Stabilization of altitude and speed in tethered flying gypsy moth males: influence of (+) and (-)-disparlure

ABSTRACT. Changes in lift and thrust were elicited in tethered male gypsy moths, Lymantria dispar L. (Lepidoptera, Lymantriidae), by visual pattern elements moving radially either towards or from the point directly beneath their body, if the sex-pheromone, (+)-disparlure, was present. The sign of these changes was such as to counteract the pattern movements, which were generated by a rotating spiral beneath the moth. By restricting the area of spiral visible to the moth to either transverse or longitudinal sectors, flight altitude was affected by the centrifugal/centripetal movements in the lateral sectors, whereas flight speed was affected by those in the frontal sector. It is deduced that in free flight these compensatory reactions are responsible for the stabilization of flight altitude and speed, respectively. Surprisingly, without pheromone present these responses were usually not detectable: a wide range of flight altitude and speed was then observed. In the presence of (+)-disparlure, however, these responses were always strongly pronounced, the animal keeping within a narrow range of speed and altitude. These compensatory reactions were blocked by the attraction-inhibiting (-)-disparlure if presented in racemic mixture with the (+) form: the range of speed and altitude shown by the moth was then the same as without any pheromone. Under closed-loop conditions, the mean flight speed was reduced by the racemic mixture as well as by (+)-disparlure alone, however.     

Oxaspiropentane derivatives as effective sex pheromone analogues in the gypsy moth: electrophysiological and behavioral evidence

A number of oxaspiropentane derivatives (OXPs) were tested as potential (+)-disparlure analogues, with the aim of identifying any possible interaction of these compounds, be it additive, synergetic, or inhibitory, with the pheromone response in the male gypsy moth Lymantria dispar. As assessed by male electroantennograms, 2 OXPs, 2-decyl-1-oxaspiro[2.2]pentane (OXP-01) and 4-(1-oxaspiro[2.2]pent-2-yl)butan-1-ol (OXP-04), were found to be effective. OXP-01 had no stimulatory effect but strongly decreased the response to (+)-disparlure in a blend in a 1:1 ratio. By contrast, OXP-04 proved to be more stimulating than (+)-disparlure and also had an additive effect in the blend. Single-cell recordings from the sensilla trichoidea showed the activity of 2 cells, one of which responded to (+)-disparlure. OXP-01 reduced the stimulating effectiveness of pheromone by silencing the pheromone-responding unit when the 2 compounds were presented in blend, whereas OXP-04 mimicked the pheromone response, evidenced by exciting the pheromone-responding neuron when tested alone. Behavioral observations are in agreement with electrophysiological results.     

Electroantennogram and behavioural responses of the gypsy moth to enantiomers of disparlure and its trans analogues

The attractive power of both enantiomers of disparlure and its trans analog was tested electrophysiologically (electroantennogram = EAG) and behaviourally (whole organism response). EAG responses correlated very well with the behaviourally determined effectiveness sequence: cis(+)-disparlure was the most effective substance; the authentic racemic disparlure came second; cis(?)-disparlure inhibited the activity of cis(+)-disparlure; and trans enantiomers were not significantly different from the control. These results allow us to conclude that cis(+)-disparlure is the natural sex pheromone of the gypsy moth, and that the male antennae have a chiral receptor system in the antenna for the reception of the sex pheromone.     

Effects of aromatic compounds on antennal responses and on the pheromone-binding proteins of the gypsy moth (Lymantria dispar)

Female gypsy moths emit a pheromone, (+)-disparlure, which the males follow until they locate the emitter. The male moths' antennae are covered with innervated sensory hairs, specialized in detection of the pheromone. The neurons in these sensory hairs are bathed by a solution rich in pheromone-binding protein (PBP). PBPs are soluble proteins that bind the pheromone and other odorants reversibly with variable thermodynamic and kinetic selectivity and are essential for olfactory responses. Here, we have studied the interaction between 2 gypsy moth PBPs with aromatic compounds that modulate the responses of male moth antennae to (+)-disparlure. The aromatic compounds do not elicit responses by themselves, but when administered together with pheromone, they inhibit, enhance, or prolong the electrophysiological response to the pheromone. Three interactions between the compounds and PBPs were studied: 1) the equilibrium binding of the compounds by themselves to the PBPs, 2) the equilibrium binding of the compounds in the presence of pheromone or a fluorescent reporter ligand, and 3) the effect of the compounds on the conformation of the pheromone-PBP complex. A subset of compounds causes a prolongation of the electroantennogram response, and from this study, we conclude that these compounds follow a structure-activity pattern and stabilize a particular conformer of the PBPs that appears to activate the olfactory response.     

