Hairpencil Research Articles

Wing pattern diversity in Eunica butterflies (Nymphalidae: Biblidinae): phylogenetic analysis implies decoupled adaptive trends in dorsal sexual dimorphism and ventral eyespot evolution.

Butterfly eyespots are wing patterns reminiscent of vertebrate eyes, formed by concentric rings of contrastingly coloured scales. Eyespots are usually located close to the wing margin and often regarded as the single most conspicuous pattern element of butterfly wing colour displays. Recent efforts to understand the processes involved in the formation of eyespots have been driven mainly by evo-devo approaches focused on model species. However, patterns of change implied by phylogenetic relationships can also inform hypotheses about the underlying developmental mechanisms associated with the formation or disappearance of eyespots, and the limits of phenotypic diversity occurring in nature. Here we present a combined evidence phylogenetic hypothesis for the genus Eunica, a prominent member of diverse Neotropical butterfly communities, that features notable variation among species in eyespot patterns on the ventral hind wing surface. The data matrix consists of one mitochondrial gene region (COI), four nuclear gene regions (GAPDH, RPS5, EF1a and Wingless) and 68 morphological characters. A combined cladistic analysis with all the characters concatenated produced a single most parsimonious tree that, although fully resolved, includes many nodes with modest branch support. The phylogenetic hypothesis presented corroborates a previously proposed morphological trend leading to the loss of eyespots, together with an increase in the size of the conserved eyespots, relative to outgroup taxa. Furthermore, wing colour pattern dimorphism and the presence of androconia suggest that the most remarkable instances of sexual dimorphism are present in the species of Eunica with the most derived eyespot patterns, and are in most cases accompanied by autapomorphic combinations of scent scales and "hair pencils". We discuss natural and sexual selection as potential adaptive explanations for dorsal and ventral wing patterns.

Involvement of GOBP2 in the perception of a sex pheromone component in both larval and adult Spodoptera litura revealed using CRISPR/Cas9 mutagenesis

General odorant-binding proteins (GOBPs) are long considered responsible for the perception of plant odorants. In this study with the important noctuid pest Spodoptera litura, we functionally characterized that GOBP2 is also involved in the perception of sex pheromone components using in vivo CRISPR/Cas9 technique. First, the GOBP2 sgRNA and Cas9 protein were injected into the newly laid insect eggs, resulting in a 35.6% target mutagenesis in G0 moths. Then, the homozygous GOBP2 knockout strain (GOBP2−/−) was obtained after the screening of three generations. The knockout male and female moths displayed a significant reduction in EAG responses to the sex pheromone components, and the knockout females also displayed a significant reduction to plant odorants. In the behavioral assay of food choice, GOBP2−/− larvae lost the preference to artificial diet added with the major sex pheromone component Z9, E11-tetradecadienyl acetate (Z9, E11-14:Ac), whereas the WT larvae highly preferred the pheromone diet. Y-tube olfactometer assay and direct pheromone stimulation assay showed that GOBP2−/− male adults reduced significantly than WT males in percentages of choice, hair pencil displaying and mating attempt to Z9, E11-14:Ac. In the oviposition test, GOBP2−/− females showed significantly reduced preference for the soybean plants compared to the WT females. Our study demonstrated that GOBP2 plays an important role in perceiving sex pheromones in adult and larval stages, providing new insight into sex pheromone perception and a potential target for sex pheromone-based behavioral regulation in the pest.

CRISPR/Cas9 mutagenesis against sex pheromone biosynthesis leads to loss of female attractiveness in Spodoptera exigua, an insect pestt.

Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.

Evidence of Male Pheromone in Conogethes punctiferalis (Lepidoptera: Pyralidae)

Conogethes punctiferalis Guenee is a polyphagous insect pest that is difficult to manage because it feeds within plant tissue. Management by mass trapping using semiochemicals, especially pheromones, represents a viable option to control such borers. Herein, pheromonal compounds were extracted from male and female moths and assessed using headspace sampling and electroantennogram response. One-d-old C. punctiferalis showed a higher response to volatile solvents than 4-d-old ones, irrespective of sex. The male antenna was found more sensitive than the female for volatile compounds. However, the female response to male headspace extract and abdominal tip extract was very high (i.e., 4.006 mV and 2.217 mV, respectively), which revealed the presence of males producing a female-attracting olfactory cue in C. punctiferalis. This was also confirmed by males calling the female before mating by extruding the hair pencils in their abdominal tip. The male pheromone extract when analyzed in gas chromatography–mass spectrometry indicated the presence of methyl acetophenone or 3-ethyl acetophenone.

