Pheromone-mediated upwind flight of male gypsy moths, Lymantria dispar, in a forest

Abstract Lymantria dispar L. males flying upwind in a pheromone plume in a forest were video-recorded at 2.5, 10 and 20 m from the source of pheromone. Males flew slower and steered more across the wind as they approached the source. In concert, their ground speed decreased and track angles increased. In contrast to these changes, their drift angles were fairly constant and the transverse component of image flow, above and/or below the moths eyes, showed almost no change. The inter-turn duration (time between sequential turns), a temporal aspect of the male flight manoeuvres, showed a consistent but relatively small increase as the distance from the source increased. The flight tracks narrowed as the males approached close (2.5 m) to the source. Because of unpredicted correlations between physical variables (i.e. temperature, wind velocity) and the distance from the source, we used principal components analysis to generate a set of completely independent variables. Greater than 90% of the variability in the data could be explained by four principal factors which corresponded well with known relationships in the flight manoeuvres. All four of these factors showed a significant regression against distance to the source. Although uncontrolled factors such as temperature and wind velocity may have contributed to changes in flight behaviour, recent data indicate that, in addition to concentration, certain temporal and spatial characteristics (i.e. burst period, burst return period) of plumes in wind vary systematically with distance from the source. We propose that L.dispar males might adjust their flight manoeuvres in response to these changes.     

Modeled global invasive potential of Asian gypsy moths, Lymantria dispar

Asian populations of gypsy moths, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), remain poorly characterized – indeed, they are not presently accorded any formal taxonomic status within the broader species. Their ecology is similarly largely uncharacterized in the literature, except by assumption that it will resemble that of European populations. We developed ecological niche models specific to Asian populations of the species, which can in turn be used to identify a potential geographic distributional area for the species. We demonstrated statistically significant predictivity of distributional patterns within the East Asian range of these populations; projecting the Asian ecological niche model to Europe, correspondence with European distributions was generally good, although some differences may exist; projecting the ecological niche model globally, we characterized a likely potential invasive distribution of this set of populations across the temperate zone of both Northern and Southern Hemisphere.     

Mitochondrial DNA variation among worldwide populations of gypsy moths, Lymantria dispar

Gypsy moth populations from Japan, mainland Asia, Europe, Tunisia, and North America were analyzed for variation in mitochondrial DNA (mtDNA) sequences from three gene regions. These samples resolve into four groups, representing gypsy moths from (1) Okinawa, Japan, (2) Hokkaido, Japan, (3) Honshu and Kyushu, Japan and mainland Asia, and (4) Europe, Tunisia, and North America. Some patterns of geographic variation observed for mtDNA (for example, the distinctiveness of gypsy moths from Hokkaido, Japan) coincide with those observed by Goldschmidt from analyses of morphology, life history, and intersexuality. Other patterns (relative sequence homogeneity across Asia, Honshu, and Kyushu and reduced levels of variation in mainland Japan) do not.     

Orientation of male gypsy moths,Lymantria Dispar (L.), to pheromone sources: The role of olfactory and visual cues

The role of olfaction and vision in the close-ranging flying and walking orientation of male gypsy moths, Lymantria dispar(L.), to females was studied in the forest and in the laboratory. In the forest, feral males found an isolated pheromone source as readily as one supplemented with female visual cues; dead, acetonerinsed females deployed without pheromone received virtually no visitations. In flight tunnel choice experiments using cylinders as surrogate trees and pheromone in different spatial configurations, visual attributes of the female did not influence either the males' choice of landing site or the efficiency with which they located the female. Rather, the presence of pheromone on the cylinder was necessary to elicit orientation as well as landing and walking on the cylinder. When a female visual model was placed in various positions around a pheromone source, walking males oriented primarily to the chemical stimulus. There were, however, indications that males would alter their walking paths in response to female visual cues over short distances (<5 cm), but only if they continued to receive pheromone stimulation. When visual and chemical cues were abruptly uncoupled by altering the trajectory of the pheromone plume, most males responded to the loss of the odor cue rather than to visual cues from the female. Temporal pheromone stimulation patterns affected male walking orientation. When stimulated by pheromone, males oriented toward the source; loss of the odor cue prompted an arearestricted local search characterized by primarily vertical and oblique movements with frequent reversals in direction. Presumably these maneuvers enhance the likelihood of recontacting the plume or serendipitously encountering the female. The apparent lack of visual response to the female is discussed in light of morphological and behavioral evidence suggesting that gypsy moths were formerly nocturnal.     

