

Aleš BUČEK1; Michal TUPEC1; Petra WENZELOVÁ2; Jiří KINDL2; Iva PICHOVÁ2*; Irena VALTEROVÁ2

1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, Czech Republic 2Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic, Czech Republic

Substrate specificities ofpheromone biosynthetic enzymes influence or directly determine insect pheromonecomposition. The research of the enzymes involved in fatty acid (FA) derivedsex pheromones biosynthesis contributed to the understanding of evolution ofthe pheromone communication in e.g. moths (Lepidoptera) and flies (Diptera). Inbumblebees (Hymenoptera), we study biosynthetic enzymes to uncover themolecular basis of species- specific composition of the labial gland secretions,marking pheromones (MPs), which are used by males to attract and arrest virginqueens. MPs are blends of terpenes and FA- derivatives including fatty alcohols (FALCs). Here we address the hypothesis that distinct specificities of fattyacyl reductases (FARs), candidate biosynthetic enzymes involved in reduction ofFAs to FALCs, contribute to both quantitative and qualitative differences inthe composition of MPs in three European bumblebee species: commerciallyimportant greenhouse pollinator, the buff-tailed bumblebee (Bombus terrestris), its sister species B. lucorum, and distantly related B. lapidarius. We analyzed labial gland and fatbody (used as a reference tissue) transcriptomes of the studied species toidentify pheromone biosynthetic FAR candidates. We identified over 10 tentativeFAR orthologs in each species, several of them highly abundantly andpreferentially expressed in the labial glands. To assay the FAR substratespecificities we have cloned the candidate FAR coding regions, expressed themin yeasts and used GC/MS to identify the FALCs produced by the heterologouslyexpressed FARs. The first results indicate that there are distinctLG-abundantly expressed FAR orthologs in B.terrestris and B. lucorumpreferring very long chain fatty acid substrates, which are presumably involvedin biosynthesis of the very long chain FALCs in B. terrestris and B. lucorumMPs. Characterization of further FAR candidates which are presumably involvedin biosynthesis of major FALC MP components is in progress.Financial support: Czech ScienceFoundation (#15- 06569S).

149

SECRETIONS FROM A SPINNERET OF A SILKWORM (BOMBYX MORI) SUPPRESS GREEN LEAF VOLATILE PRODUCTION IN MULBERRY LEAVES.

Hiroki Takai1; Rika OZAWA2; Junji TAKABAYASHI2; Saki FUJII3; Jun KOBAYASHI3; Takashi KIUCHI3; Susumu KATSUMA3; Toru SHIMADA3; Kenji MATSUI3*

1University of Tokyo, Tokyo, Tokyo, JAPAN 2Kyoto University, Kyoto, Kyoto, JAPAN 3Yamaguchi university, Yamaguchi, Yamaguchi, JAPAN

In response to herbivory, plants start emitting so-called herbivore-induced plant volatiles. One of the ecological functions of the volatiles is to attract carnivorous natural enemies of currently infesting herbivores. The induction/emission of volatiles attractive to natural enemies by herbivores is clearly maladaptive to the herbivores. Thus, herbivores might have some counteraction against the attraction by plants. Here, we show that, in mulberry plant-silkworm interactions, secretions from the spinneret of a silkworm were left on the edge of the infested part of a leaf, and this resulted in the suppression of the production of green leaf volatiles (GLVs) that were, in general, emitted by leaves in response to feeding related mechanical damage. The suppression factor was elucidated to be an enzyme that converted fatty acid hydroperoxides, the intermediates in the biosynthetic pathway of green leaf volatiles, into their keto derivatives to suppress GLVs. The adaptive functions of the enzyme secreted from silkworms to leaf edges were discussed in the viewpoints of the induction of the plant defense.

150

SOME THINGS OLD, SOME THINGS NEW: AREAS OF CHEMICAL ECOLOGY THAT MERIT ATTENTION.

Jeremy N. MCNEIL1*

1The University of Western Ontario, London, Ontario, CANADA

Most chemical ecologists would agree (i) that abiotic factors play an important role modulating both intra and interspecific interactions mediated by infochemicals, and (ii) that climate change will significantly influence these important environmental factors. For example, the excessive periods of rain and drought predicted by climate change will affect relative humidity conditions (RH), yet there are few published studies examining the impact of RH on chemically mediated interactions. Similarly, the impact of temperature conditions during any developmental stages other than the adult on pheromone mediated mating has not been extensively examined. I believe that research in these areas is of interest from both basic and applied perspectives, especially in the context of climate change, and will use examples of research on the European corn borer, Ostrinia nubilalis to make the point. While it is known that females of few lepidopteran species are able to detect the sex pheromone of conspecifics, the phenomenon of autodetection is considered to be unusual rather than the norm. However, one has to ask, is autodetection really an unusual phenomenon or has it just been neglected by the scientific community? I believe it is the latter and will discuss the different ways in which autodetection could be of ecological importance to support the argument that more research effort in this area is justified.The use of GM crops is now part of IPM programmes in different parts of the world and while they offer definite advantages they are not without potential problems. I will discuss how changes in the calling behaviour of fall armyworm, Spodoptera frugiperda females that have developed resistance to Bt corn, could impact on resistance management practices and could affect the use of pheromone traps in IPM.

151

SPINOCHROMES: KAIROMONES INVOLVED IN THE SYMBIOSIS BETWEEN THE SHRIMP TULEARIOCARIS HOLTHUISI AND ECHINOMETRA MATHAEI.

Brasseur L.1; Guillaume C.1; Demeyer M.1; Gerbaux P.1; Eeckhaut I.1*

1University of Mons, Mons, Hainaut, BELGIUM

Communication is one of the oldest biological principles that can be found at every organization level in biology: from cells to populations. Among many forms of communication, the most widespread remains the chemical communication, the molecular diversity being so huge that it allows to produce a large quantity of unique signals ensuring accurate transmission of information to receptors. Seawater being an ideal dispersing agent, chemical communication stays predominant in marine ecosystems. However, if many molecules acting in chemical heterospecific communication have already been well described in terrestrial ecosystems, only two of these molecules were identified in marine ecosystems. Echinoderms and their symbiotic organisms constitute very good models to study heterospecific chemical communication because each class synthesizes a specific type of molecules and symbioses with echinoderms as hosts are very usual. Sea urchins synthesise polyhydroxynaphthoquinonic pigments, also known as spinochromes, involved in antioxidant and defence processes. In this study, we investigate the chemical communication that allows the commensal shrimps Tuleariocaris holthuisi Hipeau-Jacquotte, 1965 to live with their host Echinometra mathaei (Blainville, 1825). Prevalence of the infestation on the Great Barrier Reef of Toliara where the organisms where collected was ca 36% and the rate of infestation was of 1,5 per sea urchin. We characterised the chemoreception of the shrimp using olfactometers and we demonstrate that hosts and synthetic polyhydroxynaphthoquinones are attractive to the symbiotic shrimps. Our study is the first highlighting the ecological function of naphthoquinones as kairomones. We also performed chemical extractions on sea urchins to analyse and identify its specific naphthoquinones (spinochromes) using HPLC-ESI- MS. Accurate mass identification and elemental composition have been performed on various organs (gonads, coelomic liquid, digestive system and test) in different colored morphotypes of Echinometra mathaei for a better understanding of the molecular diversity of these semiochemicals.

152

SUB-FUNCTIONALIZATION OF A NICOTIANA SPECIFIC COI1 HOMOLOG IS REQUIRED FOR JASMONATE-DEPENDENT REGULATION OF FLORAL MATURATION AND DEFENSIVE ROOT METABOLISM.

Michael STITZ1*; Klaus GASE2; Youngjoo OH2; Ian Thomas BALDWIN2; Emmanuel GAQUEREL3

1Centre for Organismal Sciences, Heidelberg, Baden-Württemberg, GERMANY 2Max Planck Institute for Chemical Ecology, Jena, Thuringia, THURINGIA 3Centre for Organismal Sciences, Centre for Organismal Sciences, Heidelberg, GERMANY

Plants permanently have to face versatile challenges such as constant feeding pressure by herbivorous threats or direct competitors for nutrients or even pollination services. Accordingly, plants essentially depend on highly functional signaling for both their survival and their successful reproduction. Phytohormones, such as Jasmonoyl-isoleucine with its unique receptor protein CORONATINE INSENSITIVE1 (COI1), are core elements in the signaling cascades coordinating plant defenses as well as developmental processes. While in the well-studied model plant Arabidopsis thaliana only one COI protein is known, we here functionally describe a new COI receptor protein in Nicotiana attenuata. Originating from a whole genome duplication in the solanaceous, this receptor was only maintained in the Nicotiana genus. Using a transgenic silencing approach with RNA interference, we demonstrate with combined transcriptomic, targeted metabolic, and allometric analyses, that COI1 and COI2 mediate partly overlapping and partly distinct signaling events during herbivore defense and floral development. We further provide evidence that COI2 is not involved in jasmonate mediated root-growth inhibition, the susceptibility to a specialized herbivore and the emission of the main floral volatile Benzylacetone. These findings contrast with the accumulation of the defense compound nicotine and the elongation of floral corolla and style, which essentially depend on synergistic signal transmission by both functional receptor proteins. Ongoing microarray experiments and metabolom studies of dissected floral tissues further will provide knowledge about the yet elusive regulatory role of COI2 in plant organ specific metabolism.

153

TERGAL GLAND SECRETION OF THE ALEOCHARA PSEUDOCHRYSORROA CARON, MISE & KLIMASZEWSKI (STAPHYLINIDAE: COLEOPTERA).

Maycon R. SILVA1*; Diogo M. VIDAL1; Paulo H. G. ZARBIN1

1Universidade Federal do Paraná, Cutitiba, Paraná, BRASIL

Aleochara pseudochryssoroa is a very common and abundant species in Centro Politécnico UFPR (Federal University of Paraná), Curitiba, Paraná, Brazil. As in other aleocharinae beetles this species has a tergal gland with a bilobate reservoir formed by an invagination of the intersegmental membrane between the sixth and seventh abdominal tergites (Peschke & Metzler, 1981). Aleocharinae tergal gland have been studied and demonstrate to have defensive properties (Jordan, 1913; Pasteels, 1968; Holldobler, 1970, Peschke, 1982, 1987 Peschke & Metzler, 1981). Aleochara pseudochryssoroa were collected in in Centro Politécnico UFPR (Federal University of Paraná) Curitiba, Paraná, Brazil in animal carcass, the adults were feeding with first and second larval instar of Sarconesia chlorogaster (Wiedemann) (Diptera) and with puparia of Sarconesia chlorogaster as hosts for the parasitoid larvae. The extractions were performed through freezing the beetles, dissecting the gland and extracting with 100 µl of Hexane. GC analysis showed that the tergal gland secretions contains at least fourteen volatile compounds. GC-MS and GC-FTIR spectra and retention indexes were employed on structural elucidation. The major compound was determined to be related to methyl-p- benzoquinone. The minor components refers to structures related to methyl-p- benzoquinone and to unbranched short chain alkanes and alkenes and aldehydes. The double bond positions of the unsaturated compounds were determined by methiolation with DMDS and double bond configuration by FTIR. This type of compounds seems to be common for tergal glands in this subfamily, once this same type of defensive compounds were identified in other aleocharinae species (Blum et al., 1971; Peschke, 1982, Steidle & Dettner, 1993).

154

TETRODOTOXIN IN THREE NEW SPECIES OF CUTTLEFISH: METASEPIA PFEFFERI, SEPIA BANDENSIS, AND SEPIA OFFICINALIS.

Becky WILLIAMS1*; Christine BEDORE2; SaVanna ARIAS3; Daniel ROPER3

1Utah State University Uintah Basin, Vernal, Utah, USA 2Georgia Southern University, Statesboro, Georgia, USA 3Utah State University Uintah Basin, Vernal, Utah, USA

Tetrodotoxin (TTX) causes asphyxiation and death in vertebrates through nervous and muscular tissue function disruption. The deadly blue-ringed octopus is the only cephalopod known to possess TTX. Here we investigated the presence of TTX in another brightly-colored and conspicuous cephalopod. We examined both captive-born and wild- caught Flamboyant Cuttlefish (Metasepia pfeifferi; n = 34), as well as Stumpy Cuttlefish (Sepia bandensis; n = 4) and a Common Cuttlefish (Sepia officinalis; n = 1). Seventeen tissue-types were sampled, extracted based on standard methods, and assayed for TTX via an improved competitive inhibition enzymatic immunoassay. Our results revealed TTX in several tissues of all three species of cuttlefish, and in both captive-bred and wild-caught specimens. Tetrodotoxin was found in the hearts and gills (suggesting distribution via the blood), the reproductive organs (gonads, eggs, and associated glands), ink, and the skin. Tetrodotoxin was also found in the anterior and posterior salivary glands (the venom gland of cephalopods). Whether the TTX discovered here is endogenous or exogenous is currently unknown. Interestingly, based on the literature and our own unpublished data, these amounts of TTX are unlikely to subdue prey or deter common predators of the cuttlefish (maximum 100 ng total TTX in an adult wild-caught male M. pfefferi). The ecological function of TTX, if any, in these organisms requires further study.

155

THE MALE-PRODUCED SEXUAL PHEROMONE OF THE STRAWBERRY SAP-BEETLE LOBIOPA INSULARIS (CASTELNAU, 1840) (COLEOPTERA: NITIDULIDAE).

Antonioni Acácio Campos MOLITERNO1; Rodolfo Rodrigues da SILVA1; Daiane SZCZERBOWSKI1; Camila Borges da Cruz MARTINS1; Paulo Henrique G. ZARBIN1*

1Federal University of Paraná, Depart. of Chemistry, Curitiba, PR, BRAZIL

Lobiopa insularis is one of the most important pests that affect the strawberry crop in Brazil. In this scene, in the search of a specific, clean and effective control technique, semiochemicals stand out. The chemical ecology study of L. insularis started with the collection of adults in the field which were sexed and maintained under laboratory conditions ((25 ± 2°C; 70%; 12L:12D). Insects were fed with strawberry. Volatiles were collected by aeration in two different extraction times (12 to 72h) The 12 hours aerations (photophase and scotophase) were performed to determine the dynamic of production of the pheromone. The identification of compounds present on the extracts were conducted by GC-MS analysis, retention index comparisons, and co-injections with standards and synthesized compounds. Three male specific compounds were detected, a major compound was identified as 2-undecanone (RI: 1296), 2-nonanone (RI: 1087) and a minor compound was identified as 2-undecanol (RI: 1303). Analysis to determine the stereochemistry of the latter compound were conducted by GC-Chiral chromatography employing a β-ciclodextrine column. It was possible to observe that the insect produces a mixture of the enantiomers in a ratio of 3.5:1 of S:R isomer. The male specific compounds were quantified based on external standard calibration. These male-specific compounds were produced only during scotophase in a concentration of 6.03 ± 5.15, 1.37 ± 1.50 and 0.30 ± 0.15 ng per insect in 24 hours. However, preliminary bioassays in the Y-tube olfactometry revealed that extracts of males were attractive to females, indicating the effectiveness of male extracts as attractants.

156

TOWARDS THE IDENTIFICATION OF THE SEX PHEROMONE OF H. ERATO PHYLLIS (FABRICIUS, 1775) (NYMPHALIDAE, HELICONIINAE).

Eliane de O. BORGES1*; Dayana BONFANTTI1; Camila B. C. MARTINS1; Diogo M. VIDAL1; Luana HOFFMANN1; Stefan SCHULZ2; Paulo H. G. ZARBIN2

1Universidade Federal do Paraná, Cutitiba, Paraná, BRASIL 2Institute of Organic Chemistry, Technische Universität Braunschweig, Braunschweig, Niedersachsen, GERMANY

The main objective of this research is to analyze the sex pheromones presents in the anti- aphrodisiac produced by abdominal scent glands, in the androconia scales and in the pupal mating, the external morphology of the related biological structures and the behavior associated of Heliconius erato phyllis (Fabricius). In the chemical analysis methodology were adopted the extraction with solvent, the aeration, the gas chromatography, the gas chromatography associated to mass spectrometry and the infrared spectroscopy. To analyze the bioactivity of the compounds were used behavioral bioassays and electroantennography. Morphological studies were conducted using stereomicroscopy and scanning electron microscopy. The results show that the male and female abdominal scent gland presents translucent structures. The androconial scales of males are present on the forewing in a narrow strip near the A1 + 2 vein, and on the hindwing on the overlap band, under the veins Sc + R1, RS and M1. Viewed in detail, each androconial scale shows fringes on the undulating apical region. The compounds 1- hexen-3-one, 1-octen-3-one, € -β-ocimene, 3-undecanone, and (E) -2 3-dihydrofarnesoic acid were identified in the abdominal scent glands bouquet of the specie. A sixth compound was additionally detected and its structure is proposed as an unsaturated methyl branched macrolide. The synthesis of this macrolide is underway in order to confirm the proposed structure. The virgin males it has the same scent bouquet of the mature male, three days after emergence. Electroantennography results show that all the compounds are perceived by the antennae of both virgin and mated individuals of both sexes, with variation in the magnitude of perception of the compounds. Bioassays with virgin females initially proved the anti-aphrodisiac function of odoriferous bouquet of male valve. Were found possible volatile bioactive compounds in the overlapping bands of the wings of males and in the pupae of females.

157

THE ROLE OF CHEMICAL SIGNALS IN THE ADAPTIVE RADIATION OF CLEARWING BUTTERFLIES.

Melanie MCCLURE1; Centina PINIER2; Brigitte FRÉROT2; Marianne ELIAS3*

1CNRS, MNHN, Paris, Ile-de-France, FRANCE 2INRA, Versailles, Ile-de-France, FRANCE 3CNRS, MNHN, Paris, Ile-de-France, FRANCE

Reproductive barriers, including those mediated by chemical signals, are often considered to play an important role in speciation and adaptive radiation. However, whether these barriers occur early on in the speciation process, and whether this is repeatable across taxa, remains unclear. The Amazonian clearwing butterflies (Ithomiini) provide an interesting opportunity to test this, as the tribe has undergone a rapid and extensive adaptive radiation. As such, divergence between geographical races, occurring for many genera, provides an interesting comparative framework along the speciation continuum. Our results show that for some genera, chemical profiles differ between races, suggesting that pheromones play a role in maintaining reproductive isolation. However, this wasn’t the case for all the genera studied, which may suggest that other signal modalities, such as colour pattern, are of greater importance in these cases. We suggest that the role of chemical signals in reproductive isolation may be more important for those taxa where colour pattern is less divergent and where visual signals may play a lesser role. These results are discussed in regards to the ecology of clearwing butterflies.

158

THE ROLE OF VOLATILE COMPOUNDS IN THE INTERACTION DIVERSITY OF PIPER MALACOPHYLLUM AND P. GAUDICHAUDIANUM.

Mariana A. STANTON1; Lydia F. YAMAGUCHI1; Simeão S. MORAES2; André R. NASCIMENTO2; Patricia A. MACHADO2; André V. L. FREITAS2; Massuo J. KATO3*

1Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, São Paulo, São Paulo, BRAZIL 2Department of Zoology, University of Campinas, Campinas, São Paulo, BRAZIL 3Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, São Paulo, São Paulo, BRAZIL

Piper spp. are one of the most speciose clades of magnoliid Angiosperms, consisting of approximately 2000 species that belong to the highly diverse family Piperaceae (approx. 3600 species). In addition, they represent a significant portion of the understory growth in tropical forests. Despite the abundance of this group and previous characterization of the diverse secondary chemistry of many Piper species, little is known of the role of plant chemicals in mediating the specificity of plant interactions with higher trophic levels, particularly in the case of volatile organic compounds (VOCs). Among the specialist herbivores of Piper spp. are larvae of the also diverse genus Eois spp. (Lepidoptera: Geometridae). One of the possible roles of VOCs in mediating interaction specificity is through their use in host plant choice by ovipositing adult Eois spp. and in the attraction of parasitoids that feed on them. Sampling in the state of SãoPaulo, Brazil, has revealed that Piper malacophyllum and P. gaudichaudianum are two of the most susceptible hosts to herbivory by Eois spp in this region. Thus in the present study we ask whether herbivory by Eois larvae influences host plant VOC profile and adult preference for P. malacophyllum and P. gaudichaudianum, and whether larval survival and parasitism correlate with adult host plant choice. We are investigating the role of VOCs released by both plant species in the attraction of adult Eois spp using GC-EAD, and compare our results to field surveys of the natural patterns of host plant choice by these herbivores. Future steps will include comparison of the volatile profile of P. malacophyllum and P. gaudichaudianum to those of less preferred hosts or non-host Piper spp to further shed light on the role of plant VOCs and phylogeny in the specificity of interactions with herbivores and parasitoids.

159

TO GROW AND TO DEFEND: FAR-RED MODULATES NICOTIANA ATTENUATA'S SHADE-AVOIDANCE GROWTH, WITH NO COMPROMISES IN MEJA-INDUCED PLANT DEFENSES.

Variluska FRAGOSO1*; Youngjoo OH1; Sang-Gyu KIM2; Ian Thomas BALDWIN2

1Max Planck Institute for Chemical Ecology, Jena, Thuringia, Thuringia 2CENTRE FOR GENOME ENGINEERING, INSTITUTE FOR BASIC SCIENCE, DAEJEON, HOSEO, SOUTH KOREA

It has been largely accepted that under light-limiting growth conditions imposed by neighboring plants, phytochromes mediate plant diversion of resources towards outgrowing competitors, collectively referred as shade-avoidance responses (SAR), in detriment of plant chemical defenses against herbivores. In agreement, this trade-off has been evidenced at the molecular level: far-red light (FR), a signal of plant proximity detection, and jasmonic acid (JA) signaling pathways converge in an antagonistic manner modulating plant responses upon herbivore attack. Here, however, we show that under field and glasshouse conditions, FR treatment does not increase herbivore preference or impair plant capability to induce Methyl JA-responsive defenses. Nevertheless, transgenic plants displaying constitutive SAR by the silencing of phytochromes (irPhyB1B2) yield bigger caterpillars. In a realistic scenario, where PAR and drought stress are high, N. attenuata plants might benefit from lower temperatures and higher humidity provided by shade. These data suggest that, given enough herbivore pressure and PAR, shade-intolerant plants might not have evolved according to the dilemma to grow or defend; and can still maintain its inducible defenses even when displaying strong constitutive SAR.

160

UNCOVERING THE MOLECULAR BASIS OF BUMBLEBEE MALE MARKING PHEROMONE DIVERSITY VIA RNA SEQUENCING.

Aleš BUČEK1*; Jana BRABCOVÁ1; Heiko VOGEL2; Darina PRCHALOVÁ2; Jiří KINDL2; Irena VALTEROVÁ2; Iva PICHOVÁ2

1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, CZECH REPUBLIC 2Max Planck Institute for Chemical Ecology, Jena, Thuringia, THURINGIA

Male marking pheromones (MPs) are used by male bumblebees (Hymenoptera: Apidae), including a commercially important greenhouse pollinator, the buff-tailed bumblebee (Bombus terrestris), to attract conspecific virgin queens. MP biosynthetic processes in the B. terrestris male labial gland (LG) are of extraordinary complexity, presumably involving enzymes of fatty acid (FA) and isoprenoid (ISP) biosynthesis, which jointly produce more than 50 compounds with ISPs being the major MP components. By contrast, B. lucorum, a sister species of B. terrestris, uses predominantly FA-derived MP components. The two species thus represent remarkable model organisms for study of pheromone communication evolution.To gain insight into molecular basis of MP biosynthesis, we employed a differential transcriptomic approach by RNA-sequencing of B. terrestris and B. lucorum LGs and fat bodies (reference tissue) transcriptomes. We identified 12,454 abundantly expressed genes in B. terrestris LG of which 876 were substantially overexpressed in LG. By including the available B. terrestris queen head, queen ovaries and male head transcriptomic data into the analysis we identified genes potentially involved in majority of the MP biosynthetic steps, e.g. esterases and lipases, FA reductases, and enzymes involved in biosynthesis of ISPs and FAs; RNA-seq data for selected candidates were validated by qRT-PCR [1]. The comparison of the ISP gene expression in B. terrestris and B. lucorum indicates that the transcript abundances of the proposed ISP biosynthesis-regulatory genes i.e. acetoacetyl-CoA thiolase and 3-hydroxy- 3-methylglutaryl-CoA reductase are more than 17- and 66-fold, respectively, lower in B. lucorum than in B. terrestris LG, suggesting that transcriptional regulation plays a principal role in the control of ISP MP biosynthesis [2]. Acknowledgements The research was supported by project no. LO 1302 from the Ministry of Education, Czech Republic and by grant no. 15-06569S from the Czech Science Foundation. [1] Buček A et al. accepted in Insect Mol Biol [2] Prchalová D et al. (2016) ChemBioChem 17(3).

161

VOCS EMITTED BY ILEX PARAGUARIENSIS PLANTS ARE AFFECTED BY HERBIVORY OF THE LEPIDOPTERAN THELOSIA CAMINA (SCHAUS, 1920) AND THE COLEOPTERAN HEDYPATHES BETULINUS (KLUG, 1825).

Camila B. C. MARTINS1; Diogo M. VIDAL1; Sandra Mara S. GOMES1; Paulo H. G. ZARBIN1*

1Universidade Federal do Paraná, Cutitiba, Paraná, BRASIL

Leaves of the maté tree are dried and minced to produce one of the most popular brewed teas of South America. Ilex paraguariensis occurs naturally in Brazil, Argentina and Uruguay and its two main pests in Brazil are Thelosia camina (Eupterotidae, Lepidoptera) and Hedypathes betulinus (Cerambycidae, Coleoptera). Even though its essential oil and the VOCs emitted by maté beverages have already been determined it is still unknown the profile of volatiles emitted by I. paraguariensis plants. Therefore, the objective of this study was to determine the VOCs emitted by maté tree plants before and after the herbivory of its two main pests. For this purpose, larvae of T. camina and adults of H. betulinus were collected in the field and then kept under controlled temperature, humidity and photoperiod until experiments began. Young mate plants were obtained at a commercial farm and maintained at room temperature. For the headspace collection, plants were kept inside aeration chambers for 24h to 96h, and a total of four replicates were performed. Four treatments were tested: control (a), mechanical damage (b), herbivory by larvae of T. camina (c), herbivory by adults of H. betulinus (d). Extracts were analyzed by GC-MS and GC-FTIR, and identifications were based on mass spectra, infrared spectra, and KI. Statistical analyses are still underway using a PCA followed by a Multivariate Analysis of Variance and a Tukey test (program R). Among the 20 compounds detected in headspace collections, so far 15 compounds were identified as hydrocarbons, terpenes, ketones, alcohols, and aldehydes. Statistical analysis revealed that treatments c and d are different from a and b, indicating a difference between responses of the plant among treatments. DMNT, β-ocimene and linalool + n-undecane were the compounds emitted in great quantities in treatments b and c. β-ocimene was not detected in control plants.

162

WHAT DOESN’T KILL YOU MAKES YOU STRONGER? THE COMPARATIVE EFFECT OF ALIPHATIC AND INDOLIC GLUCOSINOLATES ON LEPIDOPTERAN DEVELOPMENT.

Verena JESCHKE1*; Emily E. KEARNEY1; Grit KUNERT1; Jonathan GERSHENZON1; Daniel G. VASSÃO1

1Max Planck Institute for Chemical Ecology, Jena, Thuringia, THURINGIA

Some small insect herbivores feed successfully on plants containing glucosinolates, despite their formation of a diverse array of toxic and deterrent breakdown products. Isothiocyanates (ITCs) are considered to be the most toxic glucosinolate-derived metabolites and are typically thought to be responsible for the reduced growth and delayed development observed in several generalist insects feeding on glucosinolate- producing plants. Those generalist insects detoxify a portion of ITCs via conjugation with glutathione (GSH), but a large amount of ITCs remains unmodified [1] and may lead to the observed negative effects.We compared the effects of Met- and Trp-derived glucosinolates, which lead principally to ITC and non-ITC hydrolysis products, respectively, on two generalist-feeding caterpillars with different feeding preferences for glucosinolates. The development of larvae of Spodoptera littoralis (African cotton leafworm) and Mamestra brassicae (cabbage moth) was investigated from hatching until adult emergence while the larvae were reared on Arabidopsis thaliana Col-0 and three different glucosinolate-deficient mutants: myb28myb29 (deficient in aliphatic glucosinolates), cyp79B2cyp79B3 (deficient in indolic glucosinolates), and myb28myb29cyp79B2cyp79B3 (deficient in both aliphatic and indolic glucosinolates). We found that both glucosinolate types negatively affected larval growth and development when fed separately, but in combination their effect was significantly stronger. Interestingly, the impact of the different glucosinolate classes differs between the two investigated generalist species. To our surprise, larvae fed on glucosinolates gave rise to heavier pupae and adults. Chemical analyses of the body composition of those adults confirmed changes in the protein/fat ratio derived from metabolic imbalances [2].

