Nectar-dwelling microbes of common tansy are attractive to its mosquito pollinator, Culex pipiens L.

BACKGROUND: There is widespread interkingdom signalling between insects and microbes. For example, microbes found in floral nectar may modify its nutritional composition and produce odorants that alter the floral odor bouquet which may attract insect pollinators. Mosquitoes consume nectar and can pollinate flowers. We identified microbes isolated from nectar of common tansy, Tanacetum vulgare, elucidated the microbial odorants, and tested their ability to attract the common house mosquito, Culex pipiens. RESULTS: We collected 19 microbial isolates from T. vulgare nectar, representing at least 12 different taxa which we identified with 16S or 26S rDNA sequencing as well as by biochemical and physiological tests. Three microorganisms (Lachancea thermotolerans, Micrococcus lactis, Micrococcus luteus) were grown on culture medium and tested in bioassays. Only the yeast L. thermotolerans grown on nectar, malt extract agar, or in synthetic nectar broth significantly attracted Cx. pipiens females. The odorant profile produced by L. thermotolerans varied with the nutritional composition of the culture medium. All three microbes grown separately, but presented concurrently, attracted fewer Cx. pipiens females than L. thermotolerans by itself. CONCLUSIONS: Floral nectar of T. vulgare contains various microbes whose odorants contribute to the odor profile of inflorescences. In addition, L. thermotolerans produced odorants that attract Cx. pipiens females. As the odor profile of L. thermotolerans varied with the composition of the culture medium, we hypothesize that microbe odorants inform nectar-foraging mosquitoes about the availability of certain macro-nutrients which, in turn, affect foraging decisions by mosquitoes.

Note: Evaluation of magnetometry data acquired from elongated samples.

We document and validate an analytic expression for the flux integral characterizing the response (or sensitivity) of a magnetometer equipped with second-order axial gradiometer coils to long, thin, uniformly magnetized samples. We then demonstrate an application inspired by this analysis, in which magnetometric sensitivity to weak magnetic signatures is readily and appreciably enhanced by increasing the sample volume (and hence the quantity of analyte) well beyond conventional limits.

Evidence for short-range sonic communication in lymantriine moths.

Sexual communication of nun moth, Lymantria monacha (L.), pink gypsy moth, Lymantria mathura Moore, and fumida tussock moth, Lymantria fumida Butler (all Lepidoptera: Noctuidae: Lymantriinae), is known to be mediated by pheromones. We now show that males are attracted by the sounds of conspecific females over short distances and that wing fanning male and female L. monacha, L. mathura and L. fumida produce species- and sex-specific wing beat and associated click sounds that could contribute to reproductive isolation. Evidence for short-range communication in these lymantriines includes (i) scanning electron micrographs revealing metathoracic tympanate ears, (ii) laser interferometry showing particular sensitivity of tympana tuned to frequency components of sound signals from conspecifics, and (iii) phonotaxis of male L. monacha and L. fumida to speakers playing back sound signals from conspecific females. We conclude that tympanate ears of these moths have evolved in response not only to bat predation, but also for short-range mate finding and possibly recognition.

Does larval aggregation pheromone of codling moth, Cydia pomonella, induce attraction or arrestment of receivers?

Cocoon-spinning larvae of the codling moth, Cydia pomonella, emit a pheromone that mediates aggregation by pupation site-seeking fifth-instar larvae. It was unknown and, thus, we tested whether the aggregation pheromone induces arrestment or attraction responses. In paired straight-tube experiment 1, fifth-instars moved faster and farther upwind toward cospecific cocoons compared to blank controls. In still-air cage experiment 2, fifth-instars selected more often as first and final choices of pupation sites those with cocooning conspecifics than those without. Finally, in Y-tube olfactometer experiment 3, fifth-instars anemotactically responded to, and preferred, side arms with cocooning conspecifics to those without. Our data provide evidence that codling moth larvae are attracted to, rather than merely arrested by, larval aggregation pheromone. These results help explain reported aggregations or clumped distributions of larvae on tree trunks, which would likely not occur if they were based merely on chance encounter of cocoon-spinning larvae by foraging larvae.