Enantiomer and conformer recognition of (+) and (−)-disparlure and their analogs by the pheromone binding proteins of the gypsy moth,Lymantria dispar

Adult female gypsy moths produce a sex pheromone (+)-(7R,8S)-2-methyl-7,8-epoxyoctadecane, (+)-disparlure, to attract male gypsy moths. To better understand the recognition of (+)-disparlure by the male’s olfactory system, we synthesized racemic and enantiopure oxa and thia analogs of (+)-disparlure (ee > 98%). Ab initio calculations of the conformeric landscapes around the dihedral angles C_{5–6–7–8} and C_{7–8–9–10} of (+)-disparlure and corresponding dihedral angles of analogs revealed that introduction of the heteroatom changes the conformeric landscape around these important epitopes. The energy difference between HOMO and LUMO decreased after oxygen or sulfur was introduced into the backbone. Consistent with this, an enhancement of binding affinity between sulfur analogs and the pheromone-binding proteins (PBPs) was observed in vitro. Docking of the pheromone and analogs onto models of the two known PBPs of the gypsy moth revealed that the internal binding pocket of PBP1 showed higher selectivity than that of PBP2, consistent with in vitro binding assays. Further energy analysis revealed that enantiomers adopted different conformations with different energies when docked in the internal binding pocket of PBPs, resulting in enantiomer discrimination of PBPs towards disparlure and its analogs.     

Pheromone‐trapping the nun moth,Lymantria monacha (Lepidoptera: Lymantriidae) in Inner Mongolia,China

The nun moth, Lymantria monacha L., is one of the most important defoliators of Eurasian coniferous forests. Outbreaks during 2011–2015 in the natural/planted larch, and larch‐birch mixed forests of the Greater Khingan Range in Inner Mongolia, China, caused tremendous timber losses from severe defoliation and tree mortality. A series of trapping experiments were conducted in these outbreak areas to evaluate the efficacy of a synthetic species‐specific pheromone lure based on the female pheromone blend of European nun moth populations. Our results clearly show that the nun moth in Inner Mongolia is highly and specifically attracted to this synthetic pheromone, with few gypsy moths (Lymantria dispar) captured. Flight activity monitoring of L. monacha male moths using pheromone‐baited Unitraps at 2 locations during the summer of 2015 indicated that the flight period started in mid‐July, peaking in early August at both locations. Based on male moth captures, there was a strong diurnal rhythm of flight activity throughout the entire scotophase, peaking between 22:00 and 24:00. Unitraps and wing traps had significantly and surprisingly higher catches than the gypsy moth traps. Unitraps fastened to tree trunks 2 m above ground caught significantly more male moths than those at the ground level or at 5 m height. Male L. monacha moths can be attracted to pheromone‐baited traps in open areas 150–200 m distant from the infested forest edge. Our data should allow improvement on the performance of pheromone‐baited traps for monitoring or mass‐trapping to combat outbreaks of this pest in northeastern China.     

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.     

Odor receptor responses of male gypsy and nun moths (Lepidoptera,Lymantriidae) to disparlure and its analogues

Summary The closely related species of the gypsy and the nun moth (Lymantria (Porthetria) dispar andL. monacha) were investigated with respect to their electrophysiological (electro-antennogram and single cell) responses to the sex pheromone (disparlure:cis-7,8-epoxy-2-methyl-octadecane) and 56 structurally related epoxides and the disparlure-precursor olefin. Within the limitations of reproducibility of the measurements the sequence of the effectiveness of all the tested compounds is nearly identical in both species, disparlure being every time the most effective compound. From this is deduced a high similarity in the receptor systems for female pheromones of the two moth species.     

Instrumental measurement of the gypsy moth pre-flight behavioural response to pheromone

ABSTRACT. Vibrations of the thorax and electrical activity (EMG) of gypsy moth flight muscles were recorded during wing fanning following pheromone stimulation. The percentage of positive responses and durations of bursts of flight muscle activity increased with the logarithm of pheromone dose, whereas latency decreased. The results correlated well with wing fanning responses of freely moving gypsy moths exposed to nearly identical stimulus conditions. Typical dose-response curves in the range of 0.04-400ng disparlure were obtained in both types of experiments. These methods provide an electrical analogue of wing fanning behaviour.     

