Identification and Biosynthesis of Novel Male Spec

Authors: HongLei Wang Oskar Brattström Paul M Brakefield Wittko Francke Christer Löfstedt

Publish Date: 2014/06/04

Volume: 40, Issue: 6, Pages: 549-559

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Abstract

Representatives of the highly speciose tropical butterfly genus Bicyclus Lepidoptera Nymphalidae are characterized by morphological differences in the male androconia a set of scales and hair pencils located on the surface of the wings These androconia are assumed to be associated with the release of courtship pheromones In the present study we report the identification and biosynthetic pathways of several novel esters from the wings of male B martius sanaos We found that the volatile compounds in this male butterfly were similar to femaleproduced moth sex pheromones Components associated with the male wing androconial areas were identified as ethyl isobutyl and 2phenylethyl hexadecanoates and 11Z11hexadecenoates among which the latter are novel natural products By topical application of deuteriumlabelled fatty acid and amino acid precursors we found these pheromone candidates to be produced in patches located on the forewings of the males Deuterium labels from hexadecanoic acid were incorporated into 11Z11hexadecenoic acid providing experimental evidence of a Δ11desaturase being active in butterflies This unusual desaturase was found previously to be involved in the biosynthesis of femaleproduced sex pheromones of moths In the male butterflies both hexadecanoic acid and 11Z11hexadecenoic acid were then enzymatically esterified to form the ethyl isobutyl and 2phenylethyl esters incorporating ethanol isobutanol and 2phenylethanol derived from the corresponding amino acids Lalanine Lvaline and LphenylalanineIn the insect order Lepidoptera moths and butterflies differ in their use of pheromones for mate finding and mate recognition Mate finding in moths is mediated by femaleemitted sex pheromones that elicit a finetuned response in conspecific males Wyatt 2003 Pheromone biosynthesis in most moths involves a series of more or less welldescribed enzymatic reactions including de novo synthesis of fatty acyl precursors desaturation and chain elongation or shortening to produce various carbon backbones followed by transesterification to form corresponding fatty acid esters Reduction leads to alcohols which in turn may yield acetates or aldehydes after reesterification or oxidation respectively Francke and Schulz 1999 Tillman et al 1999 So far there is no evidence for sex pheromones to be involved in longrange communication in butterflies they appear to have lost this information channel used by female moths However maleproduced pheromones are commonly involved in closerange courtship displays VaneWright and Boppré 1993 Detailed studies on the semiochemicals of butterflies include Nymphalids eg some Milkweed butterflies from the subfamily Danainae Nishida et al 1996 Schulz et al 1993 2004 Stritzke et al 2003 and some Heliconius spp from the subfamily Heliconiinae Schulz et al 2007 2008 and Pierid butterflies such as Colias eurytheme and C philodice from the subfamily Coliadinae Grula et al 1980 Rutowski 1980 and Pieris napi Andersson et al 2007 Bergström and Lundgren 1973 P rapae and P brassicae Andersson et al 2003 Yildizhan et al 2009 from the subfamily Pierinae In contrast to moths that generally use de novo synthesised compounds to produce volatile signals butterflies often utilize chemical substances acquired from plants in scent production Boppré 1984 Schulz et al 2004The tropical butterfly genus Bicyclus Lepidoptera Nymphalidae is highly speciose with over 90 species in Africa and species determination is sometimes challenging The most important classical taxonomic characters for the genus are differences in the male androconia Condamin 1973 a set of differentiated scales and hair pencils located on the surface of the wings and assumed to be associated with the release of pheromones Maleproduced courtship pheromones have been identified from the squinting bush brown B anynana Nieberding et al 2008 In this species volatile signals are as important as visual cues in female choice Costanzo and Monteiro 2007 and are most likely involved in mate quality assessment Nieberding et al 2012 Van Bergen et al 2013 