Sex pheromone of tomato fruit borer, Neoleucinodes elegantalis.

Five candidate pheromone components were identified by analyzing pheromone gland extracts by gas chromatography (GC), coupled GC-electroantennographic detection (EAD), and coupled GC-mass spectrometry (MS) : (E)-11-hexadecenol(E11-16 : OH), (Z)-11-hexadecenol (Z11-16 : OH), (E)-11-hexadecenal, (E)-11-hexadecenyl acetate, and (Z)-3,(Z)-6,(Z)-9-tricosatriene (Z3,Z6,Z9-23 : Hy). In electroantennogram (EAG) recordings, synthetic E11-16 : OH elicited stronger antennal responses at low doses than other candidate pheromone components. Field tests demonstrated that synthetic E11-16 : OH as a trap bait was effective in attracting males, whereas addition of Z11-16 : OH inhibited the males' response. Z3,Z6,Z9-23 : Hy strongly enhanced attractiveness of E11-16 : OH, but was not attractive by itself. A pheromone blend with synergistic behavioral activity of an alcohol (E11-16 : OH) and hydrocarbon (Z3,Z6,Z9-23 : Hy) component is most unusual in the Lepidoptera. The synthetic two-component pheromone is approximately 60 times more attractive than the female-produced blend and might facilitate the control of this pest.

Reproductive character displacement in Lymantria monacha from northern Japan?

Our objective was to test the hypothesis that the pheromone blend and/or diel periodicity of pheromonal communication differ in populations of the nun moth, Lymantria monacha (Lepidoptera: Lymantriidae), from eastern Asia (northern Honshu, Japan) and Central Europe (Bohemia, Czech Republic). Coupled gas chromatographic-electroantennographic detection (GC-EAD) analyses of pheromone gland extract of female L. monacha from Japan confirmed the presence of compounds previously identified in pheromone extracts of L. monacha from Bohemia, as follows: (Z)-7-octadecene, 2-methyl-(Z)-7-octadecene (2me-Z7-18Hy), cis-7,8-epoxy-octadecane (monachalure), and cis-7,8-epoxy-2-methyloctadecane (disparlure). Field experiments in Honshu suggested that (+)-monachalure is the major pheromone component of L. monacha. 2me-Z7-18Hy significantly enhanced attractiveness of (+)-monachalure. Addition of (+)-disparlure to (+)-monachalure plus 2me-Z7-18Hy in Honshu and Bohemia increased attractiveness of lures by 1.2 and 20 times, respectively, indicating that (+)-disparlure is of least and most significance in the respective L. monacha populations. Moreover, capture of male L. monacha in pheromone-baited traps between 18:00 and 24:00 hr in Bohemia and 2:00 and 5:00 hr in Honshu revealed a markedly different diel periodicity of pheromonal communication. Pheromonal communication late at night and use of (+)-monachalure, rather than (+)-disparlure, as the major pheromone component by L. monacha in Honshu may have resulted from interspecific competition with coseasonal L. fumida, which uses the early night for pheromonal communication and (+)-disparlure as major pheromone component. Whether communication channel divergence of L. monacha in Honshu indeed constitutes a case of reproductive character displacement is difficult to prove. The evolution of such divergence in sympatric populations of L. fumida and L. monacha would have to be demonstrated.

Communication ecology of webbing clothes moth: 1. Semiochemical-mediated location and suitability of larval habitat.

We tested two hypotheses: 1) that there is semiochemical-mediated attraction of male and female webbing clothes moth (WCM), Tineola bisselliella (Hum.) (Lepidoptera: Tineidae) to suitable larval habitat; and 2) that selection of optimal larval habitat has fitness consequences. In binary or ternary choice arena bioassay experiments that prevented WCM from contacting test stimuli, males and females were attracted to dried but untanned animal pelts (red squirrel, muskrat, beaver, coyote, red fox and bobcat) and preserved horseshoe crab but not to unprocessed sheep's wool, demonstrating semiochemical-based recognition of, and discrimination between, potential larval habitats. Selection of habitat has fitness consequences for ovipositing females, because significantly more male and female WCM completed development when the larval diet consisted of intact animal pelt (hide plus hair) rather than hide or hair alone. Equal attraction of male WCM to muskrat pelt volatiles in Porapak Q or solvent extracts of muskrat pelts indicated that volatile semiochemicals could be obtained by both methods.