Anemotactic orientation of gypsy moth males and its modification by the attractant pheromone (+)-disparlure during walking

ABSTRACT. Tracks of dewinged gypsy moth males, Lymantria dispar L. (Lymantriidae), walking upwind in an airstream without pheromone consist of marked alternations between more or less straight upwind segments, partly with an arcadic structure, and twisted segments. This apparently complicated behaviour can, however, simply be explained by a superposition of noise and two turning commands: an upwind turning tendency, derived from the anemoreceptive system, which represents an average of the moth's angular positions over a period of time; and an internal turning tendency which consists of strong but brief bursts. These bursts are produced intermittently; successive bursts do not necessarily alternate polarity. Amputation of one antenna increases the probability of bursts towards the amputated side; therefore a separate burst source is postulated for each antenna. In the presence of the attractant pheromone (+)-disparlure, the anemotactic signal is weighted higher; twisted segments are, therefore, less pronounced.
There is a chemotropotactical component involved in the male's orientation. The tropotactical signal, dependent on the difference of odour concentration perceived by the left and right antenna, competes with the upwind turning tendency.     

Synthesis and biological activity of conformationally restricted gypsy moth pheromone mimics

The design and synthesis of a series of conformationally constrained mimics of gypsy moth sex pheromone, (+)-disparlure (7R,8S)-2-methyl-7,8-epoxyoctadecane, are described. The core structure of the mimics is derived from 5-(2′-hydroxyethyl)cyclopent-2-en-1-ol. Substituent optimization of the analogs was accomplished through the synthesis of mini-libraries and pure individual compounds, followed by electrophysiological experiments with male gypsy moth antennae. The electroantennogram results show that the analogs elicited weak to no antennal responses themselves. There was a clear structure–activity pattern for odorant activity, with ethyl substituents being best. Further, when puffed simultaneously with the pheromone, some of the compounds gave a significant enhancement of the antennal depolarization, indicating an additive or synergistic effect. A pure pheromone stimulus following a mixed compound/pheromone stimulus was generally not affected, with two exceptions: one compound enhanced and another inhibited a subsequent stimulus. The compounds also prolonged the stimulation of the antenna, which manifested itself in widened electroantennogram peaks. We tested the hypothesis that this prolonged stimulation may be due to the stabilization of a particular conformer of the pheromone-binding protein (PBP). Compounds that caused PBP2 to adopt a similar conformation than in the presence of pheromone also caused peak widening. This was not the case with PBP1.     

Location,morphology and histology of sex pheromone glands of the female gypsy moth, Lymantria dispar (L.)

Scanning electron microscopy, histology and a male wing fanning bioassay were used in this study to locate the sex pheromone-producing glands of the female gypsy moth, Lymantria dispar. When exposed to female sex pheromone, adult males exhibit a strong wing fanning behaviour prior to take off. We found that adult males showed positive response to calling females and to tissue extract from both dorsal and ventral portions of the intersegmental membrane between 8th and 9th-abdominal segments. A typical male response usually starts with elevation of antennae, movement of head in different directions, walking, wing fanning and onset of search flight. Histological and scanning electron microscopic studies suggested that the sex pheromone glands are located on the dorsal and ventral aspects of the intersegmental membrane. The glands appear as two highly convoluted integumentary areas with hypertrophied glandular epidermal cells.     

蛾类昆虫雄性信息素及其功能

昆虫性信息素是两性通讯系统的基础,其中雄性信息素的研究相对较少。本文综述了蛾类昆虫雄性信息素的研究进展。迄今已鉴定出40余种蛾类昆虫的雄性信息素,其行为学功能主要有对雌性的引诱和激欲、对同种雄性的抑制及种间隔离等。     

Monoenyl hydrocarbons in female body wax of the yellow peach moth as synergists of aldehyde pheromone components

The non-polar components of female body wax and pheromone gland extracts of the yellow peach moth synergistically enhanced male behavioral responses from close to pheromone sources in wind tunnel tests when mixed with an aldehyde pheromone blend. When the non-polar fractions (NPFs) of female body wax were further separated by column chromatography, synergistic activities were found in the 3 and 50% ether in hexane fractions, and they additively increased male responses. The main components of the first fraction were (Z)-9-tricosene, (Z)-9-pentacosene, (Z)-9-heptacosene, (Z)-9-nonacosene and (Z)-9-hentriacontene. Only (Z)-9-heptacosene showed a significant synergistic effect in enhancing male responses, but the other components had no effect. A mixture of the five monoenyl hydrocarbons lost activity at lower doses than 5 ng. Natural ratios of these hydrocarbons in the female body wax and pheromone gland extracts were similar, but the amount of (Z)-9-heptacosene in the female body wax was significantly higher than in the pheromone gland extracts. We conclude that (Z)-9-heptacosene increases male responses to aldehyde pheromones, and unknown component(s) in the 50% ether in the hexane fraction are required for full synergistic enhancement by the NPFs of the female body wax and the pheromone gland extracts.     