Over the past 5 years we carried out a largescale analysis of chemicals found in more than 30 species of Bicyclus Bacquet PMB Brattström O Wang HL Allen CE Löfstedt C Brakefield PM and Nieberding CM unpublished data Our results strongly suggest that the use of male pheromones is generalized within the genus and that the pheromone signals are speciesspecific Among all the candidate pheromone components in the genus Bicyclus nearly two thirds of the compounds are chemically classified as saturated and unsaturated fatty alcohols aldehydes esters and hydrocarbons which are structurally similar to femaleproduced moth pheromones suggesting a close biosynthetic relationship with common moth pheromone componentsTo test the hypothesis that conserved pheromone biosynthetic pathways and related enzymes/genes are used in both moths and butterflies we carried out biosynthesis studies of volatile compounds in representative Bicyclus species In the present study we report the identification of several novel ethyl isobutyl and 2phenylethyl esters in B martius sanaos We also investigated the origin of the alcohols and the biosynthesis of the unsaturated fatty acyl moiety involving a Δ11desaturase an enzyme family that has previously been found typically in relation with moth sex pheromone biosynthesisA laboratory colony of B martius sanaos originally started in 2009 from around 50 adult females collected in Ologbo Forest N 602 E 555 20 masl in southern Nigeria was maintained at 27 ± 1 °C and 70  relative humidity under a 1212 hr LD photoperiod The larvae of different instars were separately fed on the leaves of potted wheat and pupated directly on the wheat stems at the end of their larval stage Newly emerged adults were collected daily and held in singlesex cohorts feeding on banana Adult males 7d to 10dold were used for chemical analysis and labelling experiments In the labelling experiment the adults were fed on 10  glucose solution in order to avoid the possible involvement of foodderived substancesEthyl and butyl esters of hexadecanoic acid 11E11hexadecenoic acid and 11Z11hexadecenoic acid were prepared by mixing the acids separately with ethanol or each of the butanol isomers n sec iso and tert in HCl 05 M in a ratio of 112 acid alcohol The reaction was run at 80 °C for 1 hr and stopped by adding saturated aqueous sodium carbonate solution The target ester was extracted with hexane To prepare the 2phenylethyl esters the acids were separately mixed with 2phenylethyl alcohol in a ratio of 112 and the reaction was carried out at 120 °C for 2 hr after adding one drop of concentrated sulfuric acid The two unsaturated acids were prepared by oxidation of corresponding alcohols 11E11hexadecen1ol and 11Z11hexadecen1ol from our laboratory stock with pyridinium dichromate PDC in dimethylformamide DMF at 25 °C for 10 hr The synthesis of 11Z1313141415151616162H911hexadecenoic acid d 9Z1116Acid has been described in Löfstedt et al 1994 Deuterium substituted 1616162H3hexadecanoic acid d 316Acid 23332H4Lalanine d 4Ala 234444′4′4′2H8Lvaline d 8Val and Lphenyl2H5alanine2332H3 d 8Phe were purchased from Cambridge Isotope Lab Larodan Fine Chemicals Limhamn and Malmö SwedenFor the initial investigation of the compounds found in individual androconia of B martius sanaos butterflies were collected in the field at the location where collections were made for setting up the laboratory colony Traps baited with fermented bananas or hand netting were used Three males and two females were sampled individually The thorax was pinched prior to sampling and then samples of different part of the wings ie the patchlike tissue and several brushlike androconia Fig S1 were cut out using microscissors cleaned in 99  alcohol between each cut to avoid contamination across samples All sampling were done within 15 min to minimize possible postmortem changes to the chemical composition The wing tissue samples then were extracted individually in a 15 ml glass vial with 100 μl of redistilled heptane containing 1 ng/μl of 8Z8tridecen1yl acetate as internal standard After the androconia had been removed the remaining wing tissue of the hind and forewing were extracted separately with 300 μl of heptane containing the same internal standard in a concentration of 033 ng/μl

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