Communication ecology of webbing clothes moth: 2. Identification of semiochemicals mediating attraction of adults to larval habitat.

Our objective was to identify the semiochemicals that mediate attraction of the webbing clothes moth (WCM), Tineola bisselliella (Lepidoptera: Tineidae), to suitable larval habitat. Coupled gas chromatographic-electroantennographic detection (GC-EAD) analyses of Porapak Q-captured bioactive volatiles from horseshoe crab, and dried but untanned vertebrate pelts revealed numerous EAD-active volatiles. These volatiles were identified by comparative GC-mass spectrometry and GC-EAD analyses of natural and synthetic compounds. A blend of 28 synthetic candidate semiochemicals attracted both male and female WCM. Experiments deleting various components determined that saturated aldehydes--but not unsaturated aldehydes, saturated hydrocarbons, saturated alcohols, or ketones--were essential for blend attractiveness. A blend of nonanal, the single most attractive aldehyde, in combination with geranylacetone was more attractive to WCM than the 28-component blend or dried, untanned animal pelt. Selection of larval habitat resides more with male than female WCM, as indicated by stronger EAD responses from male than female antennae to habitat-derived semiochemicals, and more selective and early response to habitat cues by males than females. Exploitation of nonanal and geranylacetone as resource-derived semiochemicals by both adult WCM and its larval parasitoid, Apanteles carpatus, is an example of convergent semiochemical parsimony.

Does pheromone biology of Lambdina athasaria and L. pellucidaria contribute to their reproductive isolation?

Recently, 7-methylheptadecane and 7,11-dimethylheptadecane have been reported as sex pheromone components of both spring hemlock looper (SHL), Lambdina athasaria, and pitch pine looper (PPL), Lambdina pellucidaria. Our objective was to test the hypothesis that SHL and PPL are reproductively isolated, in part, through species specificity in: (1) absolute configuration of pheromone components, (2) diel periodicity of pheromonal communication, and/or (3) seasonal flight period. In coupled gas chromatographic-electroantennographic detection (GC-EAD) analyses of stereoselectively synthesized (7S)- and (7R)-7-methylheptadecane [7S; 7R] as well as (7S,11S)-, (7R,11R)-, and (meso-7,11)-7,11-dimethylheptadecane [7S,11S; 7R,11R; meso-7,11], only 7S and meso-7,11 elicited responses by male SHL and PPL antennae. In field experiments, male SHL and PPL were attracted only to lures containing 7S plus meso-7,11. In hourly recordings of trap-captured males, SHL and PPL in their respective habitats were trapped between 24:00 and 03:00 hr. Capture of both SHL and PPL in pheromone-baited traps throughout June indicated overlapping seasonal flight periods. These findings of identical absolute configuration of pheromoal components, diel periodicity of pheromonal communication, and overlap of seasonal flight periods support synonymy of SHL and PPL. Finite taxonomic classification of PPL and SHL must await careful assessment of further criteria, such as morphometrics, molecular comparisons and ecological analyses.

Sex pheromone of orange wheat blossom midge, Sitodiplosis mosellana.

Pheromone extract of the female orange wheat blossom midge, Sitodiplosis mosellana (Géhin) (SM) (Diptera: Cecidomyiidae), was analyzed by coupled gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometry (MS), employing fused silica columns coated with DB-5, DB-210, DB-23 or SP-1000. These analyses revealed a single, EAD-active candidate pheromone which was identified as 2,7-nonanediyl dibutyrate. In experiments in wheat fields in Saskatchewan, traps baited with (2S,7S)-2,7-nonanediyl dibutyrate attracted significant numbers of male SM. The presence of other stereoisomers did not adversely affect trap captures. Facile synthesis of stereoisomeric 2,7-nonanediyl dibutyrate will facilitate the development of pheromone-based monitoring or even control of SM populations.

Association of nickel versus transport of cadmium and calcium in tonoplast vesicles of oat roots.