[1] Schramm, K. et al. (2012). Insect Biochem Mol Biol 42, 174-182. [2] Jeschke, V. et al. (2016). Insect Biochem Mol Biol 71, 37-48.

163 INTERNATIONAL COLLABORATIVE RESEARCH ON CHEMICAL ECOLOGY IN LATIN AMERICA

EFFECTS OF DEFENSES OF DEVELOPING SEEDS OF FIELD-GROWN SOYBEAN ON STINK BUGS (NEZARA VIRIDULA)

Jorge A. ZAVALA1

1Catedra de Bioquimica, Facultad de Agronomia, Universidad de Buenos Aires, Instituto de Investigaciones em Biociencias Agricolas y Ambientales, Conicet

Southern green stink bugs (Nezara viridula) invade field-grown soybean crops, where they feed on developing seeds and inject toxic saliva that causes yield reduction. Although leaf responses to herbivory is well studied, no information is available about the regulation of defenses in reproductive structures, which are the main components of fitness in natural species and the final product of many field crops. This study demonstrated that mitogen-activated protein kinases (MPK) 3, MPK4 and MPK6 are expressed and activated in developing seeds of field-grown soybean and regulates a defensive respond after stink bug damage. Although 10-20 min after stink bug damage of developing seeds induced expression of GmMPK3, GmMPK6 and GmMPK4, only MPK6 was phosphorylated after damage. Herbivory induced an early peak of jasmonic acid (JA) accumulation and ethylene (ET) emission after 3 h in developing seeds, whereas salicylic acid (SA) was also early induced and with increasing levels up to 72 h after damage. Damaged developing seeds up-regulated defensive genes and increased the production of chemical defenses typically modulated by JA/ET or SA, such as proteinase inhibitors and isoflavonoids. Plant defenses decreased the activity of digestive enzymes in the gut of stink bugs and reduced their survivorship. Induced seeds were less preferred by stink bugs. This study shows that stink bug damage is regulated by seed defenses, which is perceived early by MPKs that activate defense metabolic pathways in developing seeds of field grown-soybean, the main components of yield crop.

165

FLORAL SCENTS IN THE ORCHID GENUS CATASETUM: DO SCENT PATTERNS PREDICT POLLINATING EUGLOSSINE BEE GENERA?.

Paulo MILET-PINHEIRO1*; Manfred AYASSE2; Daniela M.A.F. NAVARRO3; Stefan DÖTTERL4; Günter GERLACH5; Roman KAISER6

1Federal University of Pernambuco, Department of Chemistry, Recife, Pernambuco, BRAZIL 2University of Ulm Institute of Evolutionary Ecology and Conservation Genomics; Department, Ulm, Baden-Württemberg, GERMANY 3Federal University of Pernambuco, Department of Chemistry, Recife, Pernambuco, BRAZIL 4University of Salzburg, Department of Ecology, Evolution and Plant Ecology, Salzburg, Salzburg, AUSTRIA 5Botanical Garden of Munich, Munich, Munich, GERMANY 6Research Center of Givaudan, Dübendorf, Zurich, SWITZERLAND

The orchid genus Catasetum comprises about 170 fragrance-producing species pollinated exclusively by male euglossine bees, mainly of the genera and Eulaema. Interestingly, species pollinated by Eulaema are rarely visited by Euglossa, and vice versa, and floral scents are assumed to play a pivotal role in this selective attraction. Recent data suggests that the evolution of floral scents in Catasetum is shaped by pre- existing sensory and/or behaviour biases of pollinators, however, experimental support is scarce. Here we used literature and own data to test whether scent traits differ between species pollinated by Eulaema and Euglossa. We focused on the composition of floral scents and on daily fluctuations in total scent emission. In 34 Catasetum species, we found 264 floral scent components, mainly monoterpenes, aromatics, and sesquiterpenes. Comparative and multivariate analyses of relative scent patterns showed that the Catasetum species group according to pollinator genera, i.e. Eulaema and Euglossa. The scent composition of species pollinated by Eulaema strongly differed from scent of species pollinated by Euglossa (dissimilarity of 94%). Most responsible for this difference were (E)-carvone epoxide, α-pinene, and benzyl acetate, dominant in species pollinated by Eulaema, and eucalyptol, myrcene, and ipsdienol, dominant in species pollinated by Euglossa. With respect to daily fluctuation in scent emission, we found that the peak of emission in species pollinated by Eulaema (N=3) and Euglossa (N=3) occur between 06:00h and 09:00h and between 09:00h and 12:00h, respectively. This pattern matches with the peak of foraging activity of Eulaema and Euglossa species in nature. We conclude that scent traits in Catasetum predict the genus of their pollinating bees, suggesting that the plants adapt to pre-existing sensory and behaviour biases of one of the euglossine genera. This study might serve as basis for future research on the ecology and evolution of floral scents in other fragrance-producing plants.

166

FIRST REPORT OF SCENT MIMICRY IN FOOD DECEPTIVE FLOWERS: THE CASE OF CEPHALANTHERA RUBRA (ORCHIDACEAE).

Paulo MILET-PINHEIRO1*; Stefan DÖTTERL2; Manfred AYASSE3

1Universidade Federal de Pernambuco, Recife, Pernambuco, BRAZIL 2University of Salzburg, Salzburg, Salzburg, AUSTRIA 3University of Ulm, Ulm, Baden-Württemberg, GERMANY

Food deceptive flowers are assumed to mimic visual, but not olfactory floral signals of model flowers. Evidence in the literature suggests that food deceptive flowers of the European orchid Cephalanthera rubra mimic the colour, but not the scent, of Campanula flowers, thereby attracting bees of Chelostoma rapunculi, which are Campanula-pollen specialists. Campanula flowers produce unusual floral volatiles (i.e. spiroacetals) involved in the olfactory recognition of host flowers by Ch. rapunculi. Since these Campanula-typifying volatiles are emitted in extremely low amounts, their role in this food deceptive system may have been previously overlooked. Here, we investigated the floral mimicry of Ce. rubra using a multidisciplinary approach that included colorimetric measurements of flowers, chemical analyses of floral scents and behavioural experiments. Colour measurements confirmed a high similarity across species of Campanula (14 investigated spp.) and Ce. rubra. The analyses of floral headspace samples of Ce. rubra revealed a scent bouquet composed of eight compounds, including the spiroacetal E-conophthorin, which was also reported in samples of nine out of the 11 investigated Campanula species. Two-choice bioassays performed with flower-naive bees of Ch. rapunculi showed that 1) visual and olfactory floral signals of Ca. trachelium and Ce. rubra are equally attractive to bees when tested against one another, 2) E- conophthorin attracts the same number of bees as both a synthetic mixture simulating the floral scent bouquet of Ce. rubra and an inflorescence of Ce. rubra. Our results experimentally confirm that Ce. rubra is a food deceptive species, which, however, do not only mimic visual but also olfactory floral signals of Campanula species to lure Ch. rapunculi pollinators. This work reports for the first time a case of scent mimicry in food deceptive flowers and suggests that the assumption that floral scents play a minor role in food deceptive systems might be considered carefully.

167

IDENTIFICATION OF EUSCHISTUS CONSPERSUS EGG VOLATILES AND KAIROMONAL ACTIVITY BY THE PARASITOID TELENOMUS PODISI.

Roberta TOGNON1*; Jeffrey R. ALDRICH2; Jocelyn G. MILLAR3; Qing-He ZHANG4; Josué SANT’ANA5; Frank G. ZALOM6

1PPG-Fitotecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, BRAZIL 2Jeffrey R. Aldrich consulting LLC, Santa Cruz, California, USA 3Department of Entomology, University of California, Riverside, CA, USA 4Sterling International, Inc., Spokane, Washington, USA 5PPG-Fitotecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, BRAZIL 6Department of Entomology and Nematology, University of California, Davis, California, USA

The consperse stink bug, Euschistus conspersus Uhler (Heteroptera: Pentatomidae), is the most important native pest that feeds on tomatoes in California. Telenomus podisi Ashmead (Hymenoptera: ) is a cosmopolitan egg parasitoid of many pentatomids, particularly Euschistus spp, being chemical compounds released by eggs its main cue for host location. This study was undertaken to identify chemical compounds on the E. conspersus egg masses and test their kairomonal activity for T. podisi. Fresh E. conspersus eggs (1g) were extracted with hexane or tert-butyl methyl ether in glass vials for 5 min and used for laboratory bioassays and GC-MS chemical analyses, respectively. Methyl (2E,4Z)-2,4-decadienoate, the male-produced aggregation pheromone of E. conspersus, was the major volatile identified from eggs extracts. Parasitoids were presented with a choice between egg extract (5 µl=10 eggs equivalent) and methyl (2E,4Z)-2,4-decadienoate (5 µl=1 and 10 ng), all versus the same amount of hexane (control), in tissue paper by using a Y-tube olfactometer. Olfactometer data were analyzed by χ2-tests (P E,4Z)-2,4-decadienoate (both doses). The volatile male-produced pheromone on E. conspersus eggs is an important kairomone for T. podisi to find and recognize E. conspersus host eggs. This information may help to improve the biological control by using egg parasitoids against stink bug pests in agroecosystems.

168

IDENTIFICATION OF HALYOMORPHA HALYS EGG VOLATILES AND KAIROMONAL EFFECT ON THE PARASITOID TELENOMUS PODISI.

Roberta TOGNON1*; Jeffrey R. ALDRICH2; Jocelyn G. MILLAR3; Qing-He ZHANG4; Josué SANT’ANA5; Frank G. ZALOM6

1PPG-Fitotecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, BRAZIL 2Jeffrey R. Aldrich consulting LLC, Santa Cruz, California, USA 3Department of Entomology, University of California, Riverside, CA, USA 4Sterling International, Inc., Spokane, Washington, USA 5PPG-Fitotecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, BRAZIL 6Department of Entomology and Nematology, University of California, Davis, California, USA

Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is an invasive, polyphagous fruit and vegetable pest from eastern Asia that has spread throughout Europe and North America. BMSB parasitism by endemic natural enemies in North America and Europe is typically low (Telenomus podisi Ashmead (Hymenoptera: Platygastridae) is a cosmopolitan egg parasitoid of many pentatomid species. Chemical compounds on eggs are important for natural enemies to find and recognize hosts. This study aimed to identify chemical compounds on H. halys eggs and test their kairomonal activity on T. podisi. Fresh H. halys eggs (1g) were extracted with hexane or tert-butyl methyl ether both in glass vials for 5 min, and used for behavior bioassays and GC-MS chemical analyses, respectively. Telenomus podisi were reared in laboratory on Euschistus conspersus (Heteroptera: Pentatomidae) eggs and the kairomonal activity observed with a Y-tube olfactometer. Females were given a choice between a synthetic aldehyde blend (5 µl=0.1 ng) or egg extract (5 µl=4.5 eggs equivalent), all contrasted with filter paper with the same volume of hexane (control). Olfactometer data were analyzed by χ2-tests (PH. halys egg extracts, and their identities were confirmed with a synthetic aldehyde mixture. Females of T. podisi, a native parasitoid in California, were significantly repelled to both natural egg extract and to synthetic C16,18,20 aldehyde blend.

169

LESSONS FROM THE FIRST INTERNATIONAL COOPERATIVE BIODIVERSITY GROUP (ICBG) IN BRAZIL ON MICROBIAL SYMBIONTS OF INSECTS.

Monica T. PUPO1*; Cameron R. CURRIE2; Jon CLARDY3

1Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São PauloRibeirão Preto,, Ribeirão Preto, SP, BRAZIL 2Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

The International Cooperative Biodiversity Group (ICBG) program is supported by the Fogarty International Center (FIC)/National Institutes of Health (NIH) to address the interdependence of biodiversity exploration for potential applications in health, with investments in research capacity that support sustainable use of these resources, The knowledge to conserve them, and equitable partnership frameworks among research organizations in the U.S. and low- and middle-income-countries.1 For the first time Brazil takes part in an ICBG project with co-support from the São Paulo Research Foundation (FAPESP).The Brazil-USA ICBG aims to discover novel therapeutic agents from bacterial symbionts of insects. The project aligns ecological function of bacterial natural products with their possible therapeutic applications, rationally increasing the chances of discovering new drug candidates. During this presentation I will discuss the rationale of the ongoing ICBG, some initial results, the importance of this collaboration for the Brazilian group as well as the legal steps involved to set up this cooperative group on biodiversity studies.

Finnacial support: FIC/NIH Grant U19TW009872; FAPESP Grant 2013/50954-0

1 http://www.fic.nih.gov/programs/pages/biodiversity.aspx

170

THE PROTECTIVE ROLE OF FOLIAR ENDOPHYTIC FUNGI IN EMBOTHRIUM COCCINEUM (PROTEACEAE).

Marcia GONZÁLEZ-TEUBER1*

1Universidad de La Serena, La Serena, La Serena, CHILE

Fungal endophytes colonize living internal tissues of plants without causing any visible symptoms of disease. Endophytic fungi associated with healthy leaves can play an important role in the protection of hosts against herbivores and pathogens. In the present study, the diversity of foliar endophytic fungi (FEF) of the southern temperate tree Embothrium coccineum (Proteacea), as well as their role in plant protection in nature and in planta was determined. Fungal endophytes were isolated from 40 asymptomatic leaves by the culture method for further molecular identification of the 18S rRNA gene. A relationship between FEF frequency in plants (evaluated with real-time PCR analyses) and plant protection in nature was evaluated in juveniles of E. coccineum. Additionally, different fungal endophytes were inoculated in seedlings of E. coccienum in order to evaluate their protective role against a bacterial pathogen. A total of 178 fungal isolates were obtained and identified, with sequence data revealing 34 different operational taxonomic units. Only a few common taxa dominated the fungal endophyte community, whereas most taxa qualified as rare. A significant positive correlation between plant protection (evaluated as the percentage of leaf damage) and FEF frequency was found in nature. In planta, endophytes inhibited effectively the growth of Pseudomonas syringae. Data here show a relatively high diversity of fungal endophytes associated with leaves of E. coccineum, and suggest that fungal endophytes in leaves contribute to better plant protection in nature.

171

TRANSGENERATIONAL EFFECTS OF TUBER HERBIVORY ON POTATO COMPENSATION AND RESISTANCE.

Katja POVEDA1*; Etzel GARRIDO1; Pavan KUMAR1; Erandi VARGAS1; Georg JANDER1

1Department of Entomology, Cornell University, Ithaca, NY, USA

The maternal environment of a plant has been proposed to influence the phenotype of its offspring. Evidence for this hypothesis comes from plant-insect interactions, where the exposure of maternal plants to herbivores induces offspring to express a more resistant phenotype. In addition to resistance traits, plants are known to express compensatory traits in response to herbivore damage. However, it remains unknown whether herbivory in the parental generation could also affect these compensatory traits in the progeny. In one Colombian potato variety (Solanum tuberosum L. cv Pastusa Suprema) we previously found that plants attacked by Guatemalan potato moth (Tecia solanivora, Povolny) larvae produce up to a two-fold higher yield than undamaged plants, providing a model case of herbivore-mediated overcompensation responses. To test if herbivory in the parental generation change the compensatory response and resistance of the progeny, we exposed potato progeny from control and damaged parents to the tuber moth. We found that while tuber herbivory in the parental generation leads to compensatory responses in the progeny, independent whether the progeny were exposed to herbivores or not, it also induced their susceptibility, being the most resistant plants those that did not experience damage in either generation. We also found that the amount of sugars in the stolon was lower in the progeny of damaged parents, indicating that there could be differential allocation of sugars in compensating plants. Measurements of proteinase inhibitors and secondary metabolites, including solanine and chlorogenic acid, in the progeny, did not provide a clear mechanism for the induction of susceptibility in these plants. We show, for the first time, that herbivore pressure in the parental generation induce compensatory responses in the progeny even in the absence of herbivores, suggesting that yields could increase by manipulating the induced responses of the parental generation when producing seed tubers.

172

THE SEX PHEROMONE OF THE CITROPHILUS MEALYBUG: FROM IDENTIFICATION TO THE DEVELOPMENT OF APPLICATIONS IN PEST MANAGEMENT.

Jan BERGMANN1*; M. Fernanda FLORES1; Alda ROMERO2; M. Soledad OYARZUN2; Tania ZAVIEZO2; Vaughn BELL3; Ashraf El-SAYED3; Rikard UNELIUS3; David M. SUCKLING3

1Instituto de Química, Pontificia Universidad Católica de Valparaíso, Valparaíso, Valparaíso, CHILE 2Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Santiago, Santiago, CHILE 3The New Zealand Institute for Plant and Food Research Limited, Hastings, Hastings, NEW ZEALAND

The citrophilus mealybug, Pseudococcus calceolariae (Maskell) (Hemiptera: Pseudococcidae), is a pest of fruit crops in many regions of the world. Recently, its sex pheromone has been identified and synthesized by our teams based in New Zealand and Chile. Following this joint effort, field experiments were carried out independently in both countries, envisaging the use of the pheromone in agricultural applications. In Chile, experiments were carried out to assess the potential of the pheromone in monitoring and control by mating-disruption, while in New Zealand the focus was on the development of mass trapping. This presentation will summarize the joint work on the citrophilus mealybug pheromone and highlight recent advances towards its applications.

173

ONTOGENETIC AND GEOGRAPHIC VARIATION IN CYANOGENIC DEFENSES AGAINST HERBIVORES OF TURNERA VELUTINA.

Karina BOEGE1*; Paulina ZEDILLO-AVELLEYRA1; Sergio SORIA-TAPIA1

1Instituto de Ecología. Universidad Nacional Autónoma de México, México, México, México

Plant defense has traditionally been studied at particular developmental stages of plants. However, in the past decades evidence has accumulated demonstrating that ontogenetic trajectories in plant defense are common in nature. To further investigate the mechanisms and factors influencing the evolution of ontogenetic changes in plant defense, we have studied the tropical myrmecophitic shrub Turnera velutina, in which we have previously reported high contents of cyanogenic compounds early during ontogeny and an absence of these compounds later when plants reach maturity. We have also described how these ontogenetic trends seem to be correlated with simultaneous changes in other plant defenses: as chemical defense decrease, physical and biotic defense increase. In the current work we describe a detailed ontogenetic trajectory (based on the number of leaves of each plant within different genetic families) in the production of cyanogenic compounds. We report that rather than a discrete on/off switch in the production of these compounds, there is a steady decrease in their concentration from the cotyledon stage, where the maximum contents were observed, to the 10th-leaf stage, when a two-fold drop in the production of cyanogenic compounds was observed reaching close to zero values. We suggest that at this ontogenetic stage a down-regulation of the cyanogenic metabolism occurs and as a consequence, this defense is absent at older stages. However, we also report genetic and geographic variation of these trajectories, as well as the herbivore and ant communities associated with such variation. Interestingly, we found geographic variation in the ontogenetic trajectories of different defenses, with some populations maintaining a cyanogenesis at mature stages. We suggest that, indeed, the ecological context and the effectiveness of ant defenders seem to play an important role in the evolution and variation of ontogenetic trajectories in plant defense.

174

OTHER

A PORTABLE SYSTEM FOR PLANT VOLATILES COLLECTION.

Milton F. CABEZAS-GUERRERO1*; Luiza C. F. ZAZYCKI1; Natalia NARANJO- GUEVARA1; José M. S. Bento1

1Laboratory of Chemical Ecology and Insect Behavior, Department of Entomology, Luiz de Queiroz College of Agriculture (ESALQ), University of São Paulo, Piracicaba, São Paulo, BRAZIL

Choosing a method for plant volatile analysis requires an attempt for compiling the authentic chemical profile emitted by a plant. The choice for an adequate system for collection and analysis of plant volatiles usually depends both on the biological context and the plant material to be investigated. Up to now many studies have been conducted with plants grown in greenhouses, but it is becoming more important to investigate the volatiles emitted by plants under field conditions, since they are in their natural physiological state. Thus, we developed a portable system for outdoor sampling of volatile compounds. Maize plants were infested with Spodoptera frugiperda (Lepidoptera: Noctuidae) for 12 hours. Then, the volatile profiles were obtained by two dynamic headspace methods, operating at 1 L.min-1(pushing) and 0.8 L.min-1(pulling): portable and volatile collection system (ARS®). Subsequently the volatiles were analyzed by GC/FID and compared. The compounds profiles found on infested-maize plants were similar for both systems. In conclusion, the portable method is a reliable and practical method to obtain plant volatiles in the field conditions.

176

A NEW APPROACH TO INVESTIGATING DIVERSE PHYTOCHEMICAL PROFILES AND ECOLOGICAL INTERATIONS.

Lora RICHARDS1*; Celso OLIVEIRA2; Lee DYER2; Craig DODSON2; Christopher JEFFREY3

1University of Nevada, Reno, Reno, Nevada, UNITED STATES 2University of Nevada, Reno, Reno, Nevada, UNITED STATES 3UNIVERSITY OF NEVADA, RENO | Cidade: Reno, Reno, Nevada, UNITED STATES

Since Ehrlich and Raven’s (1964) influential paper on the coevolution between plant and herbivore, many studies have focused on understanding the causes and consequences of phytochemical mixtures on ecological interactions. However the challenge remains in deciphering the ecologically and biologically important compounds from diverse phytochemical mixtures. Using a network based approach, we analyzed binned 1H-NMR data from 200 artificial mixtures of commonly studied secondary metabolites including, alkaloids, amides, terpenes, iridoid glycosides, saponins, phenylpropene, flanvanoids and phytosterols. Within these mixtures we manipulated multiple dimensions of chemical diversity, including molecular complexity, mixture complexity and differences in the relative concentrations of each compound. Our analyses guided identification of modules, or clusters of chemical shifts within the network that were associated with specific compounds and shared compound structures with in the mixtures. This approach was then applied to crude extracts of 20 species in the phytochemically diverse tropical plant genus Piper. Using these extracts in multiple organism bioassays enabled us to identify a prenylated benzoic acid from these mixtures that exhibits antifungal properties. In addition, we identified small structural changes that had a large effect in the antifungal bioactivity. In sum, this approach allowed us to combine chemical and ecological datasets and identify potential biologically important compounds from crude extracts. We continue to use this approach to elucidate the causes and consequences of phytochemical mixtures on ecological interactions.

177

BEHAVIOURAL CONSIDERATIONS ON THE IDENTIFICATION OF COPITARSIA UNCILATA UNDER LABORATORY CONDITIONS.

Hugo Fernando RIVERA-TRUJILLO1*; Pilar ALTAMAR-VARON2; Daniel RODRÍGUEZ-CAICEDO2; Nancy BARRETO-TRIANA3

1Colombian Corporation of Agricultural Research, Tibaitatá, Mosquera, COLOMBIA 2Nueva Granada Military University, Cajica, Cajica, COLOMBIA 3Colombian Corporation of Agricultural Research, Tibaitatá, Mosquera, COLOMBIA

Basic studies on reproductive behavior support taxonomical defferentiation between species and provide critical information for the implementation of pest control programs based on the use of semiochemicals. In Colombia, molecular, chemical and behavioral studies, that help on the identification of Copitarsia uncilata are needed to accurately manage insect pest populations in economically important crops. The aim of this research was to study the copulation behavior of C. uncilata under laboratory conditions. The experimental setup was conducted at 20.6°C, 60% RH, and photoperiod of 12:12 L:D, where a small colony was stablished from field-collected eggs and larvae. The specimens were reared on artificial diet and, once they reached the pupa stage, they were sexed. Sixty one male-female adult pairs were confined in 300 ml plastic cages. The behavior of each pair was recorded every 10 minutes and the eggs layed were counted daily for 8 days. We found that C. uncilata sexual activity was concentrated towards the first third of the scotophase. The highest rate of calling behavior occurred on the second day after adult emergence. Oviposition was maximum on day seven, when it prevailed over mating, despite the courtship of males and their repeated attempts to mate. Oviposition period had its peak on the fourth day after emergence. The information found in this study provides ethological information that can be used for both future assessments on the efficacy of formulated pheromone mixtures for C. uncilata control programs, and taxonomical differentiation between nearby species. Project funded by Research Vice- Rectory of Nueva Granada Military University UMNG-CIAS 1570 validity- 2015.

178

CITRUS VOLATILE COMPOUNDS AS PUTATIVE KAIROMONES MEDIATING DIAPHORINA CITRI OVIPOSITION.

María Eugenia AMORÓS1; Virginia PEREIRA DAS NEVES2; José BUENAHORA2; Carmen Rossini3*

1Facultad de Química, UdelaR, Montevideo, URUGUAY 2Instituto Nacional de Investigación Agropecuraria, INIA SG, Salto, URUGUAY, Salto, Salto, Uruguay 3Facultad de Química, UdelaR, Montevideo, URUGUAY, Montevideo, Montevideo, Uruguay

Huanglongbing, currently the most destructive citrus disease worldwide, is dispersed by the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae). Management programs have concentrated on vector control [1]. During host plant search, ACP can integrate information from flushing shoots including visual and odor cues [2]. The objective of this study was to contribute to the knowledge of volatile organic compounds (VOCs) that could potentially be used in behavior management of this insect; and to establish a possible correlation between VOCs' profile emissions and insect host preference. An oviposition preference greenhouse experiment was carried out using 6 citrus species: Citron (Citrus medica), Eureka lemon (Citrus limon), Cravo lemon (Citrus limonia), Rough lemon (Citrus jambhiri), Sweet orange (Citrus sinensis) and Duncan grapefruit (Citrus paradisi). In vivo VOCs´ emissions were also collected from these hosts using aeration chambers (72h; N=5-6/species) and adsorbing the products on HaySep-Q (50 mg, 80/100 mesh). VOCs were analyzed by GC-MS. The results showed an oviposition preference of D. citri for Duncan grapefruit, Rough lemon and Sweet orange. Quantitative and qualitative differences were also observed among VOCs profiles (MANOVA, p D. citri.

1. Grafton-Cardwell, E.E., L.L. Stelinski, and P.A. Stansly, Biology and management of Asian citrus psyllid, vector of the huanglongbing pathogens, in Annual Review of Entomology. 2013. p. 413-432.

2. Wenninger, E.J., L.L. Stelinski, and D.G. Hall, Roles of olfactory cues, visual cues, and mating status in orientation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) to four different host plants. Environmental Entomology, 2009. 38(1): p. 225-234.

179

CARDENOLIDES AS A MODEL FOR THE EVOLUTION OF MULTITROPHIC INSECT-PLANT INTERACTIONS.

Georg PETSCHENKA1*

1Institute for Insect Biotechnology, Giessen, Hesse, GERMANY

Classic coevolutionary theory predicts bitrophic interactions between insect herbivores and their hostplants. In the scenario of a reciprocal arms-race, plants evolve defensive traits and insects develop countermeasures to use defended plants as a dietary resource. We have studied insect adaptations to plant cardenolides in several parallel systems such as milkweed butterflies (Danaini), milkweed bugs (Lygaeinae), and leaf miner flies (Agromyzidae). With mechanistic physiological approaches including studies of the molecular target site as well as tissues and whole organisms, we have dissected the complexity of the insect resistance syndrome. Our work revealed a high level of convergence on the one hand, but also evolutionary lability on the other. In the course of our studies we realized that using a toxic diet is not the only evolutionary driver of cardenolide resistance in insects. In fact, additional selective pressures such as sequestration of cardenolides as a defense against predators led to the evolution of specific adaptations which are different from those required to cope with dietary toxins. Our work thus revealed that sequestration can spur coevolutionary escalation and implies that the third trophic level (predators and parasitoids) is an important driver of insect resistance to host plant toxins. Sequestration of plant toxins may even cause specific insect-plant associations which are driven by the availability of plant toxins. Specialization and sequestration thus form an important mechanistic bridge connecting the third and the first trophic level in coevolutionary interactions between insects and their host plants.

180

CHEMICAL ECOLOGY AS PLANETARY MEDICINE.