Pheromone-modulated optomotor response in male gypsy moths,Lymantria dispar L.: Directionally selective visual interneurons in the ventral nerve cord

In response female pheromone the male gypsy moth flies a zigzagging path upwind to locate the source of odor. He determines wind direction visually. To learn more about the mechanism underlying this behavior, we studied descending interneurons with dye-filled micro-electrodes. We studied the interneuronal responses to combinations of pheromone and visual stimuli.

Neurological influences on pheromone release and calling behaviour in the gypsy moth, Lymantria dispar

ABSTRACT. Surgical removal of the brain or disconnection of the last abdominal ganglion from the ventral nerve cord prevented sex pheromone release in female Lymantria dispar (L.) (Lymantriidae), as assayed by the male wing-fanning response. The calling behaviour continued to occur in individuals whose terminal abdominal ganglion had been thus isolated, however, indicating that the neural mechanisms controlling calling function independently in the last abdominal ganglion.     

Discrimination of pheromone enantiomers by two pheromone binding proteins from the gypsy moth Lymantria dispar

The gypsy moth, Lymantria dispar, uses (7R, 8S)-cis-2-methyl-7, 8-epoxyoctadecane, (+)-disparlure, as a sex pheromone. The (-) enantiomer of the pheromone is a strong behavioral antagonist. Specialized sensory hairs, sensillae, on the antennae of male moths detect the pheromone. Once the pheromone enters a sensillum, the very abundant pheromone binding protein (PBP) transports the odorant to the sensory neuron. We have expressed the two PBPs found in gypsy moth antennae, PBP1 and PBP2, and we have studied the affinity of these recombinant PBPs for the enantiomers of disparlure. To study pheromone binding under equilibrium conditions, we developed and validated a binding assay. We have addressed the two major problems with hydrophobic ligands in aqueous solution: (1) concentration-dependent adsorption of the ligand on vial surfaces and (2) separation of the protein-bound ligand from the material remaining free in solution. We used this assay to demonstrate for the first time that pheromone binding to PBP is reversible and that the two PBPs from L. dispar differ in their enantiomer binding preference. PBP1 has a higher affinity for the (-) enantiomer, while PBP2 has a higher affinity for the (+) enantiomer. The PBP from the wild silk moth, Antheraea polyphemus (Apol-3) bound the disparlure enantiomers more weakly than either of the L. dispar PBPs, but Apol-3 was also able to discriminate the enantiomers. We have observed extensive aggregation of both L. dispar PBPs and an increase in pheromone binding at high (>2 microM) PBP concentrations. We present a model of disparlure binding to the two PBPs.     

The interaction of edge-fixation and negative phototaxis in the orientation of walking gypsy moths,Lymantria dispar

Summary The visual orientation towards single black stripes and more complex patterns, comprising smooth gradients of brightness was studied in walking gypsy moths. Depending on the width of a black stripe, up to three walking directions are preferred within one stimulus situation: towards the centre of the stripe and towards a region within the stripe closer to each edge. The observed responses are explained by a compromise between edge-fixation and negative phototaxis. This hypothesis turned out to be also applicable to more complex patterns.     

Host tree influences on the dispersal of first instar gypsy moths, Lymantria dispar (L.)

Abstract. 1. In laboratory tests, first instar gypsy moths attempted dispersal more frequency when exposed to less acceptable foliage.
2. First instars from small eggs attempted dispersal less frequently than larvae from large eggs when exposed to foliage from highly acceptable or marginally acceptable hosts. Dispersal rates of larvae from medium sized eggs were intermediate.
3. These results (1–2) confirm and expand upon the findings of Capinera & Barbosa (1976).
4. In the field, data on the relative densities of larvae on different host species support the conclusion that the frequency of dispersal attempts is inversely related to host acceptability.
5. The implications of these findings for the population dynamics of the gypsy moth are discussed.     

Effects of pheromone plume structure and visual stimuli on the pheromone-modulated upwind flight of male gypsy moths (Lymantria dispar) in a Forest (Lepidoptera: Lymantriidae)

The pheromone-modulated upwind flight ofLymantria dispar males responding to different pheromone plume structures and visual stimuli designed to mimic trees was video recorded in a forest. Males flying upwind along pheromone plumes of similar structure generated tracks that were similar in appearance and quantitatively similar in almost all parameters measured, regardless of the experimentally manipulated visual stimuli associated with the pheromone source. Net velocities, ground speeds, and airspeeds of males flying in point-source plumes were slower than those of males flying in the wider, more diffuse plumes issuing from a cylindrical baffle. The mean track angle of males flying in plumes issuing from a point source was greater (oriented more across the wind) than that of males flying in plumes issuing from a transparent cylindrical baffle. Males flying in point-source plumes also turned more frequently and had narrower tracks overall than males responding to plumes from a cylindrical baffle. These data suggest thatL. dispar males orienting to pheromone sources (i.e., calling females) associated with visible vertical cylinders (i.e., trees) use predominantly olfactory cues to locate the source and that the structure of the pheromone plume markedly affects the flight orientation and the resultant track.     