The plant vacuole has long been suspected of being a site for accumulation of Ni in plant roots, but testing this hypothesis directly by vacuole isolation is technically difficult and has not been reported. Here, we have attempted to determine if Ni can be transported into isolated oat (Avena sativa L.) root tonoplast vesicles as an alternative approach toward understanding the importance of the vacuole in Ni accumulation in roots. We found that, in contrast to Ca and Cd, Ni did not affect the proton gradient of vesicles (MgATP energized or artificially created), and further, that Cd/H antiport activity was not affected by the presence of Ni. Nickel was associated with vesicles, but relative rates of accumulation/association of metals with vesicles were Ca > Cd >> Ni. Protonophores and the potential Ni ligands citrate and histidine, and nucleoside triphosphates or PPi did not stimulate Ni association with vesicles. Comparison of Ni versus Ca and Cd associated with vesicles using various membrane perturbants indicated that while Ca and Cd are rapidly and principally antiported to the vesicle sap, Ni is only slowly associated with the membrane in a not-easily dissociated condition. Our results indicate the absence of an Ni/H antiport or Ni-nucleotide-dependent pump in oat root tonoplasts, and support the contention that the vacuole is not a major compartment for Ni accumulation in oat roots.

Sex attractant pheromone of the pecan nut casebearer (Lepidoptera: Pyralidae).

A female-produced sex pheromone for the pecan nut casebearer, Acrobasis nuxvorella Neunzig, has been identified from pheromone gland extracts of calling female moths. The compound (9E,11Z)-hexadecadienal [(9E,11Z)-16:Ald] was identified by coupled GC-EAD and retention time matches with a synthetic standard on four capillary GC columns of different polarities. Corroboration of the identification of (9E,11Z)-16:Ald by other analytical chemistry methods was not possible due to the minute quantities of pheromone extracted (< 1 picogram/female). In field studies, gray rubber septa impregnated with 100 micrograms of synthetic (9E,11Z)-16:Ald were attractive to male moths, whereas higher and lower doses were less attractive. The homologous (9E,11Z)-15:Ald was also slightly attractive while the more highly conjugated analogues, (9E,11Z,13Z)- and (9E,11Z,13E)-16:Ald, were not.

Identification and synthesis of new bicyclic acetals from the mountain pine beetle, Dendroctonus ponderosae Hopkins (Col.: Scol.).

Head-space volatiles obtained from male mountain pine beetles, Dendroctonus ponderosae, were analyzed by coupled GC-MS and chiral gas chromatography. 5-Ethyl-7-methyl-6,8-dioxabicyclo[3.2.1]octane (6) was found as a new naturally occurring isomer of brevicomin (1). In addition, several stereoisomers of 7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octan-2-ol (11) and 1-(5-methyl-6,8-dioxabicyclo[3.2.1]octyl)ethanol (12) could be identified. Relative and absolute configurations of the compounds were determined by unambiguous syntheses, which are described.

Transformation of presumptive precursors to frontalin and exo-brevicomin by bark beetles and west Indian sugarcane weevil (Coleoptera).

(Z)-6-Nonen-2-one (1) has recently been shown to be the biosynthetic precursor for the aggregation pheromone exo-brevicomin (2) in mountain pine beetle (MPB) males, Dendroctonus ponderosae (Hopkins). We tested the hypotheses that (1) 6-methyl-6-hepten-2-one (3) is the biosynthetic precursor for the aggregation pheromone frontalin (4) in the spruce beetle (SB), Dendroctonus rufipennis (Kirby), and (2) that frontalin and exo-brevicomin are produced from 3 and 1, respectively, only by beetles that utilize them as aggregation pheromones. Exposure of scolytids MPB, SB, pine engraver (PE), Ips pini (Say) and Ips tridens (Mannerheim) and West Indian sugar cane weevil (WISW), Metamasius hemipterus sericeus (Olivier) to deuterio- or protio-3 invariably resulted in the production of deuterio- or protio-4. Similarly, exposure of SB, WISW and I. tridens to 1 resulted in the production of 2. We were unable to demonstrate the presence of 3 in SB volatiles, nor were we able to demonstrate the conversion of 6-methyl-5-hepten-2-one to 3 by SB. Production of enantiomerically enriched frontalin and exo-brevicomin by all the beetles exposed to respective precursors reveals widespread occurrence of nonspecific polysubstrate monooxidases in the Coleoptera.