Mark HAY1*

1Georgia Institute of Technology, Atlanta, GA, USA

Chemical cues and signals provide a blue-print of, or instruction manual for, biotic interactions. Just as chemical biologists discovered treatments for diseases by understanding cell signaling, chemical ecologists can provide new insights into population biology and community organization, and new option for curing environmental collapse by understanding chemically-mediated interactions. Coral reefs provide a dramatic example. Reefs are in global decline. In recent decades, coral cover has declined by 80% throughout the Caribbean and 50%+ throughout the tropical Pacific, with seaweeds commonly replacing corals. Much of the decline and lack of recovery can be attributed to alterations in fundamental biotic interactions that are mediated via bioactive secondary metabolites. Field experiments demonstrate that biodiversity of herbivorous fishes is critical for suppressing chemically-defended seaweeds that damage corals via allelopathic toxins, because different fishes are differentially susceptible to seaweed defenses. Of equal importance is how larvae of both coral and fishes respond to chemical cues from overfished areas dominated by seaweeds versus no-take marine protected areas dominated by corals. Chemical cues from these areas create feedbacks, locking each area into its respective state; once reefs degrade past a critical point, they may not recover due to the chemical cues they generate. Recruiting fish and coral larvae chemically sense and are attracted to coral-dominated areas protected from fishing while being chemically repulsed by seaweed-dominated areas that are overfished. Attraction and repulsion are cued by odors from specific corals and seaweeds that best predict reef quality. These, and other, chemically-cued behaviors can close the open nature of marine populations, suppress larval export from protected areas to degraded reefs, and prevent recovery of coral and fish populations once reefs degrade. Translating this “chemical language of life” provides novel options for ecosystem restoration and for restoring the ecosystem services upon which we all depend.

181

COMBINED USE OF HERBIVORE-INDUCED PLANT VOLATILES AND SEX PHEROMONES FOR MATE LOCATION IN BRACONID PARASITOIDS.

Hao XU1; Gaylord DESURMONT1; Thomas DEGEN1; Guoxin ZHOU2; Diane LAPLANCHE2; Henryk LUKA3; Ted TURLINGS3*

1University of Neuchâtel, Neuchâtel, Neuchâtel, SWITZERLAND 2Zhejiang A&F University, Hangzhou, Zhejiang, CHINA 3Research Institute of Organic Agriculture (FiBL), Ackerstrasse, Frick, SWITZERLAND

Herbivore-induced plant volatiles (HIPVs) are important cues for female parasitic wasps in search of hosts. Here we investigated the possibility that HIPVs may also serve parasitoids as cues to locate mates. To test this, the odour preferences of four braconid wasps, the gregarious parasitoid Cotesia glomerata (L.) and the solitary parasitoids Cotesia marginiventris (Cresson), Microplitis rufiventris Kokujev, and Microplitis mediator (Haliday), were studied in four-arm and six-arm olfactometers. All four parasitoid species showed attraction to pheromones, but each in a somewhat different way. Males of the two Cotesia species were attracted to virgin females, and females of M. rufiventris were attracted to virgin males. Males and females of M. mediator exhibit attraction to both sexes. Importantly, females and males of all four species were strongly attracted by HIPVs, independent of whether they were virgin or not. In most cases, males also showed significant attraction to the odour of intact plants. The parasitoids generally preferred the combination of HIPVs and pheromones over plant odours alone, except in the case of M. mediator, which appears to mainly use HIPVs for mate location. We discuss the different ecological contexts in which the use of pheromones and HIPVs for mate location in parasitoids can be expected. To our knowledge, this is the first study to show that braconid parasitoids use HIPVs and pheromones in combination to locate mates.

182

COMMUNICATION INTERFERENCE BETWEEN NON-RELATED SYMPATRICALLY OCCURRING NOCTUIDAE MOTH SPECIES.

María Laura JUÁREZ1*; Patricia FERNÁNDEZ2; María Josefina RUÍZ2; Lucía GOANE2; María Elvira VILLAGRÁN3; Osvaldo ARCE4; Paula Gabriela Páez JEREZ4; Astrid GROOT5; María Teresa VERA5

1Cátedra de Terapéutica Vegetal-Facultad de Agronomía y Zootecnia- Universidad Nacional de Tucumán / CONICET, Tucumán, Tucumán, ARGENTINA 2Estación Experimental Agropecuaria Delta del Paraná, INTA/CONICET, Buenos Aires, Buenos Aires, ARGENTINA 3Estación Experimental Obispo Colombres, Las Talitas, Tucumán, ARGENTINA 4Facultad de Agronomía y Zootecnica, Universidad Nacional de Tucumán, San Miguel de Tucumán, Tucumán, ARGENTINA 5Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, Amsterdam, THE NETHERLANDS

In moths, female-emitted sex pheromones allow the attraction of conspecific males. Specificity is achieved by a combination of unique components in specific ratios and sometimes also by interspecific behavioural antagonists. Heterospecific pheromone compounds that cause inhibition of male upwind flight has been detected in a few cases between taxonomically unrelated species. In Northwest Argentina, three economically important pest species co-occur sympatrically on soybean crop: Chrysodeixis includens (Walker), Rachiplusia nu (Guenée) and Anticarsia gemmatalis (Hübner). Pheromonal compounds are known for C. includens and A. gemmatalis but not for R. nu. Given the possible practical application of using antagonistic behaviour for pest management, we evaluated the response of C. includens males to its own pheromone in presence of the two pheromone components of A. gemmatalis (Z3Z6Z9-20Hy and Z3Z6Z9-21Hy). We also explored the response of A. gemmatalis and R. nu to these blends. Field experiments were conducted with traps baited with a synthetic blend that mimics the pheromone of C. includens and different ratios of the pheromone of A. gemmatalis. Experiments were set up in two soybean commercial fields in Argentina. The addition of Z3Z6Z9-20Hy and Z3Z6Z9-21Hy inhibited the catches of C. includens males by its own pheromone in one field but not in the other field. For A. gemmatalis, we found no inhibitory effect of the compounds of C. includens pheromone. Interestingly, for R. nu, we found a dose- dependent response. In both fields, the C. includens blend with high concentrations of Z3Z6Z9-20Hy and Z3Z6Z9-21Hy significantly reduced R. nu males’ captures. Our results showed an antagonistic effect of heterospecific sex pheromone compounds of A. gemmatalis in C. includens and R. nu. This inhibitory effect between taxonomically distant species can play a role in avoiding competition. To our knowledge, this is the first report on the attraction of R. nu to specific pheromonal blends.

183

CONTEXTUAL PERCEPTION AND RECOGNITION OF HOST-RELATED ODOR BLENDS - A CASE STUDY OF OLFACTORY RESPONSE IN THE PARASITOID, MICROPLITIS CROCEIPES.

Henry FADAMIRO1; Tolulope MORAWO1*

1Auburn University, Auburn, Alabama, USA

Herbivores emit complex blends of volatile organic compounds (VOCs) after feeding on plants. These plant-related VOCs can be used by parasitoids to locate their hosts. However, there is more to be understood about perception and recognition of host-related odor blends in parasitoids. Microplitis croceipes (Hymenoptera: Braconidae), a larval parasitoid of Heliothis virescens (Lepidoptera: Noctuidae) was used as model species to identify key compounds mediating attraction of parasitoids to plant-fed hosts. We used a combination of techniques to test the hypothesis that only a fraction of the blend is key to attraction of parasitoids. Comparative GC/MS analyses of cotton-fed versus artificial diet-fed hosts indicated that 10 of 15 compounds in the headspace of H. virescens larvae were exclusive to plant-fed hosts. Parasitoids showed varying EAG responses to synthetic versions of test compounds, indicating differences in biological activity at the antennal level. In order to identify key attractive compounds, full blend of 15 compounds was modified by removing each of the 10 plant-related compounds emitted by host larvae. In a series of Y-tube olfactometer bioassays testing parasitoid responses to modified blends, 1-octen-3-ol and decanal were identified as key compounds contributing to attractiveness of odors emitted by cotton-fed hosts. However, the relevance of key components was dependent on dose and context of other components, suggesting the importance of contextual perception of odor blends. α-Pinene, β-caryophyllene, α-humulene, α- farnesene and β-pinene were also important for parasitoid attraction, suggesting a ratio- specific odor recognition pattern in M. croceipes. The results showed that while various host-related compounds act in concert to serve as useful host location cues, only a fraction of the natural blend mediates attraction in parasitoids. Furthermore, the role of a compound is better assessed in the context of other components, and odor blends are better perceived as a whole rather than as individual components.

184

DIFFERENCES IN SEX PHEROMONE PRODUCTION BY SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) FEMALES RESISTANT AND SUSCEPTIBLE TO BT CORN.

Laura MACHUCA-MESA1*; Jeremy N. MCNEIL2; Janice LOPES2; Eliseu J. G. PEREIRA2; Eraldo LIMA2

1UNIVERSIDADE FEDERAL DE VIÇOSA, Viçosa, Minas Gerais, BRAZIL 2WESTERN UNIVERSITY, London, Ontario, CANADA

The development of resistance in insect pests threatens the effectiveness of transgenic crops expressing Bt toxins. In Brazil, the fall armyworm, Spodoptera frugiperda, has developed resistance to Bt corn. In the present study we assessed the sex pheromone titers in resistant and susceptible females, as part of a project investigating the possibility that costs associated with resistance affect the pheromone mediated reproductive behavior of this species. The working hypothesis is that if there is a cost to resistance then these females would have lower pheromone titers that susceptible individuals. The pheromone titers of susceptible and resistant virgin females were quantified as a function of age (1, 3, 5 and 7 day old) at the 3rd, 6th and 9th hrs of the 10h scotophase. The pheromone titers of both susceptible and resistant females changed as a function of age and time during the scotophase. However, contrary to our hypothesis, resistant females produce significantly higher concentrations of (Z)-9- tetradecenyl acetate, the major pheromone component. These findings will be discussed within the context of resistance management strategies.

185

DYNAMICS AND ORIGIN OF CYTOKININS INVOLVED IN PLANT MANIPULATION BY A LEAF MINING INSECT.

Hui ZHANG1; Géraldine DUBREUIL1; Antoine GUIGUET1; Elisabeth HUGUET1; Neil EMERY2; Anna KISIALA2; Peter ANDREAS2; Radomira VANKOVA3; David GIRON4*

1Institut de Recherche sur la Biologie de l’Insecte, Tours, Indre et Loire, FRANCE 2Department of Biology, Trent University, Peterborough, Ontario, CANADA 3Institute of Experimental Botany, Prague, Central Bohemia, CZECH REPUBLIC 4Institut de Recherche sur la Biologie de l’Insecte, Tours, Indre et Loire, FRANCE

Cytokinins are key phytohormones in many plant-insect interactions especially for plant manipulating insects. Biochemical and transcriptional changes in plant cells in contact with Phyllonorycter blancardella show that CKs are accumulated in mined areas and CKs are not produced by the plant. However, the origin and dynamics of CKs are still ambiguous. In order to fill this gap, we identified and quantified all types of CKs in green and yellow apple leaves in contact with the leaf-miner, including types of CKs that are specifically produced by symbionts (methylthio-CKs). Previous studies have demonstrated that insect bacterial symbionts are instrumental in this plant-insect interaction. HPLC-(ESI+)-MS/MS analysis revealed that there were four classical types of CKs (tZ, cZ, iP, DZ type CKs) but also their 2-methylthio-derivatives (2MeSZ and 2MeSZR) in both green and yellow leaves. Presence of bacteria-specific CKs comforts previous results suggesting that insect bacterial symbionts contribute to the observed phenotype. However 2MeSZR were found in higher quantities in mines on green leaves but in lower amounts in mines on yellow leaves in comparison to their respective controls. Temporal distribution show that mined areas are enriched in CKs both on green and yellow leaves. Composition differs between mines on green and yellow leaves with a larger amount of active forms in mine on yellow leaves. Spatial distribution of CKs within the leaf suggests the absence of CK translocation towards the insect feeding area, reinforcing our hypothesis that CKs accumulated in the mined area originate from the insect itself. The strong reprogramming of the plant phytohormonal balance by the leaf- miner is associated with increased nutrient mobilization, inhibition of leaf senescence and mitigation of plant direct and indirect defense. We currently investigate the consequences of the plant manipulation in terms of plant indirect defenses by comparing the volatile compounds before and after the leaf-miner infestation. .

186

EFFECT OF SILICON AND METHYL JASMONATE ON LARVAL PERFORMANCE OF SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) IN RICE PLANTS.

Amanda NASCIMENTO1*; Franscinely ASSIS1; Jair MORAES1

1Universidade Federal de Lavras, Lavras, MInas Gerais, Brazil

The caterpillar Spodoptera frugiperda (Lepidoptera: Noctuidae) is a polyphagous insect that significantly damages rice crops. Elicitation of plant defense may be a potential alternative for the ecological management of herbivore insects. Therefore, the aim of this study was to evaluate the performance of S. frugiperda larvae fed on rice plants treated with silicon (Si) and methyl jasmonate (MeJA). A completely randomized experimental design with five treatments and ten replicates was used as: T1- control (no Si and MeJA application), T2- tween 60, T3- Si in the soil (1%), T4- MeJA foliar application (1mM), T5- Si in the soil (1%) + MeJA foliar application (1mM). Silicic acid was used as silicon source. Fourth-instar larvae were not fed for 24 hours. Thereafter, the initial weights were measured, and the larvae were individually placed in Petri dishes and fed on rice leaves from the treatments. After 24, 48 and 72 hours, larva survival and leaf consumption were evaluated. Insects were again starved for 24 hours and their final weights were determined to estimate the biomass gain. Afterward, another experiment was carried out, in which the larvae were kept on rice plants instead of Petri dishes, following the same methodology above; the aim was to compare survival and biomass gain between experiments. Leaf consumption was lower when larvae fed on rice leaves treated with Si and/or MeJA. Larvae fed on intact plants, or Si and/or MeJA treated leaves showed lower biomass gain. However, there was not significant reduction in insect survival in both experiments. Thus, the tested methods did not differ between them for the analyzed variables. It is possible to conclude that defense induced by Si and/or MeJA on rice plants may cause adverse effects on larval performance of S. frugiperda. Enzymatic activity associated with plant defense will be analyzed in future studies.

187

ESSENTIAL OILS FROM SCHINUS POLYGAMA AND BACCHARIS SPARTIOIDES ENHANCE MATING COMPETITIVENESS OF ANASTREPHA FRATERCULUS MALES.

Flavia JOFRE BARUD1; M. Laura JUAREZ2; M. Josefina RUIZ2; Lucia GOANE2; Gabriela VALLADARES2

1Instituto de Biotecnología-Facultad de Ingeniería-Universidad Nacional de San Juan / CONICET, San Juan, San Juan, ARGENTINA 2Cátedra de Terapéutica Vegetal-Facultad de Agronomía y Zootecnia- Universidad Nacional de Tucumán / CONICET, Tucumán, Tucumán, ARGENTINA

The sexual performance of mass-reared males is a key factor for the success of the Sterile Insect Technique (SIT). For the Mediterranean fruit fly, Ceratitis capitata (Wied.), mating competitiveness is increased through the exposure of males to the volatiles of ginger root oil (GRO) and operational programs that release sterile insects implemented its use. In previous studies with essential oils of native species, we showed that the essential oils of Schinus polygama (Cav.) Cabrera and Baccharis spartioides (Hook. & Arn.) Remy confer a mating advantage over non-exposed males. The objective of this study was to evaluate whether the same effect is observed for the South American fruit fly, Anastrepha fraterculus (Wied.). Males from a laboratory colony of A. fraterculus were exposed to the volatiles of the essential oils of S. polygama, B. spartioides and GRO. For each essential oil, one exposed male and one non-exposed male were caged with a virgin female in a mating arena. The number of matings pairs obtained by each type of male, the latency to mate and the duration of the copula were recorded for each treatment. Results showed that the essential oil of S. polygama conferred a mating advantage to A. fraterculus males over non-exposed males (65.1 % vs 34.9%, respectively, p=0.0020).A similar effect was observed for the B. spartioides essential oil (65.7 vs 34.2%, p= 0.0078). GRO did not increase the number of mating pairs (p > 0.05). No significant differences were observed for latency and copula duration between exposed and non-exposed males in any of the treatments. This is the first time that essential oils from native plants are reported to sexually enhance A. fraterculus males. Results are important in the context of the sexual behaviour of A. fraterculus as well as in the development of the SIT for this species.

188

EFFECT OF STARVATION ON THE OLFACTORY RESPONSES OF THE GRAPE WEEVIL, NAUPACTUS XANTHOGRAPHUS (COLEOPTERA: CURCULIONIDAE).

Waleska VERA1*; Leonardo PARRA2; Andrés QUIROZ2; Jan BERGMANN2

1Pontificia universidad Católica de Valparaiso, Valparaiso, Valparaiso, Chile 2UNIVERSIDAD DE LA FRONTERA, TEMUCO, CHILE, CHILE

The grape weevil, Naupactus xanthographus, is a polyphagous insect present in some countries of South America, which causes considerable damage in different economically important species, such as grape, avocado, and apple. In this work, we studied the feeding performance of grape weevil on leaves of grape and avocado and evaluated the attraction to volatiles of both host plants. Feeding performance bioassays were realized with insects deprived of food 24 h before the assays (starved insects). The insect was placed in a Petri dish and provided with a grape or avocado leaf. The weight difference of the insects was determined after 1 h and 6 h of feeding, respectively. Constitutive volatiles of grape and avocado plants, respectively, were collected for 24 h. The olfactory responses to these volatiles were determined using a Y-tube olfactometer. Our results indicate that after 1 h of feeding, insects showed no significant differences in weight increase. However, after a period of 6 h of feeding, males had continued feeding on grape leaves, but not on avocado leaves. Bioassays using a Y-tube olfactometer showed that males are attracted to volatiles of both host plants. Furthermore, starved males and females showed no preference when volatiles of grape and avocado were presented simultaneously. However, non-starved males and females preferred grape volatiles over avocado volatiles. We concluded that the nutritional state is an important factor in the olfactory response of N. xanthographus to volatiles of its host plants.

References.

Ripa R, et al. (2010). Colección libros INIA N° 26. Chile.

Reisenman C, et al. (2013). J. Insect Physiol 59:717-721.

189

EXTRAFLORAL NECTAR VOLATILES AFFECT ANT CHOICE BEHAVIOR.

Adrienne GODSCHALX1*

1Portland State University, Portland, OR, USA

Facultative ant-plant mutualisms are widespread in nature. Ants provide efficient plant defense in exchange for resources such as extrafloral nectar (EFN). However, less is known about the chemical signals exchanged between plants and the defending ants. Combining EFN headspace volatile data and an ant bioassay, we show for the first time the existence of scented EFN and that ants use olfactory cues to locate their EFN food source. We designed a custom petri dish arena with an elevated screen to expose ants (Lasius niger) to volatile space above three droplets without contacting them. We conducted two series of choice experiments offering either a set of i)water, aqueous sugar solution (sucrose), and EFN droplets from a single plant species (either Phaseolus lunatus, Passiflora edulis, or Prunus avium; all facultative ant-plants), or ii)EFN from all three different source plants simultaneously. To compare different EFN scents, we used solid phase micro-extraction (SPME) and time-of-flight mass spectroscopy, (GC- TOFMS) to characterize the volatile organic compounds (VOCs) from EFN from each species. We found diverse VOC profiles among the three EFNs. Each EFN significantly attracted ants when compared to water or sucrose. However, when comparing EFN from the different plant species, ant responses to all three EFN sources depended on the plant from which ants were collected. Ants collected from P.lunatus, P.edulis, and P.avium plants spent 4.7, 2.6, and 6.02 times longer above EFN of their original host plant compared to the second most attractive EFN. Naïve ants collected from colonies not in contact with any of the plant species were most attracted to P.edulis EFN relative to the other two nectar sources. To the best of our knowledge this is the first study showing that plant volatiles directly released from EFN affect choice behavior of ants and thus play an important role in facultative ant-plant mutualisms.

190

GENERALIST PIERCING-SUCKING INSECT DAMAGE CHANGE VOLATILE PROFILE IN CORN SEEDLINGS.

Vanesa Gisela JACOBI1; Patricia Carina FERNANDEZ2; María Victoria COLL ARAOZ3; Jorge Alberto ZAVALA3*

1INBA - CONICET, Ciudad Autónoma de Buenos Aires, Buenos Aires, ARGENTINA 2Estación Experimental Agropecuaria INTA Delta del Paraná - CONICET, Ciudad Autónoma de Buenos AiresBuenos Aires, Buenos Aires, ARGENTINA 3PROIMI- CONICET, San Miguel de Tucuman, Tucuman, ARGENTINA

The emission of volatile organic compounds (VOCs) represents a source of chemical information that could be used by herbivorous insects as cues in selection and acceptance of host plants. Corn (Zea mays) seedlings emit a cocktail of volatile compounds that change under herbivore attack. Volatiles emitted by corn after damage of chewing insects are widely characterized in literature, but there is not information about piercing-sucking insects, such as stink bugs. Dichelops furcatus (Hemiptera: Pentatomidae) is a generalist neotropical stink bug that recently became a pest of corn in Argentina because it feeds an kills seedlings in the field. Here we studied D. furcatus feeding preference between two commercial corn hybrids, P1780YR (temperate) versus P30B39HR (tropical), and characterized its VOCs emission from damaged and undamaged seedlings. First, seedlings of both corn hybrids were offered to adult females of D. furcatus to assess feeding preference. Observations of feeding behavior were recorded every half an hour for 2,5hs. Volatiles released by damaged and undamaged plants were collected and analyzed by coupled gas chromatography-mass spectrometry. Adults of D. furcatus preferred to feed on seedlings with temperate genetic background during the first 2 hours (paired T test; p<0.05). Two hours after the experiment was initiated, insects did not show feeding preference between corn genotypes. After 2 and 24 hours of feeding, analysis of volatiles collected showed significant differences between genotypes in total volatiles emitted and the amounts of Linalool, Ethyl Benzoate, Methyl Salicylate, Beta Ocimene and (+) Cyclosativene between (p<0.05, Mann Whitney U test). These results suggest that corn seedlings respond to stink bugs damage by quali and quantitatively changes in the profile of VOCs emitted. Moreover the early preference found for the temperate genotype seemed to be related to the constitutive volatile compounds, which might have a role as attraction cues.

191

HOW TO MAKE AN EFFICIENT ANT-PLANT MUTUALISM: PARTNER MANIPULATION, PHENOTYPIC ADJUSTMENTS AND HONEST SIGNALLING.

Martin HEIL1*

1CINVESTAV - Irapuato, México, Irapuato, Guanajuato, MÉXICO

Mutualisms require an optimized exchange of rewards and services between the involved partners. We study an ant-plant mutualism in which Acacia plants provide food rewards (food bodies, FBs, and extrafloral nectar, EFN) and nesting space to specific Pseudomyrmex ants, in exchange for a defence service. Here, several mechanisms optimize the exchange of rewards and services. Due to 'ecological fitting', the FBs possess a biochemical composition that makes them 'exclusive rewards'1. The EFN, in turn, inhibits invertase (sucrose-cleaving) activity in the ants. As a consequence of this 'partner manipulation' effect, the ants can only feed on the sucrose-free EFN that is secreted by their host plant2. 'Partner manipulation' is mutual, though, because the mutualistic ants trigger EFN secretion by their host3 via the active manipulation of hormone signalling in the nectaries4. Finally, the co-existence of high-quality and low- quality host species in this genus5 makes partner choice by colony-founding ants a particularly difficult task. We asked whether the plants honestly signal their quality to the ants and found that the ant queens, rather than co-evolved signals, use volatile cues that indicate their host’s status of direct antimicrobial defence6. Due to general trade-offs between different defensive pathways, intensive direct anti-microbial defence indicates low rates of FB- and EFN production, and vice-versa. Classical attempts to understand the stability of mutualisms in spite of the temptation to cheat considered evolutionary mechanisms. Our studies show that mutual phenotypic adjustments represent a further level at which mutualisms can be optimized and, thus, remain the best strategy for the interacting species. 1. Orona-Tamayo, D., et al. (2013) Mol. Ecol. 22, 4087-4100. 2. Heil, M., et al. (2014) Ecol. Lett. 17, 185-192. 3. Orona-Tamayo, D., et al. (2013) Plant J. 73, 546-554. 4. Hernández-Zepeda, O., et al. (in prep.). 5. Heil, M., et al. (2009) PNAS 106, 18091–18096. 6. Razo-Belmán, R., et al. (submitted).

192

HYDROCARBONS INCORPORATED INTO THE CUTICULAR PROFILE THROUGH DIET DO NOT GENERATE AGGRESSION IN ARGENTINE ANTS (LINEPITHEMA HUMILE MAYR).

Nuria MORROW GALLI1; Patrizia D'ETTORRE2; Roxana JOSENS3*

1Laboratorio de Estudio de Insectos Sociales (INSSOC), IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Capital Federal, Buenos Aires, ARGENTINA 2Laboratoire d’Ethologie Expérimentale et Comparée (LEEC), Université Paris 13, Villetaneuse, Sena-Saint Denis, FRANCE 3Laboratorio de Estudio de Insectos Sociales (INSSOC), IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Capital Federal, Buenos Aires, ARGENTINA

Argentine ants are highly invasive in new environments, and commonly overexpress polydomy (neighboring nests show no aggression among them, thus establishing one supercolony). This species is well known to form large supercolonies that can extend for many kilometers in the invasive range. The lack of intraspecific aggression is considered one of the key factors of their success as invaders. Aggression only occurs when an ant recognizes the cuticular profile of another ant as a non-nestmate. In this conceptual framework, we investigated (1) whether eight saturated linear or methyl-branched hydrocarbons (CHs) with 23 to 30 carbon atoms could be incorporated into the cuticle through diet (2); whether the CHs that were effectively incorporated could trigger aggression; and (3) the dynamics of incorporation of the CHs up to 3 days after they were ingested. To identify which CH was incorporated into the cuticle and to analyze their dynamics after consumption, solvent extracts were analyzed via Gas Chromatography– Mass Spectrometry (GC-MS). To test if these CHs could trigger aggression, behavioral tests were performed between treated and untreated ants. We found that (1) all CHs were effectively incorporated into the cuticle 24 hours after ingestion; (2) none of the CHs tested elicited aggression; (3) all CHs remained on the cuticle 3 days after ingestion. Even though each CH was effectively incorporated into the cuticle within 24 hours after ingestion, its single presence was not enough to trigger aggression during the behavioral tests. We thus conclude that the CHs tested in this study are not key cues for nestmate recognition in L. humile.

193

INDUCED CHANGES IN THE BEHAVIOR OF RODHNIUS PROLIXUS AS AN INDICATOR OF SENSITIVITY TO DIFFERENT BITTER COMPOUNDS.

Yamila ASPARCH1*; Romina BARROZO1; Gina PONTES1; Sebastian MINOLI1

1University of Buenos Aires, Buenos Aires, Buenos Aires, Argentina

Kissing bugs (Heteroptera: Reduviidae: Triatominae) are hematophagous insects responsible for the transmission of Chagas disease in Latin America. In previous experiments carried out in our laboratory we demonstrated that the presence of bitter substances as caffeine (CAF) and quinine (QUI) on the biting surface and/or in the solution to ingest can decrease the ingestion rate of R. prolixus. Although the sensorial pathways for the detection of CAF and QUI are not known yet, our group is currently working in this subject via different approaches. The aim of the present work is to study if kissing bugs are able to distinguish between CAF and QUI (i.e. two bitter substances) or if instead if they are perceived as different compounds that generate a similar gustative aversion. Over a 2-choice experimental arena we registered the aversion produced by CAF and QUI confronted to water, and both compounds confronted to each other. Then, in a second series, we applied an operant aversive conditioning to study if CAF and QUI can be discriminated by triatomines. Briefly, every time bugs visited one side of the arena loaded with either CAF or QUI, a mechanical disturbance was applied, which constituted a negative reinforcement. Our results show that both, CAF and QUI, repel these bugs in a similar manner. Moreover, no clear preferences were obtained when presented confronted or after the aversive conditioning. We conclude that triatomines are incapable of distinguishing between CAF and QUI. Additionally, the conditioning experiments changed the behavior of these insects, demonstrating that they are capable of learning about their environments. These studies have relevance at the time to develop repellents that could decrease bites of these bugs to humans.

194

INDUCIBLE DE NOVO BIOSYNTHESIS OF ISOFLAVONOIDS IN SOYBEAN LEAVES BY SPODOPTERA LITURA (LEPIDOPTERA: NOCTUIDAE) DERIVED ELICITORS: TRACER TECHNIQUES AIDED BY HIGH RESOLUTION LCMS.