Forest edges enhance mate‐finding in the invasive European gypsy moth,Lymantria dispar

Habitat type, fragmentation, and edge effects can play important roles in the mate‐finding abilities of many species. These effects can be particularly pronounced in low‐density populations, which are often found at the margins of species' ranges or at the leading edge of an invasion. The European gypsy moth, Lymantria dispar (L.) (Lepidoptera: Erebidae), is a non‐native insect defoliator in the USA and Canada, where flightless females attract male moths through pheromone production and local extirpation of low‐density populations can be due to mate‐finding failure. To assess the effects of habitat edges on the ability of gypsy moths to find mates, we conducted a release experiment with male gypsy moths using female‐baited trap arrays in fields, at forest edges, and in the forest interior. Reduced mate‐finding was expected in fields and near forest edges based on geographic variation in invasion rates, male flight behavior, and pheromone plume dynamics. However, we found that mate‐finding was highest at forest edges, reduced in fields, and lowest within the forest interior. Within an array, traps closest to the forest edge also had the highest mate‐finding, suggesting that habitat characteristics can influence male flight direction in addition to pheromone cues. These results suggest that a moderate level of forest fragmentation enhances mate‐finding ability in the gypsy moth. Understanding the relationship between habitat heterogeneity and mate‐finding success in invasive species can inform predictions of future spread and assist with management plans that target mating disruption.     

Towards pheromone-based monitoring of nun moth, Lymantria monacha (L.) (Lep., Lymantriidae) populations

The research objective was to develop pheromone-based monitoring of the nun moth, Lymantria monacha (L.), an important defoliator of spruce and pine forests in central Europe. In 38 spruce or pine forests in central Europe, captures of male L. monacha in nonsaturating Unitraps and saturating Delta sticky traps baited with 0.2, 2, 20, or 200 μg of the L. monacha (pheromone) volatile blend [(±)-disparlure, (±)-monachalure, and 2-methyl- Z 7-octadecene at a 20 : 20 : 1 ratio] were compared with estimates of population densities obtained by counts of larval faecal pellets, pupal cases, and adult moths resting on tree trunks. Total captures of male L. monacha throughout the flight season in both types of trap were correlated with numbers of larval faecal pellets, irrespective of pheromone dose. Nonsaturating Unitraps baited with 2 μg of the L. monacha volatile blend seem to provide a cost-effective tool for monitoring densities of L. monacha populations. Long-term testing of this monitoring system has been initiated to substantiate the quantitative relationship between larval populations and trap captures of male L. monacha and to determine the threshold number of captured male moths that indicates an incipient outbreak.     

Release mechanism of sex pheromone in the female gypsy moth <Emphasis Type="Italic">Lymantria dispar</Emphasis>: a morpho-functional approach

A morpho-functional investigation of the sex pheromone-producing area was correlated with the pheromone release mechanism in the female gypsy moth Lymantria dispar. As assessed by male electroantennograms (EAG) and morphological observations, the pheromone gland consists of a single-layered epithelium both in the dorsal and ventral halves of the intersegmental membrane between the 8th and 9th abdominal segments. By using the male EAG as a biosensor of real-time release of sex pheromone from whole calling females, we found this process time coupled with extension movements of the ovipositor. Nevertheless, in females in which normal calling behavior was prevented, pheromone release was detected neither in absence nor in presence of electrical stimulation of the ventral nerve cord/terminal abdominal ganglion (TAG) complex. Tetramethylrhodamine-conjugated dextran amine stainings also confirm the lack of any innervation of the gland from nerves IV to VI emerging from the TAG. These findings indicate that the release of sex pheromone from the glands in female gypsy moths is independent of any neural control exerted by the TAG on the glands, at least by way of its three most caudally located pairs of nerves, and appears as a consequence of a squeezing mechanism in the pheromone-producing area.     

Comparative efficacy of two controlled-release gypsy moth mating disruption formulations

The effects of aerial applications of the gypsy moth sex pheromone, disparlure, on mating disruption and suppression of growth of populations of the gypsy moth, Lymantria dispar (L.), were investigated. Two formulations of disparlure, plastic laminate flakes applied in a