Structural and functional difference of pheromone binding proteins in discriminating chemicals in the gypsy moth, Lymantria dispar

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68 ± 0.01 μM, for (+)-D was 5.32 ± 0.11 μM, while PBP2 with OXP1 and (+)-D were 1.88 ± 0.02 μM and 5.54 ± 0.04 μM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control.     

Development of male-incubated ovaries in the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin in the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased in size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion fromation was delayed in male-grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS-PAGE analysis of the male-grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the male-incubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed.     

Identification of cuticular hydrocarbons and the alkene precursor to the pheromone in hemolymph of the female gypsy moth, Lymantria dispar

Hydrocarbons were extracted from the surface of the cuticle and from the hemolymph of adult female gypsy moths. GC and GC/MS analysis indicated that the cuticular hydrocarbons with chain lengths >21 carbons were the same as those found in the hemolymph. These consisted of mostly saturated straight chain hydrocarbons with heptacosane the major component. Methyl branched hydrocarbons were also identified including a series of tetramethylalkanes with chain lengths of 30, 32, and 34 carbons. In addition to those found on the cuticle surface, the hemolymph contained the alkene pheromone precursor, 2-methyl-Z7-octadecene and two saturated analogues, 2-methyl-octadecane and 2-methyl-hexadecane. No evidence was obtained for the presence of the pheromone 2-methyl-7, 8-epoxy-octadecane in the hemolymph. Pheromone gland extracts indicated that small amounts (<1 ng) of the alkene precursor were also present in the gland. Relatively larger amounts of the alkene precursor were found in the hemolymph at the time when pheromone titers were higher on the gland. The presence of the hydrocarbon pheromone precursor in the hemolymph is discussed in relation to possible biosynthetic pathways for producing the gypsy moth pheromone.     

Vertical transmission and overwintering of microsporidia in the gypsy moth, Lymantria dispar

Vertical transmission and the overwintering success of three different microsporidia infecting Lymantria dispar (Lepidoptera: Lymantriidae) larvae were investigated. Endoreticulatus schubergi, a midgut pathogen, was transmitted to offspring via female and male via the egg chorion (transovum transmission). Between 8% and 29% of the emerging larvae became infected. No spores of E. schubergi were found in surface-washed eggs. Nosema lymantriae, a microsporidium that causes systemic infections, was transovarially transmitted. Between 35% and 72% of the progeny were infected. Vairimorpha disparis, a fat body pathogen, was not vertically transmitted. The infectivity of spores that overwintered in cadavers of infected L. dispar varied by species, placement in the environment, and weather conditions. Spores of E. schubergi were still infective after an eight month exposure period of cadavers on the ground. Spores of N. lymantriae and V. disparis remained highly infective only when cadavers overwintered under a more or less continuous snow cover for four months.     

Ultrastructural studies of male-incubated ovaries of the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking the vitellogenin ligand; i.e., the male milieu. Transmission electron micrographs comparing the terminal oocytes of male-grown ovaries and normal ovaries showed that yolk sphere diameters were reduced in the male-grown oocytes. However, there were larger numbers of these small yolk spheres per unit area of cytoplasm, indicating that the coalescence of endosomes into yolk spheres is reduced in the absence of vitellogenin. Although there are larger numbers of yolk spheres in male-grown oocytes, the smaller diameter of yolk spheres resulted in less area being taken up by yolk spheres per unit area of cytoplasm in male-grown oocytes, yielding lowered yolk production. This lowered yolk production is a result at least in part of the lowered number of coated vesicles per unit area of submembrane space and in part of the reduced interfollicular spaces seen in male-grown ovaries.     