Ryu NAKATA1; Yuki KIMURA1; Kenta AOKI1; Naoko YOSHINAGA1; Masayoshi TERAISHI1; Yutaka OKUMOTO1; Alisa HUFFAKER2; Eric A SCHMELZ2; Naoki MORI2*

1Kyoto Univ. Graduate School of Agriculture, Kyoto, Kyoto, JAPAN 2University of California at San Diego, Section of Cell and Developmental Biology, San Diego, California, America

Isoflavonoids are characteristic family of natural products in legumes known to mediate a range of plant-biotic interactions. For example, in soybean (Glycine max: Fabaceae) multiple isoflavones display an inducible accumulation in leaves following attack by Spodoptera litura (Lepidoptera: Noctuidae) larvae. To quantitatively examine patterns of activated de novo biosynthesis, soybean (Var. Enrei) leaves were treated with a combination of plant defense elicitors present in S. litura gut content extracts and L-a- [13C9, 15N]phenylalanine as a traceable isoflavonoid precursor. Combined treatments promoted significant increases in 13C-labeled isoflavone aglycones (daidzein, formononetin and genistein), 13C-labeled isoflavone 7-O-glucosides (daidzin, ononin and genistin), and 13C-labeled isoflavone 7-O-(6’’-O-malonyl-b-glucosides) (malonyldaidzin, malonylononin and malonylgenistin). In contrast levels of 13C-labeled flavones and flavonol (4’,7-dihydroxyflavone, kaempferol and apigenin) were not significantly altered. Curiously, application of fatty acid-amino acid conjugate (FAC) elicitors present in S. litura gut contents, namely N-linolenoyl-L-glutamine and N- linoleoyl-L-glutamine, both promoted the induced accumulation of isoflavone 7-O- glucosides and isoflavone 7-O-(6’’-O-malonyl-b-glucosides), but not isoflavone aglycones in the leaves. These results demonstrate that at least two separate reactions are involved in elicitor-induced soybean leaf responses to the S. litura gut contents: one is the de novo biosynthesis of isoflavone conjugates, induced by FACs, and the other is the hydrolysis reaction of the isoflavone conjugates yielding isoflavone aglycones. Gut content extracts alone displayed no hydrolysis activity. The quantitative relative analysis of isoflavone de novo biosynthesis, with respect to both aglycones and conjugates, affords a useful bioassay system for the discovery of additional plant defense elicitor(s) in S. litura gut contents that specifically promote hydrolysis of isoflavone conjugates.

195

INSECTS’ LITTLE HELPERS: SYMBIONT-PRODUCED SECONDARY METABOLITES FOR PATHOGEN DEFENSE.

Martin KALTENPOTH1*

1Johannes Gutenberg University Mainz, Mainz, Rhineland-Palatinate, GERMANY

Symbiotic associations with microbes are important driving forces of evolutionary innovation. While many symbioses have a nutritional basis, an increasing number of defensive alliances are being discovered. The latter do not only present promising sources of novel bioactive molecules, but also allow to study their action in an ecological and evolutionary context. A group of solitary digger wasps, the “beewolves” engage in a protective symbiosis with Streptomyces bacteria that they cultivate in specialized glands in their antennae and transfer to the larval cocoon. Within the first two weeks after cocoon-spinning by the beewolf larva, the symbionts produce a complex cocktail of antimicrobial compounds, thereby efficiently protecting the larva against fungal and bacterial pathogens during the long period of hibernation in the cocoon. Phylogenetic analyses of hosts and symbionts indicate that the symbiosis already evolved in the Cretaceous. Interestingly, the host’s immune defenses not only maintain specificity in the symbiotic association, but also appear to accelerate the symbiont’s genome evolution, which is reflected in an accumulation of frameshift mutations that are indicative of ongoing genome erosion and increasing dependence on the host. The beewolf symbiosis demonstrates that animals can employ a strategy comparable to the combination treatment used in human medicine to successfully fend off a broad range of detrimental microorganisms and to prevent the evolution of resistant pathogens. Furthermore, recent insights into a taxonomically completely different symbiosis between tenebrionid beetles and proteobacterial symbionts reveal that the beewolf symbiosis is exemplary for a broader range of defensive associations that protect immature life stages of arthropods against pathogenic microbes.

196

ISOLATION AND IDENTIFICATION OF A MALE-PRODUCED ATTRACTANT PHEROMONE FOR THE INVASIVE VELVET LONGHORNED BEETLE, TRICHOFERUS CAMPESTRIS (CERAMBYCINAE: HESPEROPHANINI).

Ann RAY1*; Joseph FRANCESE2; Yunfan ZOU3; Kristopher Watson4; Jocelyn MILLAR5; Damon CROOK6; Baode WANG6

1Department of Biology, Xavier University, Cincinnati, OH, USA 2USDA-APHIS-PPQ-CPHST, Buzzards Bay, MA, USA 3Department of Entomology, University of California, Riverside, CA, USA 4Utah Department of Agriculture and Food, Salt Lake City, UT, USA 5Department of Entomology, University of California, Riverside, CA, USA 6USDA-APHIS-PPQ-CPHST, Buzzards Bay, MA, USA

The velvet longhorned beetle, Trichoferus campestris (Faldermann)(VLB; Cerambycidae: Cerambycinae: Hesperophanini), is native to east Asia where it feeds on a wide range of tree species, including orchard and timber trees. Larvae of VLB can be transported in wood packing material, and individuals are frequently intercepted in quarantine facilities. Populations of VLB have established outside of the native range of the species, including near Salt Lake City, UT USA. Adults are nondescript and nocturnal, and nothing is known of their pheromone-mediated biology, which hinders monitoring and control efforts. Beetles were not significantly attracted to traps baited with any of the known cerambycid pheromones, including 2,3-alkanediols or hydroxyketones, which are male-produced pheromone components of multiple species in the subfamily Cerambycinae. Subsequently, we isolated and identified a novel variant of the conserved 2,3-alkanediol/ hydroxyketone chemical structure from headspace volatiles of males but not females. The male-produced compound will be tested in field bioassays in 2016. In addition, histological sectioning revealed subcuticular metathoracic glands present in males, but not females. Glands were connected to pits in the cuticle. The glands and pits are diagnostic for production of volatile pheromones of the 2,3- alkanediol/hydroxyketone structure by males of other cerambycine species. Our work demonstrates the utility of the metathoracic gland trait for predicting pheromone use in cerambycines. Moreover, our results support the hypothesis that cerambycid species with novel pheromones may be more successful invaders than exotic species that share pheromone components with native species.

197

METHYL JASMONATE APPLICATION IN TOMATO REDUCES INFESTATION OF TETRANYCHUS URTICAE KOCH, 1836 (TETRANYCHIDAE).

Erika SILVEIRA1*; Paulo REIS2; Maria Fernanda PEÑAFLOR2; Luciano MOREIRA2; Mariana ANDRADE3; Martín PAREJA4

1Universidade Federal de Lavras, Lavras, Minas Gerais, BRAZIL 2EPAMIG, Lavras, Minas Gerais, BRASIL 3Universidade Federal de Lavras, Lavras, MInas Gerais, BRAZIL 4Universidade Estadual de Campinas – UNICAMP, Campinas, São Paulo, BRAZIL

Plants have a wide variety of defense mechanisms and some directly affect arthropod pests that cause damage to agriculture. The use of phytohormones, such as methyl jasmonate (MeJA), for inducing resistance of plants against herbivores is a promising option for the implementation of sustainable pest management strategies. The aim of this study was to evaluate the potential of MeJA as an elicitor of resistance in tomato against Tetranychus urticae, one of the most important tomato pests in the world. A greenhouse experiment was carried out to test the hypothesis that mite colony development is compromised on MeJA-treated plants. The experiment was set up in a completely randomized design, with untreated tomato plants, plants sprayed with a 1 mM MeJA solution in water, alcohol and Tween, or plants sprayed with the solution of water acetone and Tween. After 48 hours, 60 female T. urticae / plant were released, 20 in each third of the plant (lower, middle and upper). Fifteen days after spraying, plants treated with MeJA had significantly fewer T. urticae, with a reduction of nearly 78% when compared with controls. A greater mean number of eggs, immatures and adults was found in the lower third of all treatments. Furthermore, there was a significant reduction in oviposition on plants treated with MeJA compared to controls. We conclude that the application of MeJA induced tomato defenses against T. urticae and that this resistance was more pronounced in younger leaves (middle and upper thirds). The next step of this study will be the quantification of protease inhibitors in plants treated with MeJA, since they are metabolites known to have a negative effect on the development of mites.

198

MS/MS MOLECULAR NETWORKING AS A DEREPLICATION STRATEGY APPLIED TO FUSARIUM SP EXTRACTS ISOLATED FROM SENNA SPECTABILIS’S RIZHOSOHERE.

Denise SELEGATO1*; Rafael FREIRE2; Alan PILON3; Fausto CARNEVALE4; Ian CASTRO-GAMBOA5

1UNESP/, Araraquara, São Paulo, Brasil 2USP/, São Carlos, São Paulo, Brasil 3UNESP/, Araraquara, São Paulo, Brasil 4USP/, Ribeirão Preto, São Paulo, Brasil 5UNESP/, Araraquara, São Paulo, Brasil

Senna spectabilis is an arboreal plant (Fabaceae) widely used in folk herbal medicine. Its rhizosphere has a well-known cytotoxic potential, primarily due to the organisms that inhabit it. Over the years, NuBBE has isolated over 150 microorganisms from this root area, majorly filamentous fungi from Fusarium species. Among those, F. solani and F. oxysporum are extensively studied, producing different compound classes, such as jasmonates and enniatin. A major goal in the current bioprospecting programs is to rapidly dereplicate known metabolites, minimizing time, effort, and cost, as well as accelerating NP discovery workflow1. Molecular Networking has recently emerged as a new strategy and exploits the fact that chemically similar molecules present similar physical properties1. This approach, applied to mass spectroscopy, organizes tandem MS data based on chemical similarity, allowing the dereplication of known molecules and their analogues, a challenge not overcome by other methodologies1. This work applies molecular networking for Fusarium sp, aiming to elucidate important metabolites and understanding the complex molecular production among rhizosphere’s fungi. This strategy allowed the identification of two major class metabolites clusters, attributed to cyclohexadepsipeptide (cluster 01) and picolinic acid analogues (cluster 02), along with 12 minor clusters. Furthermore, each major group was carefully dereplicated, enabling elucidation of beauvericin (1), a known Fusarium metabolite, along with beauvericin F (2), beauvericin J (3), beauvericin G2 (4) and beauvericin D (5), all firstly described for F. oxysporum, as well as 2 novel depsipeptides (6-7) for cluster 01, all highly cytotoxic metabolites; and fusaric acid (8) 5-propylpyridine-2-carboxylic acid (9), 5-(3-buten-1-yl)- pyridine-2-carboxylic (10) and 5-(but-3-en-1-yl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid (11) for cluster 02, known as moderate mycotoxins already reported for both species. Therefore, this approach was successfully applied to fungi MS/MS matrix, providing important information regarding chemical composition and possible explanation on the the rhizosphere’s cytotoxicity.

[1] Yang et al. J. Nat. Prod. 2013, 76, 1686−1699.

199

MOLECULAR NETWORKING ON NMR - IDENTIFICATION OF COMPOUNDS CLASSES USING 1H, 13C E 13C-HSQC.

Rafael FREIRE1*; Denise SELEGATO2; Alan PILON2; Ian CASTRO-GAMBOA2; Alberto TANNÚS2

1USP/, São Carlos, São Paulo, BRASIL 2UNESP/, Araraquara, São Paulo, BRASIL

Natural products (NP) are compounds derived from natural sources such as plants, animals and microorganisms. Due to the high biosynthetic complexity, NP has been used as new sources of potential drugs, presenting several different pharmacological activities. In the reductionist view, in which classical phytochemistry studies are included, one of the most exhaustive and difficult steps is the structural elucidation and the identification of NP compounds. There are many techniques to help with rapid characterization of known compounds (dereplication), but the lack of organized data is one of the major drawbacks in NP and medicinal chemistry research1. Based on this bottleneck, the aim of this work was to create a molecular network system in order to rapidly identify the molecular class of an unknown compound. This system is based on a patented pattern recognition and dereplication software2 and applied to 1H, 13C and 13C-HSQC NMR. Applying NMR molecular networks requires NMR spectra of the NP mixture or a pure compound along with NMR spectra of known standards. In order to do that, we compile two online free NMR database, HMDB and MMCD, data from 15 years of the NMR studies from NuBBE’s group and NMR prediction data of all NuBBE Database compounds. into a final database with more than 5000 compounds. With the application of this new molecular network methodology, it became easier to identify the molecular class of molecules even if they are not present in the database. This was possible through the development of a new fingerprint technique based on the similarity profiles acquired by a new pattern recognition function and the use of network analysis.

[1] Yang et al. J. Nat. Prod. 2013, 76, 1686−1699.

[2] Freire R.T., Castro-Gamboa I.; Method of analysis' pattern recognition and computer program. WIPO PCT/BR2015/000075, 18/Mar/2015.

200

NON-TARGETED AND TARGETED METABOLOMIC APPROACHES REVEAL DIFFERENCES IN LEGUME CHEMISTRY BEFORE AND AFTER INFESTATION WITH PEA APHID HOST RACES.

Carlos SANCHEZ-ARCOS1; Jonathan GERSHENZON1; Grit KUNERT1*

1MAX PLANCK INSTITUTE FOR CHEMICAL ECOLOGY, BIOCHEMISTRY DEPARTMENT, Jena, Turingia, Germany

The pea aphid (Acyrthosiphon pisum), an insect that feeds on several host plants in the legume family (Fabaceae), is a complex of at least 15 genetically different host races. Each race prefers different host plant species and performs better on these plants than on other legumes, which makes it an attractive model insect for the study of ecological speciation. Pea aphids consume the sugar-rich phloem sap of plants through their sucking mouthparts. Phloem contains other metabolites besides sugars and aphids contact other plant tissues to reach the phloem. Thus, aphids encounter many different plant chemicals. Until now, the contribution of chemistry to the differential performance of the host races on legume plants is still largely unknown. Using a mass spectrometry-based non-targeted metabolomic approach, we found significant differences among the metabolic fingerprints of the host plants before aphid infestation and observed that aphid feeding altered the metabolome of each plant specifically. This analysis resulted in a list of candidate chemical compounds that could be responsible for the specialization of pea aphid host races on their host plants. Also, using a targeted metabolomic approach we collected information about the time course of plant hormone concentrations, including jasmonates, salicylic acid, and abscisic acid in response to pea aphid infestation. The results suggest that aphids were able to modulate the phytohormone levels on their native host plants to avoid defensive responses. Thus, we conclude that pea aphid host races use active strategies to avoid the chemical defenses of their food plants. This information opens new opportunities to understand how plant chemistry can influence aphid performance and vice-versa, as well as how plant chemistry can be modified to reduce the infestation of aphids on crop plants.

201

ONE PATHWAY IS NOT ENOUGH: GLUCOSINOLATE METABOLISM IN THE CABBAGE STEM FLEA BEETLE PSYLLIODES CHRYSOCEPHALA.

Anton SHEKHOV1*; Seung-Joon AHN1; Michael REICHELT1; Riya C. MENEZES1; Christian PAETZ1; David G. HECKEL1; Jonathan GERSHENZON1; Daniel G. VASSÃO1; Franziska BERAN1

1Max Planck Institute for Chemical Ecology, Jena, Thuringia, THURINGIA

Plants from the Brassicaceae family use a myrosinase-glucosinolate (gls) defense system to chemically protect themselves against herbivory. Yet some insects are able to feed on Brassicaceae without any obvious negative effects. Psylliodes chrysocephala, the cabbage stem flea beetle, is a serious crop pest that can successfully feed on Brassicaceae plants. We are investigating the biochemical basis of insect feeding on these gls- containing plants. We compared the metabolic profiles of flea beetle bodies and feces after feeding on leaves of the Arabidopsis thaliana myb28myb29 double mutant (lacking alkyl gls) with and without supplementation of 4msob (4-methylsulfinylbutyl gls) or sinalbin (p-hydroxybenzyl-gls). We found that P. chrysocephala sequesters 4msob-gls in high concentrations and excretes desulfo-sinalbin. The formation of desulfo-sinalbin shows that the beetles can cleave the sulfate group from p- hydroxybenzyl-gls (but apparently not from 4msob-gls), thus making it unrecognizable for plant myrosinases and preventing glucosinolate hydrolysis to toxic derivatives. These results fit well with the activities of the beetle’s gls-sulfatase enzymes in vitro. Using 14C-labeled sinalbin in feeding experiments, we observed that while a quarter of the radioactivity in the feces corresponds to desulfo-sinalbin, half of the radioactivity was excreted as an additional unknown metabolite. The in-source MS fragmentation of this major product suggests that it is formed after sinalbin activation by myrosinase. Thus, when feeding on aliphatic and aromatic glucosinolates, P. chrysocephala can 1) sequester 4msob-gls, 2) disable myrosinase substrate recognition by desulfation, and 3) detoxify sinalbin hydrolysis products. These strategies indicate that specialists such as the cabbage stem flea beetle possess biochemical and physiological adaptations to adapt to different types of glucosinolates that may facilitate their survival and persistence on Brassicaceae host plants.

202

OVIPOSITION CHOICES OF THE WILLOW SAWFLY NEMATUS OLIGOSPILUS IS AFFECTED BY CONSPECIFIC HERBIVORY IN THE NATIVE WILLOW SALIX HUMBOLDTIANA.

Gabriela A. VALLADARES1*; Celina BRACCINI2; Mariela ALDERETE3; M. Victoria COLL ARAOZ3; Teresa VERA3; Patricia C. FERNANDEZ4

1Department of Plant Health. National University of Tucumán, San Miguel de Tucuman, Tucuman, ARGENTINA 2Institute of Biological Resources. INTA Castelar, Castelar, Buenos Aires, ARGENTINA 3Natural Sciences Faculty. National University of Tucumán, San Miguel de Tucuman, Tucuman, ARGENTINA 4INTA EEA Delta del Paraná, Buenos Aires, Buenos Aires, ARGENTINA

Most herbivorous insects start attacking a plant by laying eggs on it. Host plant selection can be crucial for the survival of the new insect generation, especially when larvae lack mobility to select their own feeding places. The willow sawfly, Nematus oligospilus (Förster), is an exotic emerging pest in Salix commercial forests and has been reported worldwide. Females of this species are parthenogenetic in the southern hemisphere and must find and recognize their host plant when they emerge as adults.The objective of the present work was to evaluate the effect of conspecific herbivory on the oviposition choices of N. oligospilus females. Local and systemic effect, were studied. First, choice tests were conducted on Salix humboldtiana (Willdenow) and S. babylonica (Linneaus) var. sacramenta in undamaged potted plants vs. plants after 48 hs. of larval feeding. Results on S. humboldtiana, suggests strong repellence to plants with damage as females highly preferred branches from undamaged plants for oviposition. This was observed for both, local and systemic damage. For S. babylonica we did not find significant differences between treatments.In order to look for the chemical cues associated to this behavior, plant volatiles are being analyzed. Preliminary observations showed qualitative differences between the chemical composition of damaged and undamaged plants, suggesting the presence of plant cues elicited by larval feeding as oviposition repellents.

203

PHLOEM SAP INGESTION-RELATED PLANT - APHID TRANSFER OF CYCLITOLS AND THEIR EFFECT ON THE PEA APHID ACYRTHOSIPHON PISUM HARRIS PROBING BEHAVIOUR.

Bozena KORDAN1*; Leslaw B. LAHUTA1; Katarzyna DANCEWICZ2; Beata GABRYS2

1University of Warmia and Mazury, Olsztyn, Warmia and Mazury, POLAND 2University of Zielona Góra, Zielona Góra, Lubuskie, POLAND

Cyclitols, the hydroxylated cycloalkanes biosynthetically deriving from glucose, occur in any living cell and express a broad spectrum of biological activity: they participate in membrane biogenesis and dynamics, signal transduction, ion channel physiology, osmoregulation, and antioxidation and in environmental interactions, e.g., in conferring salt tolerance in plants and plant response to water stress or in plant-insect relationships. Leguminous plants (Fabaceae) contain an exceptionally high amount of cyclitols as compared to the majority of plant species: in some seeds, these sugar alcohols may make up to 30 % of the total content of soluble carbohydrates. The effect of cyclitols: D- pinitol, D-chiro-inositol that are naturally present in tissues of Lupinus angustifolius on the behaviour of the pea associated clone of Acyrthosiphon pisum during various stages of probing was studied. D-pinitol, D-chiro-inositol and their mixture were exogenously applied to pea Pisum sativum explants and the aphid probing behaviour was evaluated using the Electrical Penetration Graph Technique (EPG). Feeding of peas with cyclitols at 10 mM concentration caused a selective accumulation of D-pinitol and D-chiro- inositol in stems, leaf petioles, and leaf blades. In aphid bodies, both cyclitols were detected, respectively to the host plant treatment. Aphid behaviour on cyclitol-treated plants as compared to control was altered: non-probing and probing in mesophyl prevailed among aphid activities during initial period of stylet penetration, aphids on D- pinitol+D-chiro-inositol-treated plants reached phloem vessels relatively later than aphids on control and D-chiro-inositol plants, and there were recurrent switches between E1 (salivation) and E2 (sap ingestion) patterns in some aphids during the phloem phase on D-pinitol and D-pinitol+D-chiro- inositol – treated plants. This may reflect difficulties in the uptake of the phloem sap and point at lupin cyclitols as responsible, at least in part, for the rejection of L. angustifolius as a host plant by the pea clone of A. pisum.

204

PLANT LITTER VOLATILES AND THEIR INTERACTION WITH DECOMPOSER ORGANISMS.

M. Soledad MÉNDEZ1*; Adelia GONZALEZ-ARZAC1; Adriana FERNANDEZ SOUTO1; Patricia C. FERNÁNDEZ2; Lucía VIVANCO2; Amy T. AUSTIN3

1IFEVA – Facultad de Agronomía, Universidad de Buenos Aires - CONICET, Ciudad Autónoma de Buenos Aires, Buenos Aires, ARGENTINA 2INTA - CONICET, Campana, Buenos Aires, ARGENTINA 3IFEVA-CONICET-UNSaM, Buenos Aires, Buenos Aires, ARGENTINA

Plants interact with soil organisms in a myriad of ways, and this interaction is a key factor in determining the formation of soil organic matter and nutrient availability in terrestrial ecosystems. In a old-growth temperate Patagonian forest, three southern beech species (Nothofagus obliqua, N. nervosa and N. dombeyi), demonstrated faster decomposition rates in areas defined as ´tree triangles´ on the forest floor, below an intersection of three adult trees of the same speciesa and, while the abundance of soil and litter macrofauna did not differ among the tree triangles, its composition was markedly distinct. Our objective in this study was to explore possible mechanisms which could generate specialized decomposer communities at the scale of single tree species in these microsites. We hypothesized that the chemical characteristics of leaf litter input, over long periods of time, could generate conditions for a specialized decomposer community, both for microbial communities and for soil macrofauna. Therefore, we explored volatile signals from the recently-senesced leaf litter of the three plant species. We collected volatiles of unsterilized left litter of the three southern beech species, and analyzed their composition using a GC-MS. Surprisingly, each species has its own completely distinct footprint of volatile organic compounds (VOCs). We analysed the distribution of VOCs with a Principal Component Analysis which explained with its first two components 67.2% of total variance. While N. nervosa was dominated by sesquiterpenes N. dombeyi by aromatic derivatives and monoterpenes and finally N. obliqua had equal proportion of chemical compounds. These results suggest that leaf litter, through volatile signals could modulate the colonization by soil faunal community and this could drive changes in decomposition rates.a Vivanco and Austin (2008).

205

RESISTANCE TO CARDENOLIDES CARDENOLIDES – GENETIC BACKGROUNDS MATTER!

Susanne DOBLER1*; Safaa DALLA1; Fabian LIST1; Herman SWARTS2; Jan KOENDERINK2; Johannes KIRCHMAIR3

1Universität Hamburg, Hamburg, Germany 2Radboud University, Nijmegen, The Netherlands 3Universität Hamburg, Hamburg, Germany

Cardiac glycosides are potent toxins that act by blocking an essential animal transmembrane carrier, the Na,K-ATPase or sodium pump. The binding site for cardiac glycosides in the Na,K-ATPase has been well characterized and is highly conserved across the animal kingdom. Few insects and vertebrates are able to tolerate and feed on toxic plants or prey containing cardiac glycosides. Most of these species have evolved target site insensitivity through specific amino acid substitutions that lower the Na,K- ATPase binding affinity to cardiac glycosides. Yet, despite high levels of molecular convergence among insects, the resistance-conferring substitutions observed in insects differ from those observed in vertebrates.In this study we tested whether substitutions that have evolved in the genetic backgrounds of insects versus mammals are equally efficient at lowering Na,K-ATPase binding affinity to cardiac glycosides. We compared the effect of the relevant substitutions of the monarch butterfly, Q111V and N122H, with those observed in the rat Na,K-ATPase 1α, Q111R and N122D. Although the resistance- conferring abilities of both substitution pairs have been previously characterized, their effects in the genetic backgrounds of distantly related species are unknown. We used site- directed mutagenesis to introduce the combined substitutions into both the human and fruit fly Na,K-ATPase α-subunit and expressed the enzymes by baculovirus infection of Sf9 insect cells. The recombinant proteins were then tested for their cardenolide sensitivity under increasing concentrations of ouabain, a standard cardiac glycoside. These data clearly show that the genetic background strongly influences the resistance efficiencies of the substitutions. Although both the combined Q111V and N122H and the Q111R and N122D substitutions significantly increased Na,K-ATPase resistance towards ouabain, the rat substitutions proved to be superior in the mammalian Na,K-ATPase while the monarch substitutions were superior in the genetic background of the fly Na,K- ATPase.

206

SHEDDING LIGHT ON PHYTOCHEMICAL DIVERSITY.

Karissa MONTES1; Christopher JEFFREY1*

1University of Nevada, Reno, Reno, Nevada, UNITED STATES

Our group recently discovered a set of unique natural products that were isolated from Piper kelleyii, a tropical shrub that grows in the upper elevations of the cloud forests of Ecuador. These compounds displayed interesting anti-herbivore properties and were found to strongly correlate to the diversity of an associated specialist caterpillar and were likely to be biosynthetically related. Of most interest to our group was the fact that the dimeric chromane was found to occur as a racemic mixture. Further analysis led to the hypothesis that this chromane is likely the result of a hetero-[4+2] cycloaddition reaction between a o-quinone methide, which could be the result of a photo-induced electrocyclization of the chromene.This project is focused on understanding the photochromic behavior of naturally occurring chromenes and its role in the biosynthesis of a variety of natural product architectures. With established conditions for the photochromic conversion of the o-quinone methides, I have explored its reactivity under a variety of different uni-molecular and bi-molecular reactions, by irradiation under various conditions, isolation of the products, and structural characterization. Further experiments will integrate our findings from the photogenerated quinone methide and understand the patterns in selectivity and reactivity leading to a variety of natural product architectures.

207

SPECIFICITY OF PLANT RESPONSES TOWARD HERBIVORE’S TOUCH, FEEDING AND OVIPOSITION: IMPLICATION FOR EGG PARASITOIDS.

Francesca FRATI1; Antonino CUSUMANO2; Eric CONTI3; Stefano COLAZZA4*; Ezio PERI4; Roberto ROMANI5; Gianandrea SALERNO5

1University of Perugia, Perugia, Umbria, ITALY 2University of Palermo, Palermo, Sicily, ITALY 3University of Perugia, Perugia, Umbria, ITALY 4University of Palermo, Palermo, Sicily, ITALY 5University of Perugia, Perugia, Umbria, ITALY

Several phases of herbivore attack including walking, feeding and oviposition are known to induce plant defenses. Plants emit volatiles induced by herbivore stress to recruit insect parasitoids as an indirect defense strategy. Egg parasitoids respond to herbivore-induced plant volatiles (HIPVs), including oviposition-induced plant volatiles (OIPVs). So far, volatiles induced by herbivore walking and their putative role in the foraging behavior of egg parasitoids have not been investigated. In this paper we studied the response of the egg parasitoid Trissolcus basalis toward putative footprint-induced plant volatiles (FIPVs) induced on Vicia faba by the walking activity of the host Nezara viridula. Olfactometer bioassays have been carried out to evaluate T. basalis responses to plants in which the abaxial or the adaxial leaf surfaces have been treated with footprints or/and oviposition. Results showed that host female footprints on the abaxial leaf surface elicited the induction of FIPVs favouring a repellence effect in T. basalis. Remarkably, FIPV emission also occurred when the leaf is turned upside down, indicating a specificity of stress localization. This specificity of plant response toward herbivore attack is supported by the results showing that oviposition combined with feeding elicits the induction of OIPVs only when attack occurs on the abaxial leaf surface. Analyses of the volatile blends among the differently treated plants show significant differences in terms of whole volatile emissions.

208

SYNERGETIC EFFECTS OF CIS-JASMON AND METHYL SALICYLATE IN ATTRACTING DIFFERENT MEMBERS OF A COMPLEX MULTITROPHIC FLOWER-VISITING ARTHROPOD COMMUNITY.