Volatile Compounds Release by the Hair Pencils in Male Prophantis smaragdina (Lepidoptera: Crambidae: Spilomelinae)

Courtship behavior in several pyralid species is associated with the exposure to male hair pencils (HPs) or special scales that released volatile compounds. HP chemicals induce conspecific female and/or male behaviors and are therefore qualified as male pheromones. Preliminary observation on the coffee berry moth (CBM), Prophantis smaragdina Butler (Lepidoptera: Crambidae: Spilomelinae) mating behavior showed that the male displays abdominal HPs located on the last abdominal segment. The aim of the study was to identify the male volatile compounds and assess the results by electroantennography (EAG) on male and female antennae. Gas chromatography coupled to mass spectrometry analysis of male HP emissions showed five aromatic compounds identified as phenylacetaldehyde, phenylethyl alcohol, creosol, perillyl alcohol, and methyl anthranilate. EAG results showed that creosol elicited a significantly higher response than the control (hexane) on both male and female antennas. On female antennas, response to methyl anthranilate and phenylacetaldehyde was also significantly higher than the response to the control. Those results suggest that the creosol could play a behavioral role on conspecific male and female CBM during courtship behavior and mating choice. Methyl anthranilate and phenylacetaldehyde could also play a role on female behavior. Perillyl alcohol is for the first time identified as an insect product.

Evidence of Male Hair Pencil Pheromone in Choristoneura fumiferana (Lepidoptera: Tortricidae).

Male Lepidoptera often possess specialized scales, called hair pencils that emit volatiles that are critical to mating success. Spruce budworm, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae), males will display hair pencils to females before attempting copulation. The importance of volatiles on these hair pencils is, however, not clear. We compared the proportion of successful copulations in unmanipulated mating pairs to pairs where males had their hair pencils either removed or chemically washed, and to pairs where females were antennectomized. Mean proportions of successful matings were significantly lower in pairs where hair pencils had been manipulated or where females had been antennectomized compared with unmanipulated mating pairs. There was no significant difference in mating success between treatments where hair pencils had been manipulated; however, mating success was significantly lower in hair pencil treatments than in antennectomized treatments. Mean copulation proportions in hair pencil/antennectomized treatments were also significantly less than in respective sham-operated treatments. Our results suggest that volatiles are associated with hair pencils, and they may be required for mating success in C. fumiferana.

Identification and biosynthesis of novel male specific esters in the wings of the tropical butterfly, Bicyclus martius sanaos.

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 female-produced moth sex pheromones. Components associated with the male wing androconial areas were identified as ethyl, isobutyl and 2-phenylethyl hexadecanoates and (11Z)-11-hexadecenoates, among which the latter are novel natural products. By topical application of deuterium-labelled 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 (11Z)-11-hexadecenoic acid, providing experimental evidence of a Δ11-desaturase being active in butterflies. This unusual desaturase was found previously to be involved in the biosynthesis of female-produced sex pheromones of moths. In the male butterflies, both hexadecanoic acid and (11Z)-11-hexadecenoic acid were then enzymatically esterified to form the ethyl, isobutyl and 2-phenylethyl esters, incorporating ethanol, isobutanol, and 2-phenylethanol, derived from the corresponding amino acids L-alanine, L-valine, and L-phenylalanine.Electronic supplementary materialThe online version of this article (doi:10.1007/s10886-014-0452-y) contains supplementary material, which is available to authorized users.

Hairpencil Volatiles Influence Interspecific Courtship and Mating Between Two Related Moth Species

Reproductive isolation between sympatric, closely related species can be accomplished through a variety of pre-zygotic isolating mechanisms, including courtship-signaling behavior that involves pheromones. In the moths Heliothis virescens and H. subflexa, males display abdominal hairpencils (HP), which release volatile chemicals during courtship. In this study, we demonstrated that HP volatiles released by male H. subflexa function to improve mating success with conspecific females. Interspecific mating experiments were conducted to determine any influence of HP volatiles on species isolation. Female H. virescens and H. subflexa were observed during courtship with males of the other species, following either sham-operation or ablation of HPs, both with and without concurrent presentation of HP volatiles. Mating success was improved by co-presentation of HP extract from males of the same species during courtship. Ablation of HPs improved mating between H. subflexa females and H. virescens males. During interspecific matings, male H. virescens attempted copulation less frequently in the presence of H. virescens HP extract, though H. subflexa males were not affected by the presence of H. subflexa HP extract. This suggests that HP volatiles produced by males of each species may inhibit mating between species through effects on males (H. virescens) and females (H. subflexa).