Pheromone‐binding proteins in the Asian gypsy moth females,Lymantria dispar,recognizing the sex pheromone and plant volatiles

Lepidopterans are known to have different pheromone‐binding proteins with differential expression patterns that facilitate specific signal transduction of semiochemicals. Two PBPs of the Asian gypsy moth, Lymantria dispar, were reported to express in both females and males, but their physiological functions were unknown. Results showed that LdisPBP1 and LdisPBP2 were expressed in the sensilla trichodea of males and the s. trichodea and s. basiconica of females. When LdisPBP1 gene was targeted by RNA interference (RNAi) in males, the expression of LdisPBP1 and LdisPBP2 decreased by 69 and 76%, respectively, and when LdisPBP2 gene was targeted by RNAi, they decreased by 60 and 42%, respectively. In females, after treatment with LdisPBP1 dsRNA, LdisPBP1 and LdisPBP2 levels were reduced by 26 and 69%, respectively, and LdisPBP2 dsRNA reduced the relative expression of them by 4 and 62%, respectively. The expression of LdisPBP1 and LdisPBP2 was interdependent. Electroantennogram (EAG) recordings showed that LdisPBPs participate in the recognition of the sex pheromone in males, and the sex pheromone and plant volatiles in females. The function of LdisPBPs represents the sex‐specific roles.     

Structure-activity studies with pheromone-binding proteins of the gypsy moth,Lymantria dispar

Pheromone olfaction in the gypsy moth, Lymantria dispar, involves accurate distinction of compounds with similar structure and polarity. The identified sex pheromone is (7R,8S)-2-methyl-7,8-epoxyoctadecane, 1a, and a known antagonist is (7Z)-2-methyloctadec-7-ene, 4a. The first step in pheromone olfaction is binding of odorants by small, soluble pheromone-binding proteins (PBPs), found in the pheromone-sensing hairs. We have studied the molecular determinants recognized by the two PBPs found in the gypsy moth, using three pheromone/PBP binding assays. Results indicate that (i) PBPs bind analogs of the pheromone with some discrimination; (ii) PBPs experience enhancement of binding when presented with 1a or its enantiomer and 4a simultaneously; and (iii) the binding enhancement is also seen at high ligand:PBP ratios. We found no evidence of allostery, so the synergistic binding effects and the concentration effect may only be explained by multimerization of PBPs with each other, which leads to more than one population of binding sites. We suggest that the enhanced ligand binding at high ligand:PBP ratios may serve to sequester excess ligand and thereby attenuate very strong signals.     

Olfaction in the gypsy moth, Lymantria dispar: effect of pH, ionic strength, and reductants on pheromone transport by pheromone-binding proteins

The pheromone-binding proteins (PBPs) are 16-kDa abundant proteins in specialized olfactory hairs in insects. The mechanism by which the PBPs remove the pheromone from the inner surface of sensory hairs and deliver it to the sensory cell remains unclear. Existing qualitative models postulate that pheromone is released near the dendrite by a decrease in pH or by a reduced form of the PBP. This study focuses on the two PBPs from the gypsy moth and the enantiomers of the pheromone cis-2-methyl-7,8-epoxyoctadecane. The pH dependence of pheromone binding has revealed three ionizations that are important. The type of ligand influences two of these ionizations. We propose that the (-)-enantiomer of the pheromone interacts with one of the ionizable residues on the protein while the (+)-enantiomer does not. Simultaneous variation of pH and KCl concentration in the physiological range or reduction of disulfide bridges does not change the affinity of PBP for pheromone. We propose a revised model of pheromone transport from the inner surface of the sensory hair to the sensory neuron.     

Influence of pheromone concentration and ambient temperature on flight of the gypsy moth, Lymantria dispar (L), in a sustained-flight wind tunnel

Abstract. The effects of pheromone concentration and ambient temperature on male gypsy moth, Lymantria dispar (L.) (Lepidoptera), flight responses to pheromone were investigated in a wind tunnel. As the pheromone dose increased from 10 ng to 1000 ng, males flew at progressively slower airspeeds and ground speeds, and reduced their wingbeat frequencies. Furthermore, the moths steered significantly smaller course angles as the pheromone concentration increased, indicating that they were adopting a more upwind heading. The overall width of the flight tracks also decreased when males flew in more concentrated pheromone plumes. Estimation of plume dimensions using a male wing-fanning assay showed that as pheromone dosage increased, the resultant active spaces became wider, indicating that an inverse relationship existed between the dimensions of the time-averaged plume and the width of track reversals and that most turns were initiated within the plume. When males were flown at cool (20°C) and warm (26°C) ambient temperatures but to equivalent pheromone emission rates, they exhibited higher airspeeds and ground speeds at the higher temperature but steered larger course angles. Track widths, and length of track legs were, however, similar at the two temperatures. The mean turning frequency was nearly the same (c. 4 turns/s) across all the concentrations and temperatures tested even though the moths' thoracic temperature differed by 5°C when the ambient temperature was varied.