Florian ETL1*; Andreas BERGER2; Anton WEBER2; Jürg SCHÖNENBERGER2; Stefan DÖTTERL3

1University of Salzburg, Salzburg, Austria, SALZBURG 2University of Vienna, Vienna, Austria, VIENNA 3University of Salzburg, Salzburg, Austria, SALZBURG

Some plants exhibit extreme specialization for pollination in that only one or a few animal species belonging to a single functional group (e.g. bees, birds, beetles) act as their pollinators. One example of a specialized pollination system is the interaction between some Annonaceae, Magnoliaceae, and Araceae species and their Cyclocephalini (Scarabaeidae) pollinators. Recent studies have shown that flowers / inflorescences of these plants emit a strong scent, which is the most important signal for pollinator attraction in these specialized pollination systems. Dieffenbachia (Araceae) species are exclusively pollinated by cyclocephaline scarab beetles, however, some species are visited by several other non-pollinating arthropod species. Field observations on Dieffenbachia aurantiaca in a lowland rainforest in Costa Rica also showed that inflorescences are visited by a multitrophic non-pollinating insect community consisting of several florivorous species as well as parasitic flies and some of their parasitoid wasps in addition to the cyclocephaline scarab beetle pollinators. Our study aimed to identifying the chemical communication between the plant and its mutualisitc and parasitic interaction partners. By using dynamic headspace sampling and gas chromatography / mass spectrometry we identified cis-jasmone and methyl salicylate as the main inflorescence scents. Behavioural studies demonstrated that these two compounds are sufficient to attract the pollinating and non-pollinating insect community associated with this plant. Interestingly, while members of the non-pollinating community responded to the single compounds in field-biotests, the pollinating beetles did only respond to the mixture of these components. Overall, our study shows that Dieffenbachia aurantiaca attracts their mutualistic beetle pollinators by a mixture of two compounds, and this communication channel is exploited by florivorous insects and also by a parasitoid wasp of a dipteran parasite.

209

TWO SIDES OF THE SAME COIN: THE BENEFIT AND BACKLASH OF ISOTHIOCYANATE DETOXIFICATION.

Verena JESCHKE1*; Sagar S. PANDIT1; Jonathan GERSHENZON1; Daniel G. VASSÃO1

1MAX PLANCK INSTITUTE FOR CHEMICAL ECOLOGY, BIOCHEMISTRY DEPARTMENT, Jena, Turingia, GERMANY

Isothiocyanates (ITCs) are the toxic breakdown products derived from glucosinolates, which are the major plant defense metabolites present in the order Brassicales (e.g. cabbage, broccoli, mustard). While many specialist insects possess efficient detoxification strategies avoiding glucosinolate breakdown, generalist insects must deal with the toxic ITCs. Previous studies in our group revealed that a proportion of ITCs is metabolized to glutathione conjugates [1]. Nevertheless, insects ingesting ITCs suffer from decreased growth rates and delayed development when feeding on brassicaceous plants. However, the factors causing fitness reduction and the mode of action of ITCs are still poorly understood. We aimed at understanding how insect metabolism is affected by ingested ITCs, and which role the detoxification with glutathione plays. Using artificial diets, we conducted detailed investigations on the effect of the aliphatic 4- methylsulfinylbutyl-ITC (sulforaphane) on the biochemistry and metabolism of Spodoptera littoralis larvae [2]. Detoxification caused a major decrease in the glutathione content of midgut tissue and hemolymph. As a consequence, the levels of free amino acids were altered, with a sharp decrease in cysteine leading to reductions in protein content, but an increase in lipids. The importance of GSH conjugation for ITC detoxification in generalist larvae was investigated using RNAi experiments, which led to the identification of one GST-encoding gene important in ITC detoxification. To learn more about the mechanism of glucosinolate toxicity, we are now performing comprehensive studies of the proteomic and transcriptomic changes in insect herbivores upon ITC ingestion.

[1] Schramm, K. et al. (2012). Insect Biochem Mol Biol 42, 174-182.

[2] Jeschke, V. et al. (2016). Insect Biochem Mol Biol 71, 37-48.

210

THE CHEMISTRY OF MALE DOMINANCE: SECRETION OF 3-METHYL-2- BUTENYL BUTYRATE CORRELATES WITH BEHAVIOURAL STATUS IN THE BRONZE BUG, THAUMASTOCORIS PEREGRINUS.

Hernan GROBA1*; Diana VALLE1; Gonzalo MARTÍNEZ2; Andrés GONZALEZ3

1Laboratorio de Ecología Química, Facultad de Química, UdelaR, Montevideo, Montevideo, URUGUAY 2Instituto Nacional de Ingeniería Agropecuaria Tacuarembo,, Tacuarembo, Tacuarembo, URUGUAY 3Laboratorio de Ecología Química, Facultad de Química, UdelaR, Montevideo, Montevideo, URUGUAY

The bronze bug, Thaumastocoris peregrinus (Heteroptera: Thaumastocoridae), is an exotic emerging pest in Eucalyptus commercial forests. Information on its chemical ecology and reproductive behaviour may be relevant for designing management strategies. Adults usually aggregate, and males often mount on each other forming male tandems of unknown function. In addition, adult males secrete large amounts of 3- methyl-2-butenyl butyrate, which attracts conspecific males but not females. The ecological role of this compound also remains unknown. In this study, we establish a potential correlation between the two. We describe the establishment of male dominance by aggressive male-male interactions, including mounting, and its correlation with the amount of 3-methyl-2-butenyl butyrate secreted by the males. Male contests were filmed under a stereomicroscope on a leaf square of Eucalyptus tereticornis (3x3 cm). Male dyads were allowed to interact for 30 min, and a female was then added for recording male-female interactions for additional 30 min (all insects virgin, one-week old adults). We assigned the “dominant status” to the male that attempted to copulate with the female. Both males were then extracted individually, and the amount of 3-methyl-2- butenyl butyrate was quantified by GC-MS. We recorded two aggressive behaviours between males: sparring and mounting, which were added to establish an aggressive index. Dominant males showed a higher aggressive (t0.025,13=2.84, N=14, p=0.014), and almost twice the amount of 3-methyl-2-butenyl butyrate than subordinate males (t0.025,13=3.85, N=14, p=0.002). In contrast, male dyads in which none of the males attempted copulation, suggesting no dominance status, showed no differences in the aggressive index (t0.025,6=1.11, N=7, p=0.31) or 3-methyl-2-butenyl butyrate amount (t0.025,6=0.05, N=7, p=0.96). Our results suggest that T. peregrinus males establish a dominant status through aggressive male-male interactions, and that dominant males secrete more 3-methyl-2-butenyl butyrate, a compound that may hence function as a semiochemical to establish or signal male dominance.

211

TRAPPING LITERATURE FOR FOREST COLOEPTERA: A META- ANALYSIS.

Jeremy D. ALLISON1*; Richard A. REDAK2

1Natural Resources Canada, Sault Ste Marie, Ontario, CANADA 2University of California, Riverside, California, USA

A large literature on the survey and detection of forest Coleoptera and their associates exists. Identification of patterns in the effect of trap types and design features among guilds and families of forest insects would facilitate the optimization and development of intercept traps for use in management programs. We reviewed the literature on trapping bark and woodboring beetles and their associates and conducted meta-analyses to examine patterns in effects across guilds and families and observed the following general patterns: 1) panel traps were superior to multiple-funnel traps, 2) bark beetles and woodborers were captured in higher numbers in traps treated with a surface treatment to make them slippery than untreated traps, 3) panel and multiple-funnel traps equipped with wet cups outperformed traps with dry cups, 4) black traps were superior to white and clear traps, and 5) purple traps were as good or superior to green traps for Agrilus spp.

212

UTILIZING CHLOROPHYLL FLUORESCENCE IMAGING AS A TOOL TO DETERMINE THE CHEMOATTRACTANT POTENTIAL OF GINSENOSIDES IN THE GINSENG – PYTHIUM IRREGULARE PATHOSYSTEM.

Dimitre A. IVANOV1*; Jorge R.C. GEORGAKOPOLOUS1; Mark A. BERNARDS1

1UNIVERSITY OF WESTERN ONTARIO, LONDON, ONTARIO, CANADA

Ginsenosides, the triterpenoid saponins produced by American ginseng (Panax quinquefolius L.), are mildly fungitoxic; however, exposure of the ginseng root pathogen Pythium irregulare Buisman to ginsenosides enhances its growth in a dose dependent manner. This has lead to speculation that ginsenosides may function as chemoattractants and/or growth regulators in the context of the ginseng – P. irregulare pathosystem. In the presented work, the chemoattractant potential of ginsenosides for P. irregulare was evaluated through (1) an in vivo pot experiment that monitored the pathogenicity of P. irregulare toward ginsenoside-treated and -untreated one- and two- year old ginseng plants and (2) by monitoring the affects of a purified total ginsenoside extract (GSF) and pure ginsenosides (Rb1, Re and F2) on the growth of the pathogen, in vitro, using a disk diffusion assay. Disease severity and Time to Infection (TTI) were evaluated in vivo, by monitoring the chlorophyll fluorescence parameter ΦNO through non-invasive Chl fluorescence imaging in whole leaves of infected plants. Treatment of ginseng roots with a relatively high dose of ginsenosides prior to planting resulted in delayed infection by P. irregulare, of both one- and two-year old ginseng plants. Meanwhile, in vitro exposure of P. irregulare to ginsenoside Rb1 and GSF resulted in delayed hyphal progression, while enhancing aerial hyphal build-up around ginsenoside treated disks. By contrast, assays with pure ginsenoside F2 resulted in clear zones of inhibition around treated disks. While these results do not definitively show that ginsenosides act as chemoattractants for P. irregulare, they do demonstrate that these saponins serve to alter the growth habit of this organism, both in vivo and in vitro, which may lead to changes in the severity of pathogenicity.

213 PLANT COMMUNICATION

ARBUSCULAR MYCORRHIZAE FUNGI CHANGE THE RESISTANCE OF RICE PLANTS IN RESPONSE TO FEEDING WITH FALL ARMYWORM, SPODOPTERA FRUGIPERDA.

Lina BERNAOLA1; Michael STOUT1*

1Louisiana State University, Baton Rouge, Louisiana, USA

Soil-borne organisms can have significant effects on aboveground interactions. Arbuscular mycorrhizal fungi (AMF) are one example of beneficial soil microbes that play a vital role in mediating plant resistance against pathogens and herbivores. However, little is known about the underlying molecular mechanisms of these interactions [1]. We studied how AMF, which establish symbiotic associations with many plant root systems, lead to changes in rice (Oryza sativa) gene expression by up-regulating/down-regulating resistance-related genes, thereby increasing performance of fall armyworm (Spodoptera frugiperda, FAW). In a greenhouse experiment, we characterized the molecular responses to FAW-damaged and undamaged rice plants colonized or not colonized with mycorrhizae to identify candidate marker genes and gene networks involved in plant defense. Leaves were collected at 24 hours after larvae fed from rice plants subjected to four treatments: mycorrhizae FAW-damaged (MD) and undamaged plants (MUD), and non-mycorrhizae damaged (NMD) and undamaged plants (NMUD). Following the feeding, the leaves of the plant were removed and the tissue was prepared for RNA isolation, and synthesis of cDNA. Based on a transcriptome analysis, we documented differential changes in gene expression among the four treatments. Validation of altered expression levels of eighteen unigenes, which encode plant defense biosynthetic enzymes and transcription factors, was tested using quantitative real-time PCR (qRT-PCR). The primary genes of interest were plant defense genes associated with the Salicylic acid and Jasmonic acid pathways [1]. Our results indicate that AMF colonization causes changes in gene expression related to defense responses, with important consequences for rice interactions with the herbivore FAW. This study represents the first transcriptome analysis of O. sativa plants inoculated with AMF and subjected to FAW feeding. Our ongoing research in this system will provide insights into the underlying mechanisms of plant resistance in rice.

1. Lu J, Maud Robert CA, et al. Plant Physiology, 2015, 167, 1100-1116.

215

COMMUNICATING PLANTS: THE CHEMICAL LANGUAGE OF GOLDENRODS (SOLIDAGO ALTISSIMA) AND ITS ECOLOGICAL CONSEQUENCES.

Andre KESSLER1; Kimberly MORRELL1*; Akane UESUGI2; Kaori SHIOJIRI3

1Cornell University, Ithaca, NY, USA 2Monash University, Clayton, Victoria, AUSTRALIA 3Kyoto University, Kyoto, Kyoto, JAPAN

Plant chemical communication has long been discussed as a factor that mediates interactions within ecological communities. Especially volatile organic compounds emitted from herbivore-or pathogen-attacked plants have been found to mediate interactions between the plant and their enemies, the plant and the enemies of their enemies, as well as between neighboring plants. How important the chemical information transfer is between plants and their biotic environment for larger scale ecological processes and ultimately the plants’ fitness has been a topic of controversy. The tall goldenrod, Solidago altissima, has long been used as a native community ecology model system. This species is very actively interacting with its surrounding community via chemical signals, especially volatile organic compounds (VOCs). VOC-mediated interactions between plants and herbivorous insects and between plants affect herbivore population dynamics and so the distribution of herbivory within the plant population. As a consequence VOC-mediated information transfer in interaction with herbivore-induced resistance functions as a herbivory risk-spreading strategy that minimizes damage that each individual plant receives within each population. Here we present a detailed characterization of the herbivore-species and plant genotype-specific plant-plant information transfer in S. altissima, as well as the ecological consequences and micro- evolutionary aspects of plant chemical communication.

216

EFFECTS OF MULTIPLE STRESS ON VOLATILE-MEDIATED TRITROPHIC INTERACTIONS.

Martín PAREJA1*; Delia M. PINTO-ZEVALLOS2

1Departamento de Biologia Animal, UNICAMP, Campinas, São Paulo, BRAZIL 2Departamento de Ecologia, Universidade Federal de Sergipe, São Cristóvão, Sergipe, BRAZIL

It has been known for over 30 years that plants, when subjected to individual stressors, can change their VOC profile, thus obtaining indirect defence by attracting natural enemies such as predators and parasitoids. Throughout their lifetimes, however, plants are subjected to multiple stresses, ranging from attack by herbivores with different feeding modes, to attack by pathogens and stress due to abiotic agents such as UV light and atmospheric pollutants. Because the induction of VOCs depends on the identity of the stressor, there is growing interest on the effects of multiple stress on indirect plant defence. In this study, we review the effects of multiple stressors on induced, VOC- mediated, indirect plant defence. For multiple herbivores, we show that the studies so far indicate that predators are more attracted to multiply-damaged plants, while effects on parasitoids vary greatly among systems. Furthermore, when considering multiple herbivory, plants damaged by herbivores with the same feeding mode, interference in natural enemy attraction is frequently observed. When herbivores have a different mode of feeding, interference in attraction is more rarely observed. This result is counter- intuitive, and raises interesting questions in relation to how specificity in natural enemy response is generated. Studies that analysed volatile organic compounds (VOCs) have focussed on multiple herbivores, and have shown an even distribution of VOC enhancement, suppression and no blend effect. These results highlight how system- specific many effects are, and we should be cautious with intuitive hypotheses, such as the common idea that herbivores with different feeding modes will interfere more with VOC blends and tritrophic interactions than those with similar feeding modes. Studies considering multiple stress by herbivores and abiotic agents, or herbivores and pathogens are still too few to warrant conclusions and deserve further study.

217

FLORAL CO2: A BREATH OF FRESH AIR IN PLANT-POLLINATOR COMMUNICATION.

Robert RAGUSO1*; Holly SUMMERS1; Adrian POWELL1; Sally HARTWICK1

1Cornell University, Ithaca, NY, USA

Although flowering plants and their pollinators engage in mutually beneficial interactions, conflicts of interest often arise due to the asymmetry of fitness goals for each participant. Flowering plants should optimize pollinator services in terms of the quality of pollen export (male fitness) and deposition (female fitness), while minimizing the metabolic and/or ecological costs required to attract such pollinators. Foraging animals should optimize access to floral resources per unit time (profitability), while minimizing their own energetic or opportunity costs, including exposure to predators. As a result floral advertisements (color, scent) do not always accurately indicate the presence of nectar, and discriminating pollinators should attend to other sensory cues (e.g. the scent of nectar) that are less easily faked by the plant. One such cue is floral carbon dioxide (CO2). Previous studies revealed that night blooming flowers of wrightii consistently emit CO2 during the first 60-90 minutes of anthesis, when nectar is still available in such flowers, and that nocturnal hawkmoths (Manduca sexta) show innate preferences for flowers with elevated CO2. Here we extend these studies to a lineage of evening primrose flowers (Oenothera) in which flower size and mating system vary from small, autogamously selfing flowers (O. f. flava) to very large outcrossing taxa (O. acutissima). Plants were grown in a common garden to reduce environmental variance, and floral CO2 was analyzed from replicates of several independent populations using a Licor IR detector and dry floral mass to control for allometric scaling. The largest Oenothera flowers emitted copious amounts of floral CO2, well within the threshold of detectability by foraging hawkmoths. The relationship between dry mass and floral CO2 varies markedly between populations, with slopes that co-vary with pollen:ovule ratios, an indicator of dependence upon outcrossing.

218

ORCHIDS’ CHEMICAL IMITATION GAME: FLORAL SCENTS OF CATASETUM CERNUUM AND GONGORA BUFONIA SUGGEST CONVERGENT EVOLUTION TO ATTRACT A UNIQUE POLLINATOR.

Carlos Eduardo Pereira NUNES1*; Günter GERLACH2; Karen D. O. BANDEIRA3; Leonardo GOBBO-NETO4; Emerson Ricardo PANSARIN4; Marlies SAZIMA4

1State University of Campinas - Unicamp, Campinas, São Paulo, BRAZIL 2Botanical Garden München-Nymphenburg, Munich, Bavaria, GERMANY 3University of São Paulo, Piracicaba, São Paulo, BRAZIL 4University of São Paulo, Ribeirão Preto, São Paulo, BRAZIL

Floral volatile organic compounds (VOCs) are important signals in plant-pollinator communication. Thus, floral VOCs are subjected to pollinator-mediated selection that may favour individual plants attracting the most effective pollinators. Occasionally, phylogenetically unrelated plant species would converge to similar floral scent signalling patterns to attract a given pollinator representing a common adaptive pollination niche. Our field observations in Atlantic forest areas of south-eastern Brazil and reports from literature provided the information that Catasetum cernuum and Gongora bufonia are exclusively pollinated by males of a single orchid-bee species, Eufriesea violacea. In order to test the hypothesis that these two orchid species converged to similar scents attracting males of a single euglossine as pollinator, the floral scents of the two species were characterized using headspace techniques, then the similarity of scents were tested using multivariate analyses. Floral scents of both species do not differ significantly from each other, being a blend of VOCs usually found in other euglossine-pollinated species. These species share some unusual floral VOCs, such as (e)-epoxyocimene, a rare constituent of floral scents, found in few other orchid species, all of them occurring outside of the distribution range of C. cernuum,G. bufonia and E. violacea. Thereby, the authors hypothesize that (e)-epoxyocimene or a mixture of it with other floral VOCs determines the specific association between these two orchids and their exclusive euglossine pollinator.

219

PHYTOTOXITY OF EUCALYPTUS SALIGNA SM ESSENTIAL OIL ON GROWTH OF GRASSES.

Luis Henrique RUIZ DA SILVEIRA1; Eliane Regina DA SILVA1*; Geraldo Luiz GONÇALVES SOARES1

1UFRGS, Porto Alegre, Rio Grande do Sul, BRAZIL

Plants can release secondary metabolites through processes like leaching, exudation and volatilization, and with that affect the development of other neighboring plants; the study of this phenomenon is named allelopathy. Eucalyptus L’Hér species have been investigated for the possible involvement of allelopathy in difficulting the establishment of other plants. Plantations of these species are replacing grassland vegetation in South Brazil, where few species establish inside these plantations. This study aims to access the effects of essential oil of Eucalyptus saligna leaf litter on the growth of species from Campos Sulinos. Species of grasses (Poaceae) that are syntopic with the Eucalyptus plantations in the Campos Sulinos have been chosen as recipient plants, being them Paspalum notatum Fluggé (native) and Eragrotis plana Ness (invasive exotic). Seeds of each species were put on Petri dishes, with filter paper and containing distilled water. After germination, seedlings where exposed to various doses of E. saligna essential oil (0, 1 μL, 10 μL, 20 μL, 30μL, 40 μL e 50 μL). Bioassays were performed with four repetitions per group. Root and shot were measured after 96 hours in 15 seedlings per repetition. In relation to root length, both species demonstrated reduction when exposed to all doses of essential oil, compared to the control group (0 μL). Regarding shoot lenght, E. plana a presented decreased from 10 to 50 μL, while P. notatum only showed reduction from 20 to 50 μL of essential oil. Even small quantities of essential oil caused negative effects on both species. This suggests that the difficulty of establishment of others plants is related to the demonstrated inhibitory action of the volatiles present in E. saligna leaf litter. CNPQ.

220

PLANT BETA-GLUCOSIDASES RESIST DIGESTION BY A LEPIDOPTERAN HERBIVORE TO PROMOTE SUSTAINED ACTIVATION OF CHEMICAL DEFENSES.

Daniel G. VASSAO1*; Natalie WIELSCH2; Ana Maria M. M. GOMES2; Yvonne HUPFER2; Ales SVATOS2; Jonathan GERSHENZON2

1Max Planck Institute for Chemical Ecology, Jena, GERMANY, ,Jena, GERMANY, GERMANY 2Max Planck Institute for Chemical Ecology, Jena, GERMANY, ,Jena, GERMANY, GERMANY

Proteins involved in plant defense have been shown to act even after ingestion by herbivores to carry out defensive actions within the confines of an herbivore’s digestive system. These plant proteins can for example interfere with digestive processes or deplete essential nutrients before they can be absorbed. Additionally, the deglucosylation of plant chemical defenses such as the benzoxazinoids of maize or the glucosinolates of the Brassicaceae to form the corresponding toxic aglucones has been shown to be catalyzed by plant proteins that act within the insect during and after ingestion.Here, we show that selected plant defensive beta-glucosidases are particularly resistant to digestion by a generalist insect herbivore, Spodoptera littoralis Boisduval. While most plant proteins are efficiently degraded, these beta-glucosidases retain much of their enzymatic competence throughout digestion even in the resulting feces. We have studied feces from larvae fed on maize and white mustard (Sinapis alba Linn) using proteomic methods, and have detected unambiguous peptides derived from individual plant beta-glucosidases, but not from co-expressed isoforms with high sequence similarity. This suggests that particular beta-glucosidase isoforms are more relevant than others for defense in vivo, although the others may also be active in vitro. Eighteen additional maize proteins, including a beta- glucosidase aggregation factor, were also detected in feces from maize-fed insects and could play a role in post-ingestive plant defense. The resilience of these defensive proteins supports the notion that evolutionary selection for resistance against digestion has increased plant defense capacity, in this case by maximizing the plant’s ability to activate its chemical defenses upon herbivory.

221

POPLAR COMMUNICATIONS: HOW A WOODY PLANT TEXTS ITS HERBIVORES, HERBIVORE ENEMIES AND NEIGHBORS.

Jonathan GERSHENZON1*; Andrea Clavijo MCCORMICK1; Sandra LACKNER1; Andreas BOECKLER1; Andreas REINICKE1; Sybille UNSICKER1

1MAX PLANCK INSTITUTE FOR CHEMICAL ECOLOGY, JENA, GERMANY, Jena, Jena, GERMANY

The ability of plants to communicate with a wide-range of other organisms is a vibrant theme in modern chemical ecology. There is evidence for information exchange between plants and neighbors, competitors, symbionts, pathogens, parasites, herbivores, pollinators and other visitors. The presence of so many different possible receiver organisms may explain why plants often release complex mixtures of volatiles.We have been studying black poplar (Populus nigra) volatiles to learn more about communication in a wild species of woody plant. This native European taxon releases an enormous diversity of volatile compounds upon herbivore attack. I will review our findings about how herbivores, herbivore enemies and neighboring trees respond to these volatiles. Generalist caterpillars are attracted to the constitutive and herbivore-induced odors of black poplar leaves, but experienced conspecifics prefer the odor of undamaged foliage. Herbivore parasitoids are more attracted to black poplar foliage damaged by one of their hosts than to adjacent undamaged foliage. Individual trees respond to volatile emission from herbivore-damaged neighbors by increasing their own defenses against herbivores. I will discuss the significance of this communication under natural conditions, and compare the role of terpene, green leaf, aromatic and nitrogenous volatiles in conveying information to various organisms.

222

PLANT BIO-WARS: PROTEIN NETWORKS REVEAL TISSUE-SPECIFIC DEFENSE STRATEGIES.

Lina CASTANO-DUQUE1; Dawn S. LUTHE1*

1The Pennsylvania State University, State College, Pennsylvania, United States

Plant metabolic changes against insects include a wide variety of mechanisms that allow the plant to survive infestation, but a proteomics and network analyses from insect infested maize has not been done before. Using tandem mass spectrometry tags (TMT), we measured protein abundance changes in maize roots and leaves in response to leaf pest, Spodoptera frugiperda (FAW) or root pest, Diabrotica virgifera (WCR). We analyzed our TMT data by using a protein-protein interaction network and a protein co- abundance network. We detected 4677 proteins of which 794 had significant changes under FAW infestation, and 4878 proteins of which 863 had significant changes under WCR infestation. We found proteins with higher abundance under FAW or WCR infestations that are involved in the jasmonic acid (JA) pathway like lipoxygenase 5 (lox5), allene oxidase synthase 1 (aos1), and 12-oxophytodienoate reductase 2 (opr2). Other proteins with higher abundance under infestations are part of abscic acid (ABA) signaling, reactive oxygen species signaling, and ethylene (ET) biosynthesis and signaling pathways. We measured the changes in those pathways by looking at the gene expression involved in metabolism or signaling of JA, ABA and ET. Furthermore, we performed a gene ontology analysis of the protein-protein interaction network that revealed differences in oxidoreductase activity between FAW or WCR infestation. Thus, we determined the redox state and gene expression of several mitogen-activated protein kinases of the plant in leaf and root tissues under FAW and WCR infestation. Our data shows promising differences among leaves and roots of plants infested by FAW or WCR. We are exploring innovative research tools that will advance the knowledge in plant- insect signaling area.

223

PLANT COMMUNICATION IN THE RHIZOSPHERE.

Ted TURLINGS1*

1University of Neuchâtel, Neuchâtel, Neuchâtel, Switzerland

Plants excrete numerous compounds from their roots. These root exudates are not mere waste products, but can serve various functions to control abiotic and biotic processes. Particularly intriguing are root exudates that control mutualistic interactions with soil- dwelling organisms. These mutually beneficial plant-mediated signals are not only of fundamental ecological interest, but the root-produced signals may also be exploited for crop improvement and protection. This is illustrated by our work on root-feeding larvae of the beetle Diabrotoca virgifera virgifera (Western corn rootworm), which cause tremendous losses to maize growers in the USA and Europe. Entomopathogenic nematodes (EPN) are a possible solution to fight this pest. hese tiny parasitic worms kill the rootworm larvae within days and we have found that they are attracted to E-(β)- caryophyllene, a sesquiterpene that is specifically emitted from maize roots after rootworm attack. American maize lines have lost the ability to emit this signal. Using genetic transformation we restored caryophyllene emission in one such line and in field trials this was shown to result in enhanced EPN attraction and better protection against rootworm damage. Using our knowledge of these rhizosphere interactions, we are currently developing new strategies for the application of entomopathogenic nematodes to control rootworms and other soil pests. These strategies involved: 1) the selection for highly effective nematode strains, 2) the application of nematodes in beads that can be planted with maize seeds, and 3) increasing the “self life” of nematodes with the use of a plant-derived compound that puts them in a state of quiescence.

224

QUANTIFYING CHEMICAL DIVERSITY AND ITS ECOLOGICAL CONSEQUENCES.

Kaitlin OCHSENRIDER1; Josh JAHNER1; Eric TEPE2; Lora RICHARDS3; Matt FORISTER3; Lee DYER3; Angela SMILANICH3; Craig DODSON3; Christopher JEFFREY3*

1University of Nevada, Reno, Reno, Nevada, UNITED STATES 2University of Cincinnati, Cincinnati, Ohio, UNITED STATES 3University of Nevada, Reno, Reno, Nevada, UNITED STATES

Ecological interactions have driven the evolution of the complex phytochemical profiles, or mixtures of natural products, found in plants today. Recent advancements in instrumental analysis that offer increased sensitivity and resolution, such as proton nuclear magnetic resonance (1H-NMR) spectroscopy, are allowing scientists to more rapidly unravel complex mixtures of small molecules, which we apply to the study of the role of secondary metabolites in mediating interactions between organisms. Our group has initiated studies toward the development of metabolomics approaches using 1H-NMR spectroscopy that allow one to make quantitative intra- and inter-species comparisons of plant metabolic profiles. The focus of our studies is the phytochemically diverse tropical plant genus called Piper with the goal of making quantitative cross-taxa comparisons of plant secondary metabolism in order to understand the evolution of these secondary metabolites across the entire genus of Piper (Kato & Furlan 2007). A number of plant samples from the monophyletic Radula clade were collected from Brazil, Costa Rica, Ecuador, and Panama and their crude mixtures were extracted. Metabolomic data was collected using 1H-NMR spectroscopy, which provided a metabolic fingerprint for each plant sample. Spectral data were binned and analyzed with a weighted gene co- expression network analysis (WGCNA) which produced 19 modules, or clusters of co- occurring chemical shift values (Zhang & Horvath 2005). A heatmap generated from the 19 modules allowed visualization of both positive and negative correlations between clusters of chemical shift values that were shown to change together across different spectra. With further development of this tool, co-occurrence of chemical shift values could be extended to co-occurrence of specific molecules, leading to an enhanced ability to detect potential synergistic mixtures produced by plants. Mapping the modules generated by our approach onto a phylogeny will provide insight into the evolution of phytochemical diversity in Piper.