Archaeopodagrion armatumsp. nov. from Ecuador (Odonata: Megapodagrionidae)

Abstract Archaeopodagrion armatum sp. nov. is described and illustrated (holotype ♂ Ecuador, Zamora Chinchipe Prov., forest S of Zamora (4°07′18″S, 78°58′22″W), 02 iv 2008, leg. KJT; in FSCA). The new species is distinct from A. bicorne and A. bilobatum by the pair of highly recurved processes on the hind margin of the prothorax of both sexes, and a hair pencil and preapical spike-shaped process on the dorsal surface of the male paraprocts.

Molecular basis of female-specific odorant responses in Bombyx mori

Males and females of many moth species exhibit important differences in sexual behaviours. Much research in this field has focused on the male-specific behaviour, electrophysiology and molecular biology of sex pheromone reception. Female-specific behaviours have been less well studied although, like male-specific behaviours, they could provide opportunities for intervention and management of lepidopteran pests. Previously, we identified genes encoding putative odorant receptors (ORs) from the genome of the silkworm, Bombyx mori, some of which have higher levels of steady-state transcript in the antennae of adult females compared with males. We have identified the full-length cDNA sequences of some of these ORs and described a novel OR that is part of a female-biased clade. Using expression in Sf9 cells and a calcium-imaging assay, we tested a range of compounds for their ability to activate the most highly female-biased ORs, BmOR19, BmOR30, BmOR45 and BmOR47. BmOR19 responds to linalool, while BmOR45 and BmOR47 respond to benzoic acid > 2-phenylethanol > benzaldehyde. No activating ligands were found for BmOR30. RNA in situ hybridisation experiments reveal that BmOR19 is expressed in female olfactory sensory neurons that are co-located in the same sensilla as a second ORN expressing BmOR45 and/or BmOR47. Taken together the activity and expression of these receptors is likely explanatory of the observed electrophysiology of long sensilla trichoidea of female B. mori, previously shown to each contain one terpene (BmOR19) and one benzoic acid (BmOR45, BmOR47) sensory neuron. Plant volatiles such as linalool, benzoic acid, 2-phenylethanol and benzaldehyde are oviposition cues for females of some moths. These compounds have also been found in male-produced pheromone blends extracted from the hair pencils of many noctuid species. Hair pencil structures have not previously been reported for B. mori, but we have found hair pencil-like structures in adult male B. mori that are absent in female moths. It is proposed that BmOR19, BmOR45 and BmOR47 account for some of the female-specific odorant responses in B. mori, such as oviposition and/or detection of an as yet unidentified male-produced sex pheromone.

Sex pheromone components of male Tirathaba mundella (Lepidoptera: Pyralidae)

During peak calling activity by male oil palm bunch moths, Tirathaba mundella Walker (Lepidoptera: Pyralidae), their hairpencils, wings or entire body were extracted in hexane. Gas chromatographic-electroantennographic detection (GC-EAD) analyses of hair pencil extracts revealed four compounds that consistently elicited responses from female antennae. The NMR spectrum of isolated compound 1, and mass spectra and retention indices of compounds 1–4 suggested that they were (3S,6S)-2,2,6-trimethyl-6-vinyl-tetrahydro-pyran-3-ol (1), 4-hydroxy-3-methoxy-benzaldehyde (2, vanillin), 6,10,14-trimethyl-2-pentadecanone (3), and 6,10,14-trimethyl-2-pentadecanol (4). Comparative GC and GC-MS analyses of hair pencils extract and synthetic standards confirmed these structural assignments. Moreover, comparative chromatography of synthetic and hairpencil-isolated 1 on a Cyclodex-B column (which separated the four stereoisomers with baseline resolution) revealed that male T. mundella produce the SS-stereoisomer (SS-1). In field cage bioassay experiments in Palembang, Indonesia, synthetic SS-1 and vanillin in combination, but not singly, attracted female T. mundella. SS-1 plus vanillin were as effective as male T. mundella in attracting females. Compounds 3 and 4 did not enhance the blend's attractiveness.