Antennal response and field attraction of the predator Elatophilus hebraicus (Hemiptera: Anthocoridae) to sex pheromones and analogues of three Matsucoccus spp. (Homoptera: Matsucoccidae).

The predator Elatophilus hebraicus is closely associated with its prey, the pine bast scale, Matsucoccus josephi, and utilizes the M. josephi sex pheromone as a kairomone. Kairomonal activity of E. hebraicus was studied by GC-EAD and field bioassays. The sex pheromone of M. josephi [2E,5R,6E,8E)-5,7-dimethyl-2,6,8-decatrien-4-one [(R)-E-M.j.] elicited a strong EAD response and attracted large numbers of the predator. The sex pheromone of two allopatric Matsucoccus spp., Matsucoccus feytaudi, (3S,7R,8E,10E)-3,7,9-trimethyl-8,10-dodecadien-6-one [(S,R)-E-M.f.] and Matcucossus matsumurae, (2E, 4E,6R,10R)-4,6,10,12-tetramethyl-2,4-tridecadien-7-one [(R,R)-E-M.m.], were also EAD-active and attracted significant numbers of E. hebraicus in the forest. Increasing the lure load of (S,R)-E-M.f. and (R,R)-E-M.m., in order to compensate for their lower volatility relative to (R)-E-M.j., resulted in similar attraction of E. hebraicus to each of the three pheromones. Other Matsucoccus pheromone stereoisomers displayed no behavioral activity. There was a significant difference in the activity of sex pheromone analogues, (6E/Z,8E)-5,7-dimethyl-6,8-decadien-4-one (52% E + 48% Z, ANLG 1) and (6E/Z,8E)-2,4,6-trimethyl- 1,6,8-nonatrien-3-one (60% E + 40% Z, ANLG 2). The (E) isomer of ANLG 1 evoked a strong EAD response from E. hebraicus and the mixture of E/Z ANLG 1 attracted the predator in moderate numbers, whereas ANLG 2 was inactive both in EAD and field tests. Conversely, M. josephi males were not attracted to M. feytaudi and M. matsumurae pheromones or pheromone analogues. Cross-activity of E. hebraicus to M. feytaudi and M. matsumurae pheromones may be based on structural similarity of the compounds. Alternatively, E. hebraicus may respond specifically to the pheromones of two allopatric Matsucoccus spp. If true, kairomonal attraction of E. hebraicus to these pheromones may have evolved during speciation of Matsucoccidae and may have been preserved despite the allopatry of M. josephi, M feytaudi and matsumurae.

Green leaf volatiles as antiaggregants for the mountain pine beetle,Dendroctonus ponderosae Hopkins (Coleoptera: Scolytidae).

We tested the hypothesis that green leaf volatiles act as antiaggregants for the mountain pine beetle (MPB),Dendroctonus ponderosac Hopkins. In coupled gas chromatographic-electroantennographic detection (GC-EAD) analysis MPB antennae responded to 30 ng doses of all six-carbon green leaf alcohols tested [1-hexanol, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, (E)-3-hexen-1-ol, and (Z)-3-hexen-1-ol], but not to the aldehydes, hexanal or (E)-2-hexenal, or to alcohol or aldehyde homologues with more or fewer than six carbon atoms. In field trapping experiments a blend of green leaf alcohols [1-hexanol, (Z)-2-hexen-1-ol, (E)-3-hexen-1-ol and (Z)-3-hexen-1-ol] effectively disrupted the response to attractive semiochemicals; a blend of the aldehydes hexanal and (E)-2-hexenal was inactive. The two best disruptants. (E)-2-hexen-1-ol and (Z)-3-hexen-1-ol, reduced catches of both sexes to levels not significantly different from catches in unbaited control traps. They also reduced the attack on trees baited with attractive MBP pheromones to a level not significantly different from that on unbaited control trees. Neither of the clerid predators captured,Enoclerus sphegeus (F.) norThanasimus undatulus (Say), was repelled by green leaf volatiles. Our results suggest that green leaf alcohols are promising disruptants which may be used to supplement the antiaggregation pheromone, verbenone, in protecting single high-value trees as well as carefully selected stands with low-level populations of MPBs.