225

RESPONSE OF TWO MIRID PREDATORS AND COMPARATIVE ANALYSIS OF HERBIVORE-INDUCED PLANT VOLATILES FROM PEST INFESTED AND UNINFESTED TOMATO PLANTS.

Diego SILVA1*; Joop VAN LOON2; Vanda BUENO3; Joop VAN LENTEREN3; Juracy JINS JR4; Berhane WELDERGERGIS5; José Maurício BENTO5

1Laboratory of Chemical Ecology and Insect Behavior, Department of Entomology, Luiz de Queiroz College of Agriculture (ESALQ), University of São Paulo, Piracicaba, São Paulo, BRAZIL 2Wageningen University, Wageningen, Holanda, HOLANDA 3Universidade Federal de Lavras, Lavras, MInas Gerais, BRAZIL 4Centro universitário de Várzea Grande, Várzea Grande, Mato Grosso, BRASIL 5Wageningen University, Wageningen, Holanda, HOLANDA

Plants release a variety of volatile organic compounds (VOCs) that play multiple roles in the interactions with other plants and animals. Natural enemies of plant-feeding insects use these volatiles as cues to find their prey. Here we report the attraction of two mirid predators Nesidiocoris tenuis and Macrolophus pygmaeus toward plant volatiles induced by two key tomato pests (Tuta absoluta and Bemisia tabaci) in olfactometer bioassays. We subsequently compared the volatile composition of different plant sources by collecting headspace samples and analyzing them with GC-MS. Nesidiocoris tenuis responds to volatiles produced by uninfested plants only after experience with the plant, whereas naïve and experienced M. pygmaeus show positive chemotaxis to these plants. Both predators are attracted to volatiles from prey-infested plants, and we provide the first evidence that experience affects this response in M. pygmaeus. A total of 80 VOCs were recorded of which 10 compounds only occurred consistently in headspace of T. absoluta-infested plants. Plants damaged by T. absoluta emitted at least 10 times higher levels of many compounds compared to plants damaged by B. tabaci and intact plants. Our findings imply that feeding by T. absoluta and B. tabaci induced quantitative and qualitative changes in the emission of VOCs, providing a chemical basis for behavioral discrimination by two generalist predatory mirid species, natural enemies of T. absoluta and B. tabaci employed in biological control.

226

SEQUESTRATION AND TRANSPORT PROTEINS IN LEAF BEETLE LARVAE.

Wilhelm BOLAND, Antje BURSE1*

1Department of Bioorganic Chemistry. Max Planck Institute for Chemical Ecology, Jena, Thuringia, GERMANY

Leaf beetles occur worldwide; some of them are even economically important pests of of agri•culture. They raise broad ecological interest because of their host plant preferences and varying modes of life. Their larvae have developed an impressive repertoire of toxins and repellents to defend themselves against predators. Upon attack, the larvae discharge small droplets from specialized glandular reservoirs on their back (e.g. Chrysomela populi). The reservoirs represent “bioreactors” performing all late reactions of the toxin- produc•tion, starting from plant-derived or de novo synthesized glucosides of non-toxic, early precursors of the compounds in the defensive droplets. The import of the plant- derived glucosides relies on a sophisticated trans•port system. A functional network of (ABC) transporters guidethe plant derived glucosides through the larval body into the defensive system. Excess of the compounds is excreted via other transporters in the Malpighiantubules. The biosynthetic enzymes and transport proteins have been identified by sequencing and silencing by RNAi. RNAi allowed a rapid functional screening of putative enzymes and their homologs, followed by cloning and functional expression of the positively identified candidates. Due to the high efficiency and high throughput, even large protein families can be analyzed for relevant candidates, e.g. 65 ABC transporter and 68 GLUTs in C. populi and P. cochleariae (1,2,3).

(1) A.S. Strauss et al., ABC transporter functions as a pacemaker for the sequestration of plant glucosides in leaf beetles. eLife (2013), http://dx.doi.org/10.7554/eLife.01096

(2) M. Stock, R.R. Gretscher, M. Groth, S. Eiserloh, W. Boland, A. Burse. Transcriptome-wide analysis of the SLC2 sugar transporter family in the mustard leaf beetle Phaedon cochleariae. (2013) Plos one, 8(12):e84461

(3) W. Boland, Sequestration of plant-derived glycosides by leaf beetles: A model system for evolution and adaptation. (2015). Perspectives in Science, 6:38-48.

227

SYMBIOSIS WITH SYSTEMIC FUNGAL ENDOPHYTE CHANGES LEAF LITTER VOLATILES IN LOLIUM MULTIFLORUM.

Laura VENTURA1; Patricia FERNANDEZ2; Marina OMACINI3*

1Facultad de Agronomía, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Buenos Aires, ARGENTINA 2Estación Experimental Agropecuaria INTA Delta del Paraná - CONICET, Ciudad Autónoma de Buenos AiresBuenos Aires, Buenos Aires, ARGENTINA 3IFEVA – Facultad de Agronomía, Universidad de Buenos Aires - CONICET, Ciudad Autónoma de Buenos Aires, Buenos Aires, ARGENTINA

Many grass species are associated with maternally fungal endophytes that may provide chemical defenses against herbivorous insects. These chemical defenses are normally associated with the production of different types of alkaloids, which can protect plants against vertebrate and invertebrate herbivores. Recent studies showed that endophytic plants and their dead tissues (litter) conferred protection by association to neighboring plants to herbivores. The aims of this study were to characterize the volatile compounds emitted by plants with or without endophytes, and to determine the mechanism by which the presence of litter from endophyte plants affects herbivore preference. We hypothesize that the presence of fungal endophytes generates changes in litter volatile production that reduces herbivore attraction. Here the volatiles released by leaf litter from Lolium multiflorum were analyzed by coupled gas chromatography-mass spectrometry. The volatile profile of endophyte and non-endophyte leaf litter showed cuali-quantitative differences, supporting the idea that plants change their volatile profile in presence of a fungal endophyte. In order to evaluate the protective effect endophyte L. multiflorum litter on the foraging behavior of generalist hebivores on willow leaves, dual choice experiments were performed with colonies of leaf cutting ants, Acromyrmex ambiguous. We recorded feeding preference of willow leaves protected by 10 g of leaf litter from endophytic and non-endophytic plants. Ants collected irrespectively of the kind of leaf litter by which they were protected, indicating that these volatiles did not seem to affect their foraging behavior. This study showed that the presence of endophytes in leaving plants, have an afterlife effect altering the volatile profile of leaf litter but it did not seem to be the mechanism for affecting ants preference.

228

WHO’S NEXT? INSECT OVIPOSITION REVEALS THE IDENTITY OF THE UPCOMING HERBIVORES TO NICOTIANA ATTENUATA.

Sylvia DROK1*; Michele BANDOLY1; Anke STEPPUHN1

1Free University of Berlin, Berlin, Berlin, GERMANY

Several plants are known to use insect oviposition as a warning for upcoming herbivory. Oviposition by Spodoptera exigua and Manduca sexta primes the feeding-induced production of caffeoylputrescine in Nicotiana attenuata, and as a consequence their larvae endure a lower performance or a reduction in an immune parameter, respectively (Bandoly et al., 2015, 2016). We used microarrays to study how a prior oviposition shapes this plant’s transcriptional regulation upon herbivory. We analysed plants in response to their oviposition and larval feeding by both species in a full-factorial design. In coherence with previous studies, N. attenuata’s response to feeding by S. exigua and M. sexta strongly diverged. Remarkably, several analyses revealed that, when oviposited by M. sexta, the transcriptional profiles of S. exigua-fed plants became more similar to those of M. sexta-fed plants, and vice versa. These results suggest that plants use insect oviposition to prepare its herbivore-specific defence response to the feeding larvae.

Bandoly, M., Hilker, M., and Steppuhn, A. (2015). Oviposition by Spodoptera exigua on Nicotiana attenuata primes induced plant defence against larval herbivory. Plant J. 83, 661–672.

Bandoly, M., Grichnik, R., Hilker, M., and Steppuhn, A. (2016). Priming of anti- herbivore defence in Nicotiana attenuata by insect oviposition: herbivore-specific effects. Plant Cell Environ. doi: 10.1111/pce.1267

229 ROLE OF CHEMICAL ECOLOGY IN SOCIAL BEHAVIOR, AND VICE VERSA

CUTICULAR CHEMISTRY OF THE STINGLESS BEE TETRAGONISCA ANGUSTULA.

Maria Sol BALBUENA1*; Andrés GONZALEZ2; Walter FARINA2

1Laboratorio de Insectos Sociales, Departamento de Biodiversidad y Biología Experimental, IFIBYNE-CONICET, FCEyN, Universidad de Buenos Aires, Buenos Aires, CABA, ARGENTINA 2Laboratorio de Ecología Química, Facultad de Química, UdelaR, Montevideo, Montevideo, URUGUAY

In social insects, nestmate and task-related recognition play an important role in the coordination and cohesion between members of the colony. We focused our study on the stingless bee Tetragonisca angustula, which presents morphological worker subcastes1. These bees have a sophisticated system of defense involving two complementary groups of guards: hovering guards (suspended in the air close to the hive entrance) and standing guards (stand on the inner or outer surface of the entrance tube). We identified, quantified and compared the cuticular compounds of worker bees performing different tasks, namely in-hive bees, foragers and guards. In-hive bees were captured directly from inside the hive, foragers were trained to an artificial feeder and collected while feeding, and the two groups of guard bees were collected at the hive entrance. All bees were frozen immediately after capture, and cuticular compounds were extracted in dichloromethane and analyzed using gas chromatography-mass spectrometry (GC-MS). We found that, in addition to hydrocarbons (CHCs), cuticular extracts contained small amounts of fatty acids and abundant diterpenes and triterpenes that remain to be characterized. Among the CHCs, we found a mixture of normal saturated hydrocarbons with uneven carbon chains (C-25, C-27, C-28, C-31), along with minor amounts of methyl branched saturated CHCs as well as mono- and di- unsaturated hydrocarbons. The amounts of CHCs were quantified relative to an internal standard, and compared statistically by Principal Components Analysis (PCA). We found significant differences in the relative abundance of CHCs between behavioral groups. In-hive bees present lower amounts of alkanes relative to both groups with outside tasks. In addition, guard bees present higher amounts of branched saturated hydrocarbons. Different CHC profiles associated with behavioral groups suggests a mechanism for chemical recognition of nestmates of different subcastes. 1 Grüter et al. 2012, PNAS 109 (4): 1182-1186

231

CHEMICAL ECOLOGY OF POLLINATION SYSTEMS MEDIATED BY NOCTURNAL BEES: A BRAZILIAN-AUSTRIAN INITIATIVE.

Isabel Alves dos SANTOS1*

1University São Paulo, São Paulo, SP, BRAZIL

Diurnal bees are the most important pollinators of angiosperms. In several groups of bees a nocturnal/crepuscular habit was developed, yet, little is known about the role of nocturnal bees in pollination, how they communicate with their host plants, and whether some plants are adapted to these bees. Since 2014 we are working on a project entitled “Recognition of flowers in nocturnal bees”, which is supported by CNPq within the Science without Borders program. Researchers from Brazil and Austria are involved, overall c. 20 people. Our project overall aims to i) evaluate the role of nocturnal bees as effective pollinators of crop and non-crop plants, ii) identify floral scents of host plants of such bees by dynamic headspace-gas chromatography/mass spectrometry, and iii) determine biologically active compounds in the bees. Here, we characterize the floral scents of Brazilian crop plants visited by nocturnal bees, among them cambuci and guarana. We also identify floral scent compounds capable of attracting nocturnal bees in the field. Altogether, our study describes the first scent-mediated pollination systems between plants and their nocturnal bee pollinators. With the identification of the volatile floral compounds of the focal plants emphasizing the dominant ones, it is possible to test their attractiveness on the bees using biotests and electrophysiological responses of the antennae. Moreover, this project is strengthening the research area Chemical Ecology associated on Pollination in all involved working groups and consolidates the already existent cooperation.

232

CHEMICALLY MIMETIC SOCIAL PARASITE PRODUCES NON-MIMETIC BROOD.

ELIA Marta1*; LORENZI Maria Cristina2; BAGNèRES Anne-Geneviève3

1Univ. of Turin - I.R.B.I., Turin - Tours, Turin - Tours, Italy - FRANCE 2Univ. Paris 13 - Univ. of Turin, Villetaneuse - Turin, Villetaneuse - Turin, France - ITALY 3I.R.B.I. - CNRS, Tours, Tours, FRANCE

The wasp Polistes atrimandibularis is an obligate social parasite. It lacks a worker caste and instead relies on its host, the free-living social wasp Polistes biglumis, for survival. P. biglumis has an efficient nestmate recognition system, which is largely based on colony- specific chemical signatures. While both host and parasite display species-specific signatures, female parasites avoid detection by chemically mimicking the colony they invade.As a parasite takes over a nest, it progressively loses its species-specific compounds (alkenes). After a month, host and parasite chemical signatures are indistinguishable. When the parasite’s offspring emerge from the nest later in the season, they carry the alkene-rich, parasite-specific signature. Our goal was to investigate the chemical signatures of the preimaginal stages of P. atrimandibularis. Once a host foundress has been enslaved, she will accept and care for the parasite’s brood as if it were her own. This observation raises several questions. Does the parasite’s brood adopt a strategy for integrating itself into the host nest, just like adult parasites do? Does the parasite’s brood mimic the chemical signature of the host’s brood? We analyzed the chemical signatures of 160 preimaginal brood (egg, larval, and pupal stages) from 9 different parasitized and non-parasitized nests (via GC-MS).We found that the chemical signature of the two broods was generally poor in hydrocarbons, especially in branched alkanes and linear alkanes, present with a relatively lower proportion. However, the most abundant compounds in parasitic brood were alkenes (primarily nonacosene), which are absent in the signature of both host brood and adults. These results suggest that the parasite’s brood escapes host detection in a different way than do adult parasites.

233

DO QUEENS OF PRIMITIVELY EUSOCIAL INSECTS USE PHEROMONES TO DOWNREGULATE WORKER REPRODUCTION?.

Fabio S. NASCIMENTO1*; Ivelize C. TANNURE-NASCIMENTO1; Cíntia OI2; Aline C. R. ANDRADE3; Tom WENSELEERS4

1Universidade de São Paulo, Ribeirão Preto, São Paulo, BRAZIL 2University of Leuven, Leuven, Leuven, BELGIUM 3Universidade de São Paulo, Ribeirão Preto, São Paulo, BRAZIL 4University of Leuven, Leuven, Leuven, BELGIUM

Chemical communication is an important aspect for the organization of social insect colonies. In most advanced species, queen pheromones are used to signal the presence of a fertile queen and workers usually either remain sterile or do not lay eggs. However, in primitively eusocial species, where there are no morphological differences between females, the dominant female can assure her reproductive monopoly probably through behavior together with other cues as visual and chemical cues. We have studied the effect of chemical signals on subordinate females in the primitively eusocial wasps, bees and ants. Firstly, we analyzed the chemical profile of all wasps from females of Polistes satan Bequaert (Vespidae), Euglossa melanotricha Moure (Apidae) and Dinoponera quadriceps Santschi (Formicidae). We selected the cuticular hydrocarbons that were overexpressed in dominant females and applied synthetics in queenless colonies. The bioassays showed that the compounds alone did not inhibit both worker reproduction and ovary development. Therefore, we concluded that the presence of reproductive dominant females has a major negative effect to downregulate subordinate’s reproduction. Other cues such as behavioral inhibition and visual cues act to establish the hierarchy of dominance and probably, chemical cues can be used a secondary cue to advertise the reproductive potential of the dominant female.

234

EFFECT OF THE ESSENTIAL OIL OF EUPATORIUM BUNIIFOLIUM IN THE PRODUCTION OF THE HONEY BEE PHEROMONE, ETHYL OLEATE.

Federico RODRIGO1; Carmen ROSSINI1*; Belén DAVYT1; María L. UMPIÉRREZ1; Martín PORRINI2; Melisa GARRIDO2; Martín EGUARAS2

1Laboratorio de Ecología Química, Facultad de Química, UdelaR, Montevideo, Montevideo, URUGUAY 2Centro de Investigación en Abejas Sociales. Departamento de Biología. Universidad Nacional de Mar del Plata y CONICET, Mar del Plata, Buenos Aires, ARGENTINA

Task allocation in the honeybee hives is regulated by many signals including ethyloleate, a primer pheromone that delays the change from nursing to foraging. The correct balance of tasks is essential to keep the hive health. Beekeeping is threaten by many sanitary problems including parasitic mites. During a prospecting program in search for essential oils with acaricidal activity, we identified the essential oil from Eupatorium buniifolium (Asteraceae) as a candidate to control mites with no toxic effects on bees1. Since some acaricides can disturb the ethyloleate levels, we studied the effect of orally ingested E. buniifolium essential oil on the ethyloleate produced by worker bees. Two essential oils extracted from E. buniifolium plants from the same geographical location but extracted at different times were included in the honeybee diet and their effect on ethyloleate was quantified in a dose-dependent study. Hexane extracts from bees were partitioned by SPME, and the fraction containing the ethyloleate was analyzed by GCMS using arachidonic acid methyl ester as internal standard2. Results showed that the 2 essential oils applied exhibited quantitative differences in the fractions containing the monoterpene hydrocarbons (57 vs. 30%) and the sesquiterpene hydrocarbons (41 vs. 69%). Despite these chemical differences, when these essential oils were ingested no effects (MANOVA) were found on the ethyloleate levels compared to the control bees either for treatment (p = 0.494) of for doses (p = 0.532). Therefore, eventhough the use of conventional acaricides have been shown to affect ethyloleate levels3, these results suggest that the use of natural products such as essential oils to treat comb diseases may not alter the normal colony homeostasis.

(1) Parasitology Research 2013, 112, 3389. (2) J. Chem. Ecol. 2010, 36, 522. (3) In III Encuentro de la Asociación Latino Americana de Ecología Química (ALAEQ III) Bogotá-Colombia, 2014.

235

HOME IS WHERE THE POOP IS: DEVELOPMENT OF SHELTER-SEEKING BEHAVIOR IN ORIENTAL COCKROACH.

Constanza SCHAPHEER1*; Alejandro VERA1; Jan BERGMANN2; Cristian VILLAGRA3

1Instituto de Entomología, UMCE, Santiago, Regiión Metropolitana, CHILE 2Instituto de Química, PUCV, Valparaíso, Región de Valparaíso, CHILE 3Instituto de Entomología, Santiago, Región Metropolitana, CHILE uccessful sheltering is a crucial behavior for animals’ survival in general. Among the factors influencing shelter-seeking behavioral development in insects, sex and physiological maturity play a key role: both are associated with endocrine system changes where chemical stimuli operate as cues in several species. In this work we studied these phenomena in the cosmopolitan cockroach Blatta orientalis (Blattodea: Blattidae). We followed sheltering behavioral preferences three developmental stages: i) nymph stage 2 to 4, ii) 5 to 7 and iii) 7 to 9. To these nymphs we presented simultaneously their own home shelter; this treatment corresponded to a cardboard cockroach shelter where the test subjects experienced molting and living, therefore its marked with their own feces, versus control, which corresponded to unused cardboard shelters. We found that females and males preferred arena side where home shelter was present however; while early nymph females tend to remain more time inside home shelter they developed a more exploratory behavior in later developmental stages. Meanwhile, males spent significantly less time hidden inside home shelter and explored more outside area of home arena side in all ontogenetic stages tested. We suggest that the presence of conspecifics feces is an attractive cue in this cockroach, and that the response to its chemical composition will vary depending on the sex and age. We propose that B. orientalis females early stages interact with these cues expressing an aggregative behavior and as they grow they are capable of de-attach from this cue and develop more exploratory behaviors outside home shelter. Meanwhile males express this late pattern from early stages, most likely related to foraging and dispersal behaviors. We discuss on behavioral development of physiological (endocrine) influences with the developmental environment interactions and consider this information to generate integrated control strategies for these urban pests. Keywords: Synanthropic insects, Imprinting, Sexual Learning. .

236

HOSTS AND PARASITES: CHEMICAL COMMUNICATION AND SIGNAL INTERCEPTION IN TWO POLISTES SOCIAL WASPS.

ELIA Marta1*; LORENZI Maria Cristina2; CHRISTIDèS Jean-Philippe3; LUCAS Christophe3; BAGNèRES Anne-Geneviève3

1Univ. of Turin - I.R.B.I., Turin - Tours, Turin - Tours, ITALY - FRANCE 2Univ. Paris 13 - Univ. of Turin, Villetaneuse - Turin, Villetaneuse - Turin, FRANCE - ITALY 3I.R.B.I. - CNRS, Tours, Tours, FRANCE

In Polistes social wasps, nest odor is important for colony odor learning by young wasps. Usually, foundresses mark their paper nests with their own chemical signature. When obligate social parasites invade social wasp colonies, they become chemically indistinguishable from host colonies, but how they contribute to nest odor is unclear. In the present work we examine nest odor in the mountain P. biglumis social wasp and we compare changes occurring in free-living colonies with those occurring in colonies parasitized by the social parasite P. atrimandibularis, to identify the selective pressures imposed by social parasites on the chemical signature of their hosts. We analyzed the compounds of 150 nest surfaces from two populations in the Alps by collecting small pieces of nests all along the nesting season. Colony odor changes largely in quality and even more in quantity, with heavier compounds increasing in all nests through the summer. We also tracked for the first time the effect of the chemical strategy of the parasite on host nest odor and showed that, within very few days from invasion, parasite- specific unsaturated compounds appear and then disappear from the surfaces of the host nests, contributing to the integration of the parasites in host colonies and to their take over.

237

IMPACT OF A CUTICULAR COMPOUND CORRELATED WITH FERTILITY IN THE ESTABLISHMENT OF HIERARCHIES IN NEOPONERA APICALIS (HYMENOPTERA: FORMICIDAE).

Alexis LOUIS1; Stéphane CHAMERON1; Nicolas CHÂLINE2*

1Laboratoire d’Ethologie Expérimentale et Comparée (LEEC), Université Paris 13, Villetaneuse, Sena-Saint Denis, FRANCE 2Laboratorio de Etologia, Ecologia e Evolução dos Insetos Sociais (LEEEIS), Departamento de Psicologia Experimental, Instituto de Psicologia, USP, São Paulo, SP, BRASIL

In most ant species, the royal caste has the monopoly over reproduction. However, in many species, workers still have reproductive abilities and can lay unfertilized male- destined eggs. The death of the queen therefore leads to the expression of a reproductive conflict within the worker caste, which leads to the establishment of a linear hierarchy. High-ranking workers in the newly formed hierarchy reproduce when the others perform the remaining ergonomic tasks. The set-up of this hierarchy takes place over a short period (approximatively 48 h) and is characterized by virulent agonistic interactions such as antennal boxing and biting between workers. Recent studies have shown that some cuticular compounds are correlated with fertility and hierarchical position, and that they could act as honest signals of worker status and reproductive abilities. It has also been demonstrated that these signals could be perceived by the workers and modulate their social behavior during dyadic encounters. Our experiments studied the impact of a potential fertility signal on the establishment of reproductive hierarchies within the worker caste in Neoponera apicalis morph 1, one of the 11 morphs in this complex of cryptic species. To do this, we orphaned groups of workers who had undergone different treatments within the same group: increase of the putative fertility signal, increase of an hydrocarbon not-correlated with fertility and manipulated controls. By tracking individual behavior over a period of 5 days, we were able to characterize the influence of pre-existing heterogeneities in the cuticular profiles on the establishment of hierarchies. We subsequently manipulated fertility signals among high rankers of the established experimental hierarchies to observe the impact of this modification on the dynamics of group dominance.

238

INNOVATIVE APPROACHES TO CONTROLLING A THREAT TO HONEYBEES IN THE SUB-TROPICS.

RAndrew HAYES1*; Brogan AMOS2; Steven RICE3; Kate MCGLASHAN3; Diana LEEMON3

1University of the Sunshine Coast, Sippy Downs, Queensland, AUSTRALIA 2The University of Queensland, St Lucia, Queensland, AUSTRALIA 3Queensland Department of Agriculture and Fisheries, Dutton Park, Queensland, AUSTRALIA

Honeybees (Apis mellifera) and native stingless bees (Tetragonula spp.) are critical pollinators for crops throughout Australia. These bees are now under threat from the exotic small hive beetle (Aethina tumida). This beetle is a scavenger and minor pest of weakened and stressed honeybees in its native sub-Saharan range. The larval beetles feed on hive products which they contaminate with a yeast, Kodamaea ohmeri, which is responsible for the fermentation and destruction of these products. Under the warm, moist conditions in Queensland, the small hive beetle has become a serious apiary pest responsible for losses in excess of $8 million over three seasons. Current control options are only appropriate for some managed honeybee colonies. There is a critical need for a trap that can be deployed for beetle monitoring and control in both managed and unmanaged honeybee and native bee colonies. We are developing an external trap to attract and kill the small hive beetle. The beetles are attracted to a range of hive odours, especially the odours of fermenting hive products. We are investigating the use of these fermentation odours along with beetle pheromones to develop an economical synthetic lure for use in a small hive beetle specific trap. Preliminary field trials using a crude fermentation product trapped beetles both near apiaries and away from apiaries. Using gas chromatography-mass spectrometry and choice-test behavioural assays key attractive volatiles have been identified. Combinations of these key volatiles have been tested in the laboratory and field, results will be discussed.

239

MODULATION OF FEEDING BEHAVIOR FOLLOWING STARVATION IN ADULT WORKER HONEY BEES.

Rodrigo VELARDE1*; Maria Gabriela de Brito SANCHEZ2; Lucie DESMEDT2; David HALE3; Martin GIURFA4

1University of Buenos Aires, Buenos Aires, Buenos Aires, ARGENTINA 2CNRS-University Paul Sabatier Toulouse III, Toulouse, Midi-Pyrenees, FRANCE 3Wake Forest University, Winston-Salem, North Carolina, USA 4CNRS-University Paul Sabatier Toulouse III, Toulouse, Midi-Pyrenees, FRANCE

The Neuropeptide Y (NPY) signaling pathway regulates food searching and food intake across animals. In Drosophila, upregulation of this pathway by starvation increases the likelihood to feed on noxious foods. Here, we tested whether starvation increased the propensity of adult worker bees to ingest foods that would otherwise be rejected if a more palatable choice is available. We also assessed if NPY-like signaling modulates the feeding response in this context. The propensity to ingest unpalatable foods increased only in starved foragers while it remained unchanged in the starvation resilient winter bees. Starved foragers displayed an increase in NPY-like signaling, and inhibition of this signaling pathway by reducing the level of expression of snpfR, decreased their feeding response. Therefore, blocking NPY-like signaling in starved foragers induced an altered perception of the satiation state. These results confirm that NPY signaling in honey bees conserves a central role in the perception of the nutritional status that may be linked to the decision to ingest unpalatable foods after starvation. We discuss our results in light of an emerging pattern of functional divergence rather than conservation in the NPY-like pathway that reflects the diversity of feeding-related behaviors across insects.

240

MANDIBULAR GLAND PHEROMONES FROM AFRICAN HONEYBEES: NEW INSIGHTS AND PROSPECTS AS A COMPLEMENTARY TAXONOMIC TOOL.