Courtship and mating behaviour of the Mediterranean corn borer, Sesamia nonagrioides (Lepidoptera: Noctuidae)

The behavioural sequence of courtship and mating of Sesamia nonagrioides Lefèbvre males and females in laboratorycages is described. The attractant capacity of both sexes was studied by tethering males or females. The courtshipand mating sequence of tethered females was the same as untethered ones, but tethered males were absolutely inactive.Age (1 day versus 2 days) did not affect the mating rate. Differences in calling time onset and first calling agewere found between north-eastern Spanish and Greek populations. The role of hair pencils in courtship flight is discussed,rejecting that they stimulate calling behaviour of females or that they attract females. The influence of adultpopulation density was studied in laboratory and field tests, and was found to have no effect on the mating rate in thecontrols. However, moth density influenced mating rates where mating disruption treatments were applied. Several consequences for investigations into pheromone composition and its use in monitoring and population control are reported in the discussion.

Andesianidae, a new family of monotrysian moths (Lepidoptera : Andesianoidea) from austral South America

The new family Andesianidae is proposed for the genus Andesiana, consisting of three previously described species, A. brunnea Gentili, A. lamellata Gentili and A. similis Gentili, from southern Argentina and Chile. Originally described in the ditrysian family Cossidae, Andesiana has been found to possess a monotrysian female reproductive system. The principal autapomorphies for the family are the elongate second segment of the labial palpi, presence of a male tibial pouch and associated femoral hair pencil and, within the monotrysian Heteroneura, possibly the strongly dimorphic antennae (broadly bipectinate in male, filiform in female). Other significant characters of the group include a greatly reduced haustellum, male genitalia with a broad vinculum, acuminate uncus, scutiform juxta and a pronounced saccular lobe of the valva that bears a subapical, setose appendage within a shallow, mesal cavity. The female genitalia possess a short cloaca and paired anal papillae and lack signa. Although known to be retained only in the female of A. lamellata, the presence of a two-branched Rs in the hindwing may indicate an origin of the Andesianidae basal to the Nepticuloidea. In contrast, the structure of the wing coupling apparatus for the family suggests a later appearance. Because no synapomorphy is known linking the Andesianidae with any other superfamily, the new superfamily, Andesianoidea, is proposed.

Effects of host plant,Gossypium hirsutum L., on sexual attraction of cabbage looper moths,Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae)

Unmated female or male cabbage looper moths,Trichoplusia ni (Hübner), were attracted more often in a flight tunnel to a cage with moths of the opposite sex and a bouquet of cotton foliage. Increased sexual attractiveness of females with plants may be a result of stimulation of pheromone release in response to plant odor, since more males were attracted when odor of cotton foliage was passed over females than when odor of females was passed over cotton foliage before venting into the flight tunnel. Increased sexual attractiveness of males with plants is due in part to host odor enhancement of female attraction to male pheromone, since more females were attracted to synthetic male pheromone (a blend of enantiomers of linalool and isomers of cresol) and a cotton leaf extract than were attracted to male pheromone alone. A short synthesis procedure was developed for (S)-(+)-linalool, the major component of the male sex pheromone, isolated from hair pencils, used in these tests.

Constituents of wing gland and abdominal hair pencil secretions of male African sugarcane borer,Eldana saccharina walker (Lepidoptera: Pyralidae)

In addition totrans-3,7-dimethyl-6-octen-4-olide (eldanolide), vanillin, and 4-hydroxybenzaldehyde, identified by French workers in the wing gland and abdominal hair pencil secretions of the male African sugarcane borer,Eldana saccharina, we have, in an earlier note, reported the presence of several other terpenoid, aromatic, and unbranched-chain compounds such as, (Z)-3,7-dimethylocta-2,6-dienoic acid, 6,10,14-trimethyl-2-pentadecanol, 4-hydroxy-3-methoxybenzyl alcohol, 1-octadecane thiol, 16-hexadecanolide, and 18-octadecanolide in these secretions. In the present paper experimental details and spectral evidence supporting the identification of these compounds, as well as the identification of (Z)-9-hexadecenal and cw-3,7-di-methyl-6-octen-4-olide (cis-eldanolide), are reported. Using electroantennography it was found that male and female antennae reacted approximately equally strongly to both secretions. This result was confirmed in analyses of the secretions using coupled gas chromatography-electroantennography and it was found that male as well as female antennae responded to eldanolide. Vanillin, substituted phenols related to vanillin, and some oxygenated monoterpenes elicited weak responses in male and female antennae. In some analyses 6,10,14-trimethyl-2-pentadecanol, present in the secretions of the insect, gave a strong antennal response. The results obtained in dynamic and static headspace determinations showed that several of the organic compounds present in the glandular secretions are released in detectable quantities and are present in widely varying quantitative ratios in the effluvia of individual calling male moths.