Sex phermone components of mulberry looper,Hemerophila atrilineata butler (lepidoptera: Geometridae).

(6Z-9S, 10R)-Epoxy-octadecene (SR-1) and (3Z, 6Z-9S, 10R)-epoxy-octadecadiene (SR-2) are sex pheromone components of the mulberry looper (MBL),Hemerophila atrilineata Butler. Compounds extracted from female MBL pheromone glands were identified by coupled gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometry. In field experiments in China,SR-2,RS-2, or both combined were hardly attractive, butSR-2 in combination withSR-1 attracted significant numbers of MBL males. Synergistic behavioral activity ofSR-1 plusSR-2, but not of corresponding antipode mixtures, indicates enantiospecificity of MBL pheromone communication. Because blends of racemic and enantiospecific (SR)1 plus2 were similarly attractive, racemic1 plus2 may have potential for mass trapping or confusion of MBL males in commercial mulberry plantations.

Pheromone chirality of asian palm weevils,Rhynchophorus ferrugineus (Oliv.) andR. vulneratus (Panz.) (Coleoptera: Curculionidae).

Production of 4-methyl-5-nonanol, and 4-methyl-5-nonanone by two sympatric Asian palm weevils,Rhynchophorus ferrugineus (Oliv.) andR. vulneratus (Panz.) suggested that enantiospecificity of either compound could impart species specificity of pheromone communication. Weevil-produced, racemic 4-methyl-5-nonanol and 4-methyl-5-nonanone and their stereoselectively synthesized optical isomers were subjected to gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometry (MS) on a chiral Cyclodex-B column. Only theS,S stereoisomer of 4-methyl-5-nonanol was EAD active and was produced by bothR. ferrugineus andR. vulneratus. Production and EAD activity of (S)-4-methyl-5-nonanone exceeded that of its antipode in both weevils. In field experiments in Java. (4S, 5S)-4-methyl-5-nonanol and the stereoisomeric mixture were equally attractive. The 4R,5R stereoisomer was inactive. The corresponding ketone enantiomers neither enhanced nor reduced attraction to (4S,5S)-4-methyl-5-nonanol. Lack of apparent differences betweenR. ferrugineus andR. vulneratus pheromones suggests that synonomy of both weevils should be considered unless other pre- or postzygotic reproductive isolating mechanisms are disclosed in future studies.

Aggregation pheromone of coconut rhinoceros beetle,Oryctes rhinoceros (L.) (coleoptera: Scarabaeidae).

Male coconut rhinoceros beetles,Oryctes rhinoceros (L.), produce three sex-specific compounds, ethyl 4-methyloctanoate, ethyl 4-methylheptanoate, and 4-methyloctanoic acid, the first of which is an aggregation pheromone. Synthesis of these compounds involving conjugate addition of organocuprates to ethyl acrylate is reported. In field trapping experiments, (4S)-ethyl 4-methyloctanoate and the racemic mixture were equally attractive and 10 times more effective in attracting beetles than ethyl chrysanthemumate, a previously recommended attractant. Ethyl 4-methylheptanoate was as attractive as ethyl chrysanthemumate and more attractive than 4-methyloctanoic acid, but further studies are required before it can be classed as an aggregation pheromone. Compared to ethyl 4-methyloctanoate alone, combinations of the three male-produced compounds did not increase attraction, whereas addition of freshly rotting oil palm fruit bunches to pheromone-baited traps significantly enhanced attraction. With increasing dose, captures ofO. rhinoceros increased, but doses of 6, 9, and 18 mg/day were competitive with 30 mg/day lures. Newly designed vane traps were more effective in capturing beetles than were barrier or pitfall traps. Results of this study indicate that there is potential for using ethyl 4-methyloctanoate in operational programs to controlO. rhinoceros in oil palm plantations.