Abdullahi YUSUF1*; Christian PIRK1; CREWE1

1University of Pretoria, Pretoria, Gauteng, SOUTH AFRICA

Communication in eusocial insects relies mostly on pheromones and the secretion of the mandibular glands in honeybees plays an important role in regulating reproduction and other behavioural processes. The composition of the secretions from the mandibular glands of honeybees is relatively well understood, with those of the queens dominated by ώ-9 fatty acids and ώ-10 fatty acids dominating those of non-laying workers. African honeybees belong to the Apis mellifera complex and the continent is home to eleven of the thirty morphologically described sub-species in the Apis mellifera complex. Despite this morphological diversity and the importance of mandibular gland pheromones in honeybee communications, its chemistry has only being studied in four of the 11 African sub-species. Using classical chemical ecological techniques, we studied mandibular gland pheromones from two sub-species; one found in West Africa (A. m. adansonii) and another from Madagascar (A. m. unicolor). We describe the composition of mandibular gland pheromones of these sub-species for the first time and use the pheromone data to complement morphological and genetic characters to draw conclusion on the taxonomy of African honeybee subspecies. Mandibular gland profiles from both workers of A. m. adansonii and A. m. unicolor was able to differentiate workers into regions, morpho and genetic clusters. Furthermore, we conclude based on their pheromone profiles about the evolutionary relationships of the Cape honeybee A. m. capensis, savannah honeybee A. m. scutellata, the Islanders A. m. unicolor and westerners’ A. m. adansonii are related.

241

SOLENOPSIS INVICTA AND SOLENOPSIS RICHTERI HYBRIDIZATION IN THE USA AND SOUTH AMERICA – PAST AND PRESENT.

Robert Vander MEER1*; Sanford PORTER1; Daniel WOJCIK1; Juan BRIANO2

1USDA/ARS, Gainesville, FL, USA 2Fundación para el Estudio de Especies Invasivas, Hurlingham, Buenos Aires, ARGENTINA

Invasive fire ants are responsible for many billions of dollars annually in, for example, crop/livestock losses, medical and control costs. The classical taxonomy of Solenopsis species is extremely tenuous and molecular methods are not yet capable of differentiating potential cryptic species. Here we outline some of the problems in using biochemical characters, e.g, venom alkaloids, cuticular hydrocarbons, and Dufour’s gland profiles to define the range of the USA S. invicta and S. richteri in South America and how this relates to the hybridization of these two species in the USA. We also look at invasive fire ant samples collected in the late 1940s, 1960s, and 1980s in the USA to better understand the temporal progression of the hybridization event.

242

STATE OF THE ART IN HONEY BEE CHEMICAL ECOLOGY.

Yves Le CONTE1*

1INRA, AVIGNON, AVIGNON, FRANCE

Chemical communication is one of the most fascinating aspects of social insects as they use two types of pheromones, releaser and primer strongly involved in the development of the colony. When many releaser pheromones were discovered in the animal kingdom, only a few primer pheromones, modulating physiology of the recipient, have been identified, most of them in the honey bee Apis mellifera. The honey bee is probably one of the most extensively studied models in chemical ecology. Recent studies on honeybee pheromones suggest that chemical communication is much richer than we thought and deeply involved in social regulations. More than 50 chemical compounds have been identified having a pheromonal effect on the honey bee. I will present a review of findings on releaser pheromones produced by the colony giving a special emphasis on the different primer pheromones and their interactions on social regulations between the different actors of the colony. The same pheromonal compound can be produced by different actors of the colony and triggers both releaser and primer effects. I’ll show that honey bee pheromone signals can be described by complexity, synergy, and context dependency in which they are deployed, mediated through both temporal and spatial distribution. The importance of chemical communication will be describe in the framework of honey bee losses worldwide as stresses related to honeybee losses can act on chemical communication processes, modulating production or reception of the pheromonal compounds. Moreover, there are examples of the same chemical compound being used both by the host (honey bee) and the parasite (varroa).Finally, the major challenges for future research in the field of chemical communication in the honey bee will be presented for discussion.

243

THE EVOLUTIONARY ORIGINS OF SOCIAL INSECT QUEEN PHEROMONES: HONESTY AND DYNAMICS OF FERTILITY SIGNAL PRODUCTION IN A SOCIALLY POLYPHENIC HALICTID BEE.

Callum KINGWELL1*; Beryl JONES2; William WCISLO3

1Cornell University, Ithaca, New York, UNITED STATES 2University of Illinois, Urbana-Champaign, Illinois, UNITED STATES 3Smithsonian Tropical Research Institute, Panamá City, Panamá, PANAMÁ

Pheromones produced by queens are central to maintaining the characteristic reproductive division of labor seen in insect societies. The workers who make up the bulk of these societies typically respond to queen ‘fertility signals’ by suppressing their own reproductive potentials and instead contributing to that of their queen(s), yet the evolution of these signaling channels given the potential for destabilizing dishonest signal production remains unclear. Fertility signal evolution may be at a nascent stage in primitively social insect societies, and the mechanisms enforcing signal honesty may be more readily apparent in less socially derived species. However, the identities and production dynamics of chemical fertility signals in primitively social insect species are poorly known. We first identify a set of cuticular compounds linked to female fertility in a Halictid bee (Megalopta genalis) that exhibits facultative eusociality, a level of organization considered representative of the earliest stages in the evolution of complex insect social behaviour. We then examine the ontogeny, caste differentiation, and antennal sensitivities of bees to these compounds to assess their utilities as reliable signals of queen reproductive potential. Finally, we compare the cuticular and glandular chemistries of social queens and solitary reproductives from a single population to examine whether these compounds likely represent true, actively produced signals or are instead constitutively produced cues of fertility.

244

VOLATILE COMPOUNDS FROM V. DESTRUCTOR-INFESTED PUPAE ELICITED HYGIENE BEHAVIOR IN A. MELLIFERA WORKERS.

María Clara LIENDO1*; Patricia FERNANDEZ2; Romina RUSSO2; Irina MUNTAABSKI2; Silvia LANZAVECCHIA2; Jorge Luis CLADERA2; Alejandra PALACIO3; Alejandra Carla SCANNAPIECO4

1Instituto de Genética Ewald A. Favret, Instituto Nacional de Tecnología Agropecuaria (INTA)/CONICET, Castelar, Buenos Aires, ARGENTINA 2Estación Experimental Agropecuaria INTA Delta del Paraná - CONICET, Ciudad Autónoma de Buenos AiresBuenos Aires, Buenos Aires, ARGENTINA 3Unidad Integrada INTA-UNMdP, Balcarce, Buenos Aires, ARGENTINA 4Instituto de Genética Ewald A. Favret, Instituto Nacional de Tecnología Agropecuaria (INTA)/CONICET, Castelar, Buenos Aires, ARGENTINA

Varroa destructor is the most destructive ectoparasite of the western honeybee Apis mellifera. This mite feed on the haemolymph of immature and adult bees affecting its nutritional and immunological status. A. mellifera displays a social immunity mechanism against brood diseases that consists in detect, uncap and remove dead or diseased brood from the hive (hygienic behavior). Previous studies showed that chemical cues could be involved in the detection of diseased brood by worker bees and hence in triggering hygienic behavior. The aim of this research was to study the chemical stimuli that induce hygienic behavior of worker bees, exploring the volatile compounds associated with cuticle of mite-infested pupae and cell’s wax cap. Newly-capped bee brood was artificially infested with one mite. Ten days later, 15 pupae were removed from the combs and mite presence and reproductive status were confirmed (infested group). Non- infested pupae were simultaneously removed (control group). Chemical compounds were obtained by washing the pupae from each group with dichloromethane. Small filter papers that were saturated with each extract were introduced in 25 cells containing recently capped uninfected brood. In parallel, the wax caps from each group were transferred to uninfected-brood cells. For both experiments, the number of removed pupae was recorded and chemical profiles were evaluated. Worker bees removed higher percentages of brood with infested pupae extract than with control pupae extract. No differences were found in percentage of removed brood from infested vs control wax cap cells. For both experiments chemical profiles were different between groups. The present results suggest that differences in cuticle volatile compound between healthy and infested pupae could act as a signal for triggering hygienic behavior. This information contributes to a better understanding of this social mechanism against the mite and provides tools to the selection of Varroa-tolerant colonies.

245

WASTE TRAFFIC CONTROL IN ATTA SEXDENS RUBROPILOSA.

Suzana Helena LUCHESI1; Gabriela de Brito CHAVES1; Emma OTTA1; Vinicius Frayze DAVID1*

1Institute of Biosciences of the University of São Paulo-USP, São Paulo, São Paulo, BRASIL

Forager ant traffic has been extensively studied but there is still much to learn about waste-transporting ant traffic. We attempt to determine if waste-transporting ants choose the path from the colony to the waste dump randomly or following a pheromone trail. We took two similarly-sized fungus-garden containers from the same colony and placed each on a tray connected to another by either a control straight-lined bridge or an experimental sickle-shaped bridge curved (training curve) to the right in four replicas, and to the left in the next four. After 24 hours a second curve was added to the sickle-shaped bridge, giving it a C shape and providing two paths to the waste dump. 24 hours later thirty loaded ants were counted and classified by the curve they chose (left or right). During that count, the unloaded ants on the bridge were also counted and classified depending on the curve they occupied. Immediately afterwards the ants were removed from both bridges and the bridges were swapped between the sub-colonies; the counting/classifying process was repeated with both loaded and unloaded ants, but with removal of the loaded ants. We used a mixed statistical model to process the data and took the colony as a random variable, and showed a preference for the training curve (p<0.001), chosen by 93% (CI: 82-100%) loaded ants in the control sub-colony and 88% (CI: 76-98%) in the experimental sub-colony. Both sub-colonies chose similarly (p=0.119) and the colony variable accounts for 5% of the total variance. Our results indicate that the waste- transporting ants in Atta sexdens rubropilosa follow a pheromone trail to transport the waste from the colony to the waste dump.

246 THE CHEMISTRY OF CHEMICAL ECOLOGY

CIRCADIAN RHYTHM IN P. SCHEIDEANUM.

Celso OLIVEIRA1; Arran RUMBAUGH1; Lora RICHARDS1; Christopher JEFFREY1*

1University of Nevada, Reno, Nevada UNITED STATES, Reno, Nevada, UNITED STATES

Circadian oscillators are present in all forms of life, and correspond to changes in metabolism that generally follow a 24 hour period. These changes have an endogenous source, which means that they occur even in the absence of external stimuli; therefore, they likely result from a long history of adaptation to the light-dark cycles on Earth. For plants, this rhythm is especially important since sunlight is the major source of energy for the synthesis of organic molecules, but changes in plant physiology occur as a more complex response to other environmental challenges that have different intensities throughout the day (Greenham & McClung 2015). Biotic interactions play an important role in this process, as plant metabolism has diversified to serve a multitude of interactions with other organisms; accordingly, circadian rhythms must regulate the production of secondary metabolites, such as defense chemicals and pheromones. Our group is interested in understanding the factors that drive phytochemical diversity and its consequence to the diversity of interactions, which fundamentally leads to biodiversity. In particular, we focus on the model interaction of the diverse family of plants Piper (over 1000 known species) with its specialist predator Eois. In this work, we investigate circadian rhythms in the understudied species P. scheideanum from Costa Rica by analyzing the chemical profile of leaves periodically collected from several individuals in a 24 hour timespan. The chemical signature for each time point is verified by 1H-NMR analysis of acetone extracts from individual samples. With statistical tools that we developed from previous studies involving complex mixtures of natural products, we expect to identify the most important compounds related to variation in secondary metabolism. These results will serve to highlight target compounds for further isolation and characterization, and potentially provide new information for understanding the effects of ecological dynamics into plant metabolism.

248

FROM NATURAL PRODUCT TO SEXUAL PHEROMONE MIMIC – CHALLENGES BEYOND STRUCTURAL ELUCIDATION IN THE CHEMISTRY OF SEXUAL DECEPTION.

Bjorn BOHMAN1; Gavin R FLEMATTI1; Ryan D PHILLIPS2; Alyssa M WEINSTEIN2; Russell A BARROW2*; Rod PEAKALL2

1University of Western Australia, Perth, Western Australia, AUSTRALIA 2the Australian National University, Canberra, ACT, AUSTRALIA

Sexual deception is emerging as one of the most well characterised pollination systems. Yet, despite the large number of studies, relatively few cases have combined chemical investigations with behavioural bioassays to provide a direct link between specific chemicals and explicit biological function. More commonly, it is assumed that electrophysiologically active compounds, or compounds known to be active in other pollination systems, are responsible for the pollinator attraction. Here we highlight through the course of our own work that this can lead to erroneous conclusions.First, while remaining a crucial tool, it is essential to move beyond an exclusive reliance on GC-EAD as not all volatile compounds are amenable to GC analysis. Further, synergistic effects will not be detected by presenting individual compounds. GC-EAD activity without subsequent field bioassays is often of limited value, as EAD activity itself cannot be correlated with specific behaviours.Second, traditional preparative chromatography techniques such as HPLC and (preparative) GC remain helpful tools to confirm the activity of extracts, to limit the number of candidate compounds for field bioassays, and to aid chemical identification. With increasing access to modern NMR instruments employing cryoprobes and microprobes, it is possible to identify a compound from a sample of less than 1 µg post-isolation. NMR can be used in conjunction with GC-MS to help characterise new sexual attractants. Finally, emerging evidence from our work on Drakaea orchids suggests that sexual attraction could be achieved via analogous compounds, rather than exact matches to the pollinator pheromones. Therefore, the often- applied exclusion of floral volatiles absent in the females of the pollinator species from consideration or vice versa is risky.

249

GC/MS-BASED SELECTIVE ASCAROSIDE SCREENING OF NEMATODE METABOLOMES.

Stephan VON REUSS1*; Chuanfu DONG1; Franziska DOLKE2

1Department of Bioorganic Chemistry. Max Planck Institute for Chemical Ecology, Jena, Thuringia, GERMANY 2DEPARTMENT OF BIOORGANIC CHEMISTRY. MAX PLANCK INSTITUTE FOR CHEMICAL ECOLOGY | Cidade: Jena, Jena, Thuringia, GERMANY

Previous research has shown that nematode development and behavior are controlled by ascarosides, glycolipids of the dideoxysugar ascarylose linked to fatty acid derived aglycones. Ascaroside signaling is highly conserved in nematodes and plays a crucial role in intraspecies communication. Furthermore, ascarosides are involved in cross kingdom interactions between nematodes and fungi as well as plants. The large diversity of biological functions controlled by ascarosides is paralleled by the diversity of ascaroside structures, which highlights the need to develop suitable methods for their detection and identification.Here we present a novel GC/MS-based screen that facilitates selective detection of known as well as yet unidentified putative ascaroside signals in crude unfractionated exo-metabolome extracts. The scope and limitations of the new technique are evaluated by analysis of C. elegans wild-type and mutant metabolomes. Comparative analysis of various Caenorhabditis species revealed a group of highly species-specific ascaroside components, which demonstrates the potential of the selective MS screen. The structures of the new compounds were elucidated by NMR analysis and confirmed by total synthesis.

250

GLUTATHIONE-DEPENDENT DETOXIFICATION OF SINALBIN IN SPODOPTERA LITTORALIS.

Anton SHEKHOV1*; Christian PAETZ1; Michael REICHELT1; Riya C. MENEZES1; Jonathan GERSHENZON1; Daniel G. VASSÃO1

1Max Planck Institute for Chemical Ecology, Jena, Thuringia, THURINGIA

Glucosinolates (GLS) are important secondary metabolites in plants of the Brassicaceae family and play an important role in chemical defense against herbivores. Upon tissue rupture, GLS are hydrolyzed by thioglucosidase enzymes called myrosinases, forming bioactive toxic products such as isothiocyanates (ITCs), nitriles and thiocyanates. Since plants can accumulate up to 40 different GLS with different side chains (aliphatic, benzenic and indolic), the types of breakdown products are equally diverse chemically. Thus, mechanisms preventing the damage and toxicity caused by these GLS hydrolysis products are critical for herbivores feeding on crucifers. We are investigating the metabolic processing of distinct GLS groups in a generalist caterpillar, the Egyptian cotton leafworm Spodoptera littoralis, which has over 40 host plant families. We performed feeding experiments with S. littoralis fed 14C-labeled sinalbin (p- hydroxybenzyl-GLS, a benzenic glucosinolate) to determine the major detoxification pathways for this plant pro-toxin. Additionally, we compared the products and their abundance with the previously described metabolites of the aliphatic glucosinolate glucoraphanin, sulforaphane, its glutathione conjugates and its mercapturic acid derivatives (Schramm et al., 2012). Surprisingly, the direct conjugates of p- hydroxybenzyl-ITC with glutathione and its derivatives were not observed. However, in the feces the major product of 14C-sinalbin digestion was identified as being S-(p- hydroxybenzyl)cysteine. In addition, analysis of minor conjugates pointed to the presence of other mercapturic acid pathway metabolites. Therefore, glutathione also seems to be involved in the detoxification of sinalbin by S.littoralis, but via a different underlying mechanism than that in glucoraphanin detoxification. This shows that the structural differences between benzenic and aliphatic glucosinolates may lead to differences in detoxification by insect herbivores.

Schramm, K., Vassao, D. G., Reichelt, M., Gershenzon, J. and Wittstock, U. (2012). "Metabolism of glucosinolate-derived isothiocyanates to glutathione conjugates in generalist lepidopteran herbivores." Insect Biochemistry and Molecular Biology 42(3): 174-182.

251

HIGH SOWING DENSITY INHIBITS THE INDUCTION OF DEFENSIVE COMPOUNDS DAIDZEIN (4’, 7-DIHYDROXYIFLAVONE) AND GENISTEIN (4’, 5, 7-TRIHYDROXYISOFLAVONE) IN SOYBEAN LEAVES UNDER ANTICARSIA GEMMATALIS H. HERBIVORY.

Lucía G. BARRIGA1*; Carlos L. BALLARÉ2; Hugo D. CHLUDIL2; Jorge A. ZAVALA2

1Facultad de Agronomía, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Buenos Aires, ARGENTINA 2IFEVA – Facultad de Agronomía, Universidad de Buenos Aires - CONICET, Ciudad Autónoma de Buenos Aires, Buenos Aires, ARGENTINA

Generally, plants react to insect attack through diverse chemical mechanisms. One of the most interesting responses in leguminous species is the group of secondary metabolites known as isoflavonoids. There is enough evidence supporting the antiherbivore roll of isoflavonoids from soybean (Glycine max L.) leaves after lepidopteran attack. But it has been seen that the synthesis of defensive compounds can compromise plant’s ability to grow/develop and compete with other plants. We lack of knowledge about how this plant responses function under different resources availability. The aim of this work was to study the effect of soybean plants intraspecific competition of soybean plants over the inducible concentration of leaves isoflavonoids, as a response to Anticarsia gemmatalis H. herbivory. Don Mario 4670 cultivar was planted at three densities: 20, 40, and 60 cm between plants. After 24 and 72 hours of A. gemmatalis H. feeding, attacked and non- attacked leaves were harvested and conserved under -80°C. Isoflavonoid identity and quantification was studied trough a methanol extraction followed by HPLC-UV analysis. Our results demonstrated that A. gemmatalis H. attack induced daidzein (4’, 7- dihydroxyiflavone) and genistein (4’, 5, 7-trihydroxyisoflavone) concentrations at low plant densities but this response decreases as long as plant density increases. These results lead us to test two hypotheses: plants adjust investment in these defensive compounds according to the perception of competition risks through some aerial signaling (photoreceptors) or through some underground chemical compound.

252

IDENTIFICATION AND SYNTHESIS OF MALE-SPECIFIC COMPOUNDS OF CHAULIOGNATUS IGNACIOUS.

Leonardo FIGUEIREDO1; Diogo M. VIDAL1; Gabriel BIFFI2; Ann RAY3; Paulo H. G. ZARBIN3*

1Federal University of Paraná, Depart. of Chemistry, Curitiba, PR, BRAZIL 2Institute of Biosciences of the University of São Paulo-USP, São Paulo, São Paulo, BRASIL 3Department of Biology, Xavier University, Cincinnati, OH, USA

Beetles belonging to the Cantharidae family are referred to as soldier beetles or leatherwings. Chauliognathus ignacious frequently occurs in southern and southwest Brazil, along with other species of the genus. C. ignacious beetles were collected on nature reserves at UFPR campus in Curitiba. GC-MS analyses of aeration extracts of both sexes revealed the presence of four male-specific compounds. Molecular ions and fragmentation pattern of mass spectra suggested that these compounds would be related to linear C23 hydrocarbons. The first of these compounds was identified as tricosane (MW: 324g.mol-1). Two additional compounds were proposed as being related to Z- tricosenes due to possible molecular ions at m/z 322 on MS spectra and the presence of a characteristic Z-double bond band at 3005cm-1 on FTIR spectra. GC-MS analysis of the natural extract derivatization employing DMDS suggested the double bond position at carbon 7 (m/z 98 and 224) for the first monoene and 9(m/z 126 and 196) for the second. Synthetic routes based on Wittig reactions were proposed in order to obtain standards of Z-tricos-7-ene and Z-tricos-9-ene. The fourth male specific compound is related to a non- conjugated diene, and its structural determination and synthesis are underway. Laboratory and field bioassays are still necessary in order to observe the biological function of the identified male-specific compounds.

253

IDENTIFICATION OF A NOVEL PHEROMONE COMPOUND FROM CHILECOMADIA VALDIVIANA (PHILIPPI) (LEPIDOPTERA: COSSIDAE).

Heidy Herrera MUÑOZ1*; Wilson Barros PARADA2; Eduardo Fuentes CONTRERAS2; Marcela RODRIGUEZ3; Paulo ZARBIN4; Jan BERGMANN5

1Pontificia universidad Católica de Valparaiso, Valparaiso, Valparaiso, CHILE 2Universidad de Talca, Talca, Talca, CHILE 3Universidad de Concepcion, Concepcion, Concepcion, CHILE 4Universidad Federal do Paraná, Curitiba, Curitiba, BRAZIL 5Universidad Católica de Valparaiso, Valparaiso, Valparaiso, CHILE

Chilecomadia valdiviana (Philippi) (Lepidoptera: Cossidae) is an insect native to Chile. Its immature stages feed on the wood of shrubs and trees, among them economically important species such as apple, pear, olive, cherry, avocado, and eucalyptus trees. Apart from the direct damage caused by the larvae, economic consequences result from quarantine restrictions on exports of wood logs. Because of the lack of effective control methods, we identified the female sex pheromone of this species, in order to set the basis for the development of pheromone-based control strategies. Pupae were collected from infested trees in agricultural areas in the region of Maule, Chile, and were kept in the laboratory until the emergence of adults. The abdominal glands of 2-3 d old virgin females were removed and extracted with hexane. The analysis of the extracts by gas chromatography coupled to mass spectrometry (GC/MS), and gas chromatography coupled to electroantennography revealed the presence of several pheromone candidate compounds. Here, we report the identification of the major bioactive compound as (7Z,10Z)-hexadecadienal (Z7,Z10-16:Ald) by means of GC/MS analyses, microderivatization reactions, and comparison of the analytical data of the natural compound with those of a synthetic reference compound. In field bioassays, synthetic Z7,Z10-16:Ald was highly attractive to male Ch. valdiviana. This compound is reported for the first time as a sex pheromone from Lepidoptera.

254

IDENTIFICATION AND SYNTHESIS OF THE SEX PHEROMONE OF THELOSIA CAMINA (LEPIDOPTERA:EUPTEROTIDAE).

Emir Bolzani SAAD1; Diogo Montes VIDAL1; Miryan Denise Araújo CORACINI2; Maria Pilar BOSCH3; Ángel GUERRERO3; Paulo Henrique Gorgatti ZARBIN3*

1Universidade Federal do Paraná, Cutitiba, Paraná, BRASIL 2UNIOESTE, Cascavel, Paraná, BRAZIL 3Institute of Advanced Chemistry of Catalonia (CSIC), Barcelona, --, SPAIN

Yerba mate (IIex paraguariensis) is a culture which has social and economical importance in Latin America. The moth Thelosia camina occurs as an important pest of this culture. Defoliation caused by this Lepidoptera occurs on the most important phase of plant grown, affecting seriously the production. The pheromone was extracted from female pheromonal glands by dissection and solvent extraction. GC-EAD assays employing male antenna showed the presence of seven bioactive compounds in female extracts. The chemical structure of these compounds was studied by analyzing their chromatographic profiles, mass (GC-MS) and infrared spectra (GC-FTIR), as well as analysis of microderivatization products. The spectroscopic and chromatographic information obtained for the three major compounds indicated that the compounds referred to an aldehyde, an alcohol and an ester both linear dienes, with 18 carbon atoms. The FTIR spectra of all these three dienes showed a pair of well conserved bands at ~3010 and ~3040cm-1 characterizing the double bonds as conjugated and containing a Z,Z configuration. The structure of the four additional compounds were proposed as stereoisomers of the Z,Z dienes based on their MS and FTIR spectra. The structure of the three major compounds were proposed as (13Z,15Z)-octadeca-13,15-dienal, (13Z,15Z)- octadeca-13,15-dien-1-ol and (13Z,15Z)-octadeca-13,15-dien-1-yl acetate. The 13Z,15Z; 13E,15E; 13E,15Z and 13Z,15E isomers of the alcohol, aldehyde and acetate were synthesized by classical approaches, with Sonogashira couplings as key steps. The synthetic compounds were used to prove the proposed structures and also to perform initial field bioassays which showed the attractiveness of the synthetic blends to T. camina males for only the Z,Z isomers or the mixture containing the other stereoisomers.

255

IDENTIFICATION OF NEW SEMIOCHEMICALS - ACCIDENTS, PITFALLS, AND SUCCESS.

Wittko FRANCKE1*

1University of Hamburg, Hamburg, Hamburg, GERMANY

Unambiguous structure elucidation of small amounts of volatile organic compounds in complex multicomponent mixtures is always a challenge, because no NMR-data are available. GC/MS-data are similar to a puzzle with several possible solutions, but only one is correct. On the other hand, mass spectra of different compounds may be (almost) identical. On the way to a target structure, the analytical chemist will have to pass several hurdles at different levels which will be exemplified in the lecture. A library search is helpful, however, it is indispensable to check the validity of the suggested structure: e.g. are trifluoroacetic acid anhydride or its cyclopentyl ester true natural products?1 Not everything that glitters is gold - and not all components of a natural extract are natural products. Particularly widespread contaminants are plasticizers such as phthalates or adipates of 2-ethylhexanol; today, the latter alcohol is omnipresent and even found in drinking water2. “Isobutene chemistry”gives rise to another group of ubiquitous contaminants, which becomes obvious in compounds with tert.butyl sub-structures such as 2,6-di-tert.butyl-4-methylphenol or texanol, an isobutyrate of 2,2,4-trimethylpentan- 1,3-diol (with 1+2 oxygenated isobutenes). Special accidents are obvious in rows of hydrocarbons following a Boltzman-distribution that continuously include even numbered congeners, which indicates (gas)oil- contamination. The second part of the lecture will deal with misinterpretations of spectroscopic data that lead to wrong structures, which have been corrected by independent synthesis - the only solution! A third part will present unpublished compounds and the way to elucidate their structures.

1.Rosenstiel TN, Shortlidge EE, Melnychenko AN, Pankow JF, Eppley SM (2012) Sex- specific volatile compounds influence microarthropod-mediated fertilization of moss. Nature 498: 431-433

2Vitali M, Leoni V, Chiavarini S, Cremisini C (1993) Determination of 2-ethyl-1-hexanol as contaminant in drinking water. J AOAC Int 76:1133-1137.

256

IDENTIFICATION OF THE AGGREGATION PHEROMONE OF HOMALINOTUS VALIDUS (COLEOPTERA: CURCULIONIDAE).

Diogo Montes VIDAL1; Sandra Mara da Silva GOMES1; Paulo Henrique Gorgatti ZARBIN1*

1Universidade Federal do Paraná, Cutitiba, Paraná, BRASIL

Homalinotus validus (Coleoptera: Curculionidae) occurs on Latin America including the Brazilian northern region, leading to financial losses to babassu and coconut production. Insects were collected in babassu fields in Rondonia state and reared under laboratory conditions (fed with banana, 28ºC, 85%RH, 12hL:12hD) at UFPR. GC analyses of aeration extracts revealed the presence of two male-specific compounds (1 and 2) in a 99:1 ratio. MS and FTIR spectra and retention index datasets suggested the identity of the major compound as grandisol (1) which was confirmed by co-injection with an analytical standard. The mass spectrum showed by the minor compound (2) was closely related to the spectrum obtained from coumpound 1 with an increasing of 200 units in its retention index. Analysis of the FTIR spectrum suggested the presence of an carboxyl group related to an acetate (1244 and 1740cm-1). The structure of compound 2 was proposed and confirmed as related to grandisyl acetate after synthesis and comparison of MS spectra and co-injection. To the best of our knowledge this the first case of occurrence of grandisyl acetate (2) while its diastereomer, fragranyl acetate, was described only as a component of essential oils in plants. The absolute configuration of compounds 1 and 2 were determined based on chiral GC analyses as (1R,2S)-1 and (1R,2S)-2. Bioassays in laboratory and field conditions employing the natural extracts and the synthetic male specific compounds are underway, in order to confirm their respective biological roles. However, previous experiences related with sex-specific volatiles produced by Curculionidae beetles suggest that compounds 1 and 2 are related with aggregation behavior.1

(1) Ambrogi, B. G.; Vidal, D. M.; Zarbin, P. H. G.; Rosado-Neto, G. H. Química Nova 2009, 32, 2151.