Interspecific Hybridization between Heliothis virescens and H. subflexa (Lepidoptera: Noctuidae) Affects the Presence and Structure of Hair-Pencil Glands of Males

The effect of interspecific hybridization on the inheritance of hair-pencil glands was studied by crossing Heliothis virescens (F.), whose males have hair-pencil glands, with H. subflexa (Guenee), whose males lack hair pencils. Males from reciprocal interspecies crosses had hair-pencil glands the structure of which resembled those of H. virescens males. Backcross males from crosses between ( H. virescens female × H. subflexa male) F1 × H. subflexa ♂ lacked hair-pencil glands and were morphologically similar to H. subflexa males. The hair-pencil glands of first-generation backcross males from all other combinations resembled those of H. virescens . Second-generation backcross males from crosses, in which both Z chromosomes were inherited from H. subflexa , lacked hair pencils. However, second-generation backcross males that inherited at least one of the Z chromosomes from H. virescens were phenotypically similar to H. virescens . These results indicated that the presence of hair-pencil glands is regulated by a dominant allele of a locus or a group of genes located on the Z chromosome of H. virescens .

Foreleg stridulation in male Urania moths (Lepidoptera: Uraniidae)

Males of the uraniid moth genus Urania possess a stridulatory organ on the prothoracic leg. This organ represents an autapomorphy for the genus. The sound-producing mechanism consists of a peg formed of specialized scales on the coxa which produces a train of high pitched clicks when scraped against scales on the proximal end of the femur. The proximal end of the femur is enlarged compared with that of the females and would serve to amplify the sound pulse. Stridulation occurs during a series of rapid forward jerks of the forelegs. The sound produced by Urania consists of a low amplitude pulse audible to humans at short range but with a strong ultrasound component. It has not yet been observed in the wild and its function in these moths is unknown, but it may be used during intrasexual or perhaps intersexual communication. This organ is unlikely to have been evolved originally as a defence against nocturnal predators since the genus is a member of an exclusively diurnal clade and females lack the organ. Stridulation occurs in conjunction with fanning-out of the foreleg femoral hair pencil and this invites speculation as to a route for evolution of the organ.

EVIDENCE OF A MALE SEX PHEROMONE IN THE EUROPEAN CORN BORER, OSTRINIA NUBILALIS (HÜBNER) (LEPIDOPTERA: PYRALIDAE)

AbstractEuropean corn borer males have hair pencils located ventrally on the 8th sternite and these are extruded when a male approaches a calling female. The fact that (i) antennectomized females mated significantly less than both intact controls and individuals subjected to other forms of surgery, and (ii) males with hair pencils removed had a significantly lower mating success than control males, suggests that a male pheromone is involved in the mating system of the European corn borer.

Cyclic Glycerol Acetals from the Abdominal Hair Pencil Secretion of the Male African Sugarcane Borer Eldana saccharina (Lepidoptera: Pyralidae)

Four constituents of the hair pencil secretion of the male African sugarcane stalk borer, Eldana saccharina, having a molecular mass of 312 and peculiar El mass spectra with an exceptionally abundant base peak at m /z 103, were isolated preparatively from an extract of the secretion. Using 1H and 13C NMR spectral analysis, these constituents were identified as fiveand six-membered cyclic glycerol acetals of Z-9-hexadecenal, viz. cis- and trans-2-(Z- 8-pentadecenyl)-4-hydroxymethyl-1,3-dioxolane, and cis- and trans-2-(Z-8-pentadecenyl)- 5-hydroxy-1,3-dioxane. These compounds are related to the 2-alkenyl-4-hydroxymethyl-1,3- dioxolane dihydrogen phosphate esters, known to be the active constituents of the smooth muscle contracting acidic phospholipid (Darmstoff) which was isolated from the intestine of mammals. The presence of these acetals in the tail brush secretion of E. saccharina could possibly be the first evidence that com pounds related to the active principle of Darmstoff, may also be present in the insect kingdom. The possibility that these four compounds or their dihydrogen phosphate esters might play a part in the eversion or retraction of the tail brushes of the male insect, is briefly discussed.

Temporal Partitioning of Dual Sexual Attraction Strategies in Trichoplusia ni (Lepidoptera: Noctuidae)

In a field cage, male cabbage looper attraction to females and female attraction to males were temporally distinct. Male visitation and hair penciling to confined, unmated females occurred mainly throughout the third to eighth hours of a 10-h night. Unmated females visited confined males principally during the first hour of the scotophase. Female attraction to males at dusk was not associated with the male display of hair pencils that are the source of a previously identified male pheromone. The cabbage looper apparently uses both a male-produced female attractant near dusk and a female-produced male attractant later at night as part of its mate-finding strategies.