257

IDENTIFICATION OF THE HOST MARKING PHEROMONE IN THE AFRICAN INDIGENOUS FRUIT FLY CERATITIS COSYRA.

Xavier CHESETO1; Baldwyn TORTO1*

1International Centre of Insect Physiology and Ecology (icipe), Behavioral and Chemical Ecology Unit, Nairobi, Nairobi, KENYA

Some African Tephritid fruit flies in the genus Ceratitis are known to mark their oviposition sites, commonly referred to as ‘host marking behavior’ with a pheromone. This behavior is often a form of defensive strategy exhibited by the mother to protect her progeny from competition for resources from other ovipositing females of the same or another species. For example, host marking behavior has been recorded in Ceratitis capitata, Ceratitis cosyra, Ceratitis fasciventris and Ceratitis rosa. Whereas the host marking pheromone of C. capitata has been identified, those of C. cosyra, C. fasciventris and C. rosa are yet to be identified. Typically, the host-marking pheromone is produced in the posterior half of the midgut and often released along with the frass of the female. Here, we show using bioassays that extracts of the frass of C. cosyra deter its conspecifics and even the sibling species, C. fasciventris and C. rosa. Further, we used bioassay-guided fractionation and chemical analyses (liquid chromatography (LC) and LC-coupled to quadrupole time of flight mass spectrometry (Qtof-MS) of bioactive fractions of aqueous extract of the frass from the females to identify the host marking pheromone of this species and confirmed its identity with an authentic standard. This presentation will highlight the identification and potential use of the host marking pheromone in fruit fly management.

258

KETONES AS PHEROMONE ANTAGONISTS: LIGHTS AND SHADOWS.

Angel GUERRERO1*

1Institute of Advanced Chemistry of Catalonia (CSIC), Barcelona, --, SPAIN

It is well known that the catabolism of insect sex pheromones occurs in Lepidoptera by the action of enzymes present in insect antennae. These enzymes, mainly esterases, degrade pheromone components in more polar and inactive metabolites, and their inhibition may lead to the disruption of the chemical communication between sexes representing an interesting approach with potential use in pest management programs. The trifluoromethyl ketones are known to be good inhibitors of the pheromone degrading enzymes in male olfactory tissues. In the last years, we have shown that methyl and trifluoromethyl ketones structurally analogs to the pheromone are good antagonists of the natural attractant both in the laboratory and in the field. Because of their high structural similitude with the pheromone and their reversible mechanism of action, these compounds display low toxicity, decrease the electrophysiological response of the antennal receptors to the pheromone, disrupt the male flight to the pheromone in a wind tunnel and reduce pheromone catches in the field. In this talk, I will present the synthesis and activity of representative examples of these chemicals as antagonists of the pheromone in different moth species and the lights and shadows of their prospective use in pest management.

259

METABOLISM OF COMPOUNDS FROM GLYCINE MAX (L.) MERRILL LEAVES BY ANTICARSIA GEMMATALIS (HÜBNER)

Rodolfo R. SILVA1; Paulo h. G. ZARBIN1*

1UFPR, CURITIBA, PARANÁ, BRAZIL

Plants and insects have been competed between them during their evolutionary history. Plants are capable to produce a broad amount of compounds to its benefit, insects in response developed strategies to overcome the toxicity of these compounds. Chemical ecology hence has a fundamental role of understanding the relationship between plants of economic interesting and pests to avoid harm to agriculture. In Brazil the production of Glycine max (L.) Merrill (soybean) is reduced by different species of leaf-feed insects manly during its vegetative stage. Among these species we may highligth the velvetbean caterpillar Anticarsia gemmatalis Hübner that consume approximately 30% of foliage of soybean. Therefore, the aim of this study was to determine which compounds are metabolized by A. gemmatalis The methanolic extract of convencional soybean leaves and faeces of A. gemmatalis were obtained, derivatizated with N,O-bis (trimethylsilyl trifluoroacetamide) and thrimethyl chlorosilane and analized by CG-MS. It was found that aspartic acid and compounds derivated from D-glucose 6-phosphate were metabolized, however the D-pinitol was not metabolized when we compared the chromatograms of leaves and faeces. This result is due to the fact that A. gemmatalis does not possess enzymes in its midgut capable to metabolized the D-pinitol that initially is in the phytic acid form and subsequently is methylated in response to salinity stress by SAM (S-adenosyl Methionine). The biosynthesis of this compound by soybean is related to store of inorganic phosphate in the phytic acid form during the growing seedling. To confirm this proposition we need more experiments with new leaf-feed species of soybean and conditions.

260

PHEROMONES OUTSIDE THE BOX: UNEXPECTED PHEROMONES FROM CERAMBYCID BEETLES.

Jocelyn MILLAR1*; Lawrence HANKS2

1University of California, Riverside, California, USA 2University of Illinois at Urbana-Champaign, Urbana, Illinois, BRAZIL

Research on cerambycid pheromones has advanced rapidly since 2004, when pheromones were known for fewer than 10 species in only one of the 8 cerambycid subfamilies. Since then, pheromones have been identified for >100 species in the 5 major subfamilies, with likely pheromones identified from many more species based on their attraction to known pheromones in field trials. To date, the attractant pheromones are split along taxonomic lines, with all known pheromones of species in the subfamilies Lepturinae and Prioninae being female-produced sex pheromones, whereas all known pheromones in the subfamilies Cerambycinae, Lamiinae, and Spondylidinae are male- produced aggregation pheromones. Within related taxonomic groups, pheromone components are often highly conserved. However, it is also becoming apparent that there is a tremendous diversity of pheromone structures within the family overall, with compounds being produced from a variety of different biosynthetic pathways. Some of the more recently identified structures will be described and contrasted with previously known cerambycid pheromones. In parallel, recent results describing interesting and unusual aspects of how the beetles are using these pheromones will be discussed.

References: 1. Millar JG, Hanks LM (2016) Chapter 5: Chemical ecology of cerambycid beetles. In: Wang Q (ed) Cerambycidae of the World: Biology and Pest Management. CRC Press/Taylor & Francis, Boca Raton (in press)

2. Hanks LM, Millar JG (2016) Semiochemistry of cerambycid beetles: Basic science and practical applications. J Chem Ecol, submitted.

261

PYRANOCOUMARINS PRODUCTION EVALUATION IN DIFFERENT GENOTYPES OF NAVEL ORANGE (CITRUS SINENSIS (L). OSBECK) AND RANGPUR LIME (CITRUS LIMONIA OSBECK).

Jéssica Cristina AMARAL1; Michelli Massaroli da SILVA1; Edieidia Souza PINA2; Adriana Aparecida LOPES2; Ana Maria Soares PEREIRA2; Maria Fatima das Graças Fernandes da SILVA2*

1Universidade Federal de São Carlos - UFSCAR, São Carlos, São Paulo, BRASIL 2Universidade de Ribeirão Preto, Ribeirão Preto, São Paulo, BRAZIL

Brazil since the 80s, holds the world's largest producer of citrus title, followed by the US at Florida region. Citrus is a culture widespread throughout the country, having great economic and social importance for several Brazilian states. For phytosanitary control, the use of pesticides contributes to increasing agricultural yield, but has also been responsible for adverse effects on the environment and human health. Today, with the advancement of the agroecological agriculture, new means to control pests and diseases presented highlight, as the search for plants and rootstocks resistant and are therefore less susceptible to pathogen attack. Coumarins are important compounds for the adaptation of plants to biotic or abiotic stress and are important protectors of plants against pathogens and insects. Within this class the pyranocoumarins are a sub-group of natural coumarins 1500 mainly found in four families of higher plants: Apiaceae, Rutaceae, Fabaceae and Moraceae and appear as major compounds in citrus roots. The objective of this study is the quantification of pyranocoumarins; xanthyletin and seselin in navel orange and rangpur lime roots for determining a genotype with increased production of this class of compounds. Among the plants evaluated navel orange genotype LP144 and LC214 rangpur lime presented pyranocoumarins as major substance, producing 21.49 and 55.52 µg/mL respectively, while the genotypes of lower production LP107 and LC217 have 6.80 and 14,72 µg/mL of these substances in the roots. It is observed, therefore, a significant decrease in the production of these compounds among the genotypes of the two species after 4 months. However, in rangpur lime (C. limonia Osbeck) the production of the pyranocoumarins is 1.13% higher than in navel orange (C. sinensis (L). Osbeck).

262

RECENT RESULTS IN PHEROMONE SYNTHESIS.

Kenji MORI1*

1Toyo Gosei Co. Ltd. & The University of Tokyo, Inzai & Bunkyo-ku, Chiba & Tokyo, JAPAN

My ongoing projects in pheromone synthesis will be discussed emphasizing the analytical and synthetic methodologies employed in the works. The following three topics will be treated. (1) The beetle pheromones ethyl 4-methylheptanoate,4- methyloctanoic acid and 4-methyl-1-nonanol. (2) Methyl-branched hydrocarbons in the female secretion of the Korean apricot fly, Eurytoma maslovskii. (3) A monoterpene aldehyde with a cyclopentane ring as the female sex pheromone of the pineapple mealybug, Dysmicoccus brevipes.(Please note that this is a plenary lecture).

263

SEXUAL COMMUNICATION OF PAYSANDISIA ARCHON. AN INTERESTING CASE OF EVOLUTIONARY CONVERGENCE.

Carmen QUERO1*; Victor SARTO I MONTEYS2; Gloria ROSELL3; Marc PUIGMARTÍ3; Angel GUERRERO3

1Institute of Advanced Chemistry of Catalonia (CSIC), Barcelona, --, SPAIN 2Institute of Environmental Science and Technology, Bellaterra, Barcelona, SPAIN 3University of Barcelona, Barcelona, Barcelona, SPAIN

Paysandisia archon Burmeister (Lepidoptera, Castniidae) is a 'butterfly moth' which was introduced into Europe from South America in the 90s and became one of the major pests of palm trees in the Mediterranean basin. Larvae develop inside palm trees making their control very difficult by conventional insecticides. During the last years, notable efforts have focused on the study of the chemical communication of this species [1-3] since a better knowledge of it could give us the cues for designing new tools for pest management.P. archon is a moth with diurnal habits. Males use visual stimuli for detecting and pursuing conspecific females. So far, female-released long-range sex pheromones have not been detected. The latter are used by all known moths, with nocturnal and diurnal habits to attract males from long distance. In contrast, we have found that males produce a variety of pheromone components similar to that produced by other species' female moths. Analysis of extracts from midlegs revealed the presence of large quantities of (E,Z)-2,13-octadecadienol, whereas analysis of the wings showed the presence of both isomers of farnesal. Also, we identified other compounds in other body parts. However, the biological role of these compounds has not yet been determined. The lack of long-range pheromones to attract conspecific males and the presence of multiple pheromone components in males together with the low olfactory capacity and high visual ability compared to those of other moths will be presented as a clear example of evolutionary convergence with butterflies.

[1] (a) Sarto i Monteys V., et al. (2012). PLoS One 7, e29282. (b) Sarto i Monteys V., et al. (2016). Bull. Entomol. Res. http://dx.doi.org/10.1017/S0007485316000158 [2] Frérot B. et al. (2013). Chemoecology 23: 137-142. [3] Riolo P. et al. (2014). J. Econ. Entomol. 107: 1009-1021.

264

SHEDDING LIGHT ON PHYTOCHEMICAL AND INSECT DIVERSITY: EXPLORING THE PHOTOCHROMISM OF CHROMENES ISOLATED FROM PIPER KELLEYI.

Christopher S. JEFFREY1*

1University of Nevada, Reno, NV, USA

Natural products diversity has recently been established as an important aspect of shaping plant-insect interactions. Piper kelleyi, a recently described tropical shrub that grows in the Eastern Andes of Ecuador, hosts a large diversity of specialist geometrid caterpillars. Phytochemical studies of this host plant uncovered three related natural products, including a prenylated benzoic acid, a chromene, and a dimeric chromane. Collaborative studies established that host plant chemical variation of these natural products and elevation shape both community and population structure of these specialist caterpillars. Further studies of the photochemical behavior of the chromene isolated from Piper kelleyi revealed that differential toxicity and phytochemical variation can be explained by the photochromic properties of the chromene metabolite. Discussion of the photochemical, ecological, and synthetic studies of the natural products isolated from Piper kelleyi and the implications in understanding the origins of phytochemical diversity will be discussed.

265

STUDIES ON THE PHEROMONE CHEMISTRY OF THE CITRUS BORER, (COLEOPTERA: CERAMBYCIDAE).

María Eugenia AMORÓS1; Carmen ROSSINI1; Viviana HEGUABURU2; Andrés GONZÁLEZ3*

1Facultad de Química, Universidad de la República, Montevideo, Montevideo, URUGUAY 2Centro Universitario de Paysandú, Universidad de la República, Paysandú, Paysandú, URUGUAY 3Facultad de Química, Universidad de la República, Montevideo, Montevideo, URUGUAY

The citrus borer, Diploschema rotundicolle (Serville), is a Neotropical cerambycid that can become a serious pest in citrus plantations of southern Brazil, Uruguay and Argentina. The larvae bore galleries from the branches into the main trunk, causing direct and indirect damages to the tree. Several cerambycid species of the sub-familiy Cerambycinae produce male aggregation pheromones that may be used for mass trapping of male and female adults. Moreover, many species share the main pheromone chemistry, usually short chain aliphatic diols or hydroxyl ketones. To begin pheromone studies in this species, we synthesized two well know pheromones of Cerambycinae species: 2,3- hexanediol and 3-hydroxy-2-hexanone in their racemic forms, and tested them in cross- vane traps in two citrus orchards in southern Uruguay during the summer of 2015/16. We also setup light intercept traps and mesh covers around highly infested tree trunks, in order to capture adult D. rotundicolle for chemical and behavioral studies. All traps captured females only, and pheromone baited traps were not highly efficient. Males were only captured in mesh covers around trees, likely upon adult emergence. Captured males and females were studied for their volatile chemistry by air entrainment and GC-MS analysis. We found that males produce large amounts of 3-hydroxy-2-hexanone, and some minor compounds yet unidentified. Determination of chirality in 3-hydroxy-2- hexanone is underway by chiral GC analysis. Both females and males respond to the synthetic and natural compounds in GC-EAD, suggesting that the compound acts as an aggregation pheromone. Behavioral and field-catch studies will be continued in the 2016- 17 summer season, aiming at the development of a mass trapping device for D. rotundicolle. While the same pheromone chemistry has been reported in several species of Cerambycinae, this is the first species of the tribe to be reported in this regard.

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SYNTHESIS OF FOUR STEREOISOMERS OF METHYL 4,8,12- TRIMETHYLPENTADECANOATE, AIMING TO DETERMINE THE ABSOLUTE CONFIGURATION OF THE MAJOR COMPONENT OF THE SEX PHEROMONE OF EDESSA MEDITABUNDA (HEMIPTERA: PENTATOMIDAE).

Daiane SZCZERBOWSKI1; Paulo H. G. ZARBIN1*; Stefan SCHULZ1

1Universidade Federal do Paraná, Cutitiba, Paraná, BRASIL

Brazil is the second world producer of soybean and anually 20% of the production is lost due to pests, mainly moths and stink bugs. Edessa meditabunda is part of a important complex of stink bugs that occur in soybean, it used to be a secondary pest but now represent a primary pest. A study previously developed in Laboratório de Semioquímicos identified two male specific compounds for the species as methyl 4,8,12- trimethylpentadecanoate as major component and methyl 4,8,12-trimethyltetradecanoate as minor compound. Bioassays in Y olfactometer showed that the major compound is as atractive to females as the natural extract. The minor compound didn’t show any enhancement of the attraction and none of the compounds were attractive for males, characterizing a sex pheromone. As is well known stereochemistry plays a prime role in chemical ecology, so the aim of the present study was to determine the absolute stereochemistry of the major compound produced by males of E. meditabunda. It was performed the synthesis of four stereoisomers of the major component of the sex pheromone. Starting from comercial avaiable compounds with defined stereochemistry, (R) or (S)-citronellol and methyl (S)-(-)-3-hydroxi-2-methylpropionate, three building blocks were obtained. These intermediates were coupled through sequences of Grignard reactions, leading to the obtainment of the stereoisomers methyl 4R,8S,12S, 4R,8R,12S, 4S,8S,12S and 4R,8R,12R-4,8,12-trimethylpentadecanoate. The determination of the absolute stereochemistry of the natural compound is still in course. This study is being performed through analysis by HPLC of anthracene derivatives (Ohrui reagent) of the racemate, the synthesized stereoisomers and the natural compound. After comparison of the LC profile it will be possible to rationalize the stereochemistry of the naturally occuring compound.

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SYNTHESIS AND BIOLOGICAL EVALUATION OF JASMONOIDS.

Florencia PARPAL1*; Luis Amilton FOERSTER2; Enrique PANDOLFI3; Viviana HEGUABURU4

1Facultad de Química, UdelaR, Montevideo, URUGUAY, Montevideo, Montevideo, URUGUAY 2Laboratório de Controle Integrado de Insetos , Universidade Federal de Paraná, Curitiba, Paraná, BRAZIL 3Departamento de Química Orgánica, Facultad de Química, UdelaR, Montevideo, Montevideo, URUGUAY 4Departamento de Química del Litoral, Centro Universitario Regional Litoral Norte, Paysandú, Paysandú, URUGUAY

The use of phytohormones and semiochemicals is an ecologic alternative for the development of Integrated Pest Management technologies. Jasmonic acid and its derivatives have the potential to induce pest resistance. These compounds, found in plants when harmed, trigger direct and indirect responses. An example of an indirect response is the release of volatile compound mixtures that attract natural predators of the harming pests. Our work involves the design and synthesis of jasmonoids analogs to use them in biological control plans.In order to synthesize jasmonic acid and jasmonoid analogs, a versatile synthetic methodology was developed, based on our previous work. The designed route involves the functionalization of cyclopentenone cores. According this methodology, the first jasmonic acid analog and many jasmonoid precursors were synthesized. The route takes advantage from Stille cross-coupling reactions to attach the alkenyl side chain. The carboxylic side chain is introduced by a Michael addition reaction. By varying the structure of the alkenylic side chain precursor and the Michael nucleophile, a wide variety of analogs can be prepared.A preliminary biological evaluation of the jasmonoids was carried out in collaboration with the Laboratory of Integrated Insect Control (LCII) of the Universidade Federal de Paraná, Brazil, using high incidence soybean and wheat pests as targets. Experiments on the defense effects induced by methyl jasmonate in the development of Pseudaletia Sequax were performed. In addition, studies of the effect of direct application of methyl jasmonate on an artificial diet were performed to study the development of Anticarsia gemmatalis and Chrysodeixis includens. Preference tests on Anticarsia gemmatalis were also performed using soy leaves treated with a simplified analog of jasmonic acid synthesized in our laboratory. These studies yielded promising results regarding the effects on insect development when natural and synthetic jasmonoids are used.

268

TENUAZONIC ACID PRODUCED BY A FUNGUS ISOLATED FROM COLONY OF THE STINGLEES BEE FRIESEOMELITTA VARIA.

Amanda H CAVALHEIRO1; Fábio S NASCIMENTO1; Mônica T PUPO1*

1USP -University of São Paulo, Ribeirão Preto, São Paulo, BRAZIL

Chemical Ecology studies are needed to better understanding the relationships between species and how the production of secondary metabolites interferes in these mechanisms. The symbiotic relationship between insects, fungi and bacteria has been investigated and new findings in the area have shown how these relationships have evolved and which substances are involved in this cooperation. It is known that ants, termites and bees have a symbiotic relationship with fungi and bacteria and many of the products produced by these microorganisms may have pharmacological or agricultural importance. The aim of this work is to isolate and to identify natural products produced by microorganisms found in colonies of the stingless bee Frieseomelitta varia. Microorganisms from the bee, different parts of the colony and garbage were isolated using ISP2 medium. The microorganisms were tested against Staphylococcus aureus ATCC6538 in antagonism assay. One bioactive filamentous fungus isolated from the garbage was selected for further bioassay-guided fractionation using Staphylococcus aureus. The fungus was cultured in large scale in liquid medium and the obtained extract was fractionated using SPE column with different concentrations of methanol and water. The active fraction was further purified in semi-preparative HPLC with C6-Phenyl column using acetonitrile/water as mobile phase. The bioactive compound was identified using NMR and HRESIMS-MS/MS as tenuazonic acid, a previously described mycotoxin. This fungus and its bioactive compound may be pathogenic to the bee and probably for that reason the fungus was only found in the garbage of the colony.

269

THE CHEMICAL ANALYSIS OF BUFADIENOLIDES IN DEFENSIVE GLANDS OF THE ASIAN RHABDOPHIS SNAKES.

Tatsuya YOSHIDA1; Teppei JONO2; Hirohiko TAKEUCHI3; Naoko YOSHINAGA3; Li DING4; Yezhong TANG4; Akira MORI5; Naoki MORI5*

1Kyoto Univ. Graduate School of Agriculture, Kyoto, Kyoto, JAPAN 2TBRC Univ. Ryukyus, Nakagami, Okinawa, JAPAN 3FSERC, Kyoto, Kyoto, JAPAN 4CIB, Chengdu, Sichuan, CHINA 5Kyoto Univ. Graduate School of Science, Kyoto, Kyoto, JAPAN

The Japanese snake Rhabdophis tigrinus has defensive glands on its neck, called nuchal glands. The major chemical components of the glands are steroidal toxins known as bufadienolides (M.W., 398 ~ 434). Rhabdophis tigrinus sequesters the toxins from toads that it consumes as prey. The nuchal glands have been reported from about ten species of Asian Rhabdophis snakes, but except for R. tigrinus chemical components of the glandular fluid have not been investigated. To identify the compounds in the glands, we conducted chemical analysis on the glandular fluid from seven species of Rhabdophis and compared with bufadienolides from R. tigrinus. The analysis showed that the glandular fluid from all species contained bufadienolides and that two of them, R. nuchalis and R. pentasupralabialis, had some heavier bufadienolides (M.W., 448 ~ 564) than R. tigrinus had. Then we purified three bufadienolides from the glandular fluid of the Chinese snake R. pentasupralabialis. Through NMR spectroscopic analysis, we identified one of them as a bufadienolide xyloside, which has been reported from the North American firefly Lucidota atra, and the others also had same planer structures as bufadienolides from L. atra. These results suggest the possibility that R. pentasupralabialis preys on Chinese fireflies related to L. atra and sequesters bufadienolides from the fireflies into the glands. Although R. pentasupralabialis is considered to mainly eat earthworms, similarity of habitats between earthworms and the fireflies implies that fireflies are also exploited by R. pentasupralabialis as natural diet. In the course of the evolution of Rhabdophis species, the dietary origin of bufadienolides sequestered in the glands may have shifted from toads to fireflies after the shift of prey.

270

THE RELATIONSHIP BETWEEN ENDOPHYTES AND OXYLIPINS IN A. BRASILIANA AND A NEW WOUNDING SIGNALLING MOLECULE.

Marilia Almeida TRAPP1*; Natalia Ritez RIVABEN1; Axel MITHÖFER2; Edson Rodrigues FILHO3

1Laboratório de Bioquímica Micromolecular de Microorganismos, Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo, BRAZIL 2Department of Bioorganic Chemistry Max Planck Institute for Chemical Ecology, Jena, Jena, GERMANY 3Laboratório de Bioquímica Micromolecular de Microorganismos, Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo, BRAZIL

Alternanthera brasiliana is a plant widely used in Brazil as a folk medicine for the treatment of numerous diseases, especially due to its antibiotic properties. Usingdereplication strategies, we identified the antibiotic compounds as modified fatty acids, which are closely related to oxylipins.1 Since some of these compounds have already been assigned either as signaling molecules involved in oxidative stress response or bacterial metabolites,2 we hypothesize that they would be related with endophytic bacteria living in A. brasiliana tissues. We have shown that endophytic bacteria isolated from A. brasiliana are able to produce some of the oxylipins isolated from this plant.1 In order to understand the relationship between endophytes and oxylipins in A. brasiliana, we have established axenic cultures of this plant and developed methods for quantification of bacterial cells, oxylipins and phytohormones in plant tissues.3 We observed that both oxylipins and bacterial cells are present in smaller amounts in axenic plants compared to those cultured in normal conditions. These results emphasize the influence of endophytic bacterial on antibiotic production in A. brasiliana tissues. Moreover, re-inoculation experiments provided some intriguing results concerning the concentration of oxylipins after tissue damage. Among the 17 oxylipins analyzed during re-inoculation experiments, only the concentration of 9Z-octadecendioc acid had been increased after wounding in A. brasiliana. It is the first time that this compound is reported as signaling molecule after tissue damage.

271

TRAPPING CAPACITY AND BREAK-THROUGH VOLUMES OF AERATION COLUMNS.

Göran BIRGERSSON1*

1Department of Plant Protection, Swedish University of Agricultural Sciences, Alnarp, N/A, SWEDEN

Trapping devices for collection of volatiles are often referred to as "filters". By using the word "filter" we assume that the volatile molecules that goes into the filter stays on the filter, until these organic compounds are desorbed or eluted. With this as a background many volatile collections continue for many hours with a high air flow. But is this true? Unfortunately not! Our trapping devices, called "filters", are not trapping and holding everything until desorption or elution. Instead we are actually running a very slow and inefficient chromatography: the organic compounds that goes into the adsorbent slowly migrates through the "column", based on the basic for all chromatography: affinity to the stationary phase and solubility in mobile phase. Therefore, our trapping devices should be referred to as "aeration columns" rather than "filters". Small teflon® columns were packed with three different adsorbents, Porapak Q, Chromosorb 102 and Tenax GR. In addition two different mesh sizes of Tenax GR was tested. A test mixture of 22 different compounds, with different vapor pressures, polarities and molecular weights, was continuously released from open capillary tubes into each test column. Air was pumped through the test column and breakthrough-compounds were trapped in a collection column, connected to the test column. The collection columns were changed after one, two, four and six hours after the test was started. Aeration extracts were analyzed on GC- MS. Quantification were based on standard curves for each compound in the test mixture. The different compound classes showed a variety in affinity to the different adsorbents. In general, Porapak Q has the best trapping capacity, followed by Chromosorb 102, while Tenax GR has the lowest trapping capacity, especially so for monoterpene hydrocarbons. Tenax GR with mesh 20-35 show earlier breakthrough and therefore smaller breakthrough volumes than do Tenax GR with mesh 60-80.

272

Z-SELECTIVE OLEFIN CROSS-METATHESIS IN THE SYNTHESIS OF ECOLOGICALLY RELEVANT COMPOUNDS: JASMONATES AS A CASE STUDY.

Guillermo H. JIMENEZ-ALEMAN1*; Selina SEÇINTI1; Wilhelm BOLAND1

1Department of Bioorganic Chemistry. Max Planck Institute for Chemical Ecology, Jena, Thuringia, GERMANY

The biological activity of a natural product is often determined by the stereochemistry of double bonds present in the molecule. Therefore, stereoselective access to the “Z” or “E” isomer is of great importance for the synthesis of such compounds. Recently olefin cross- metathesis (CM) has become a powerful tool to prepare Z-alkenes with high stereoselectivity. This is mainly due to the availability of the relatively inexpensive Z- selective Grubbs type of catalyst.[1]Jasmonates are plant hormones with a single Z- double bond in a pentenyl side chain. -substituted (12-modified) jasmonates are ubiquitous to plants and the biological function of this compound class is yet unclear. Availability of 12-modified jasmonates is of interest to carry out biological assays. In this study, we describe a short (two to four steps, depending on the target molecule) and simple (no special conditions required) synthesis of Z-enriched 12-modified jasmonates based on the CM of alkenes. On the one hand, the Z-selective Grubbs catalyst (1) (catalyst loading 2-5 mol%) produces the Z-isomer in excellent yields and high stereoselectivity, typically more than 90%. On the other hand, both the Grubbs (2) and the Hoveyda-Grubbs (3) second generation catalysts are more active (catalyst loading as low as 0.1%, and shorter reaction times) than the catalyst 1. Although the stereoselectivity is considerably reduced using catalysts 2 and 3, both produce a mixture of isomers containing 20-30% of the Z-alkene which is acceptable in order to conduct bioassays. Additionally, the synthetic approach proceeds smoothly under solvent-free conditions which makes the synthesis environmentally friendly. The above-mentioned highlights the potential of alkenes’ CM not only in the synthesis of jasmonates, but also in other natural products where the stereochemistry of double bonds is crucial for bioactivity, e.g. in insect pheromones.

[1] M. B. Herbert, R. H. Grubbs, Angew Chem Int Ed Engl 2015, 54, 5018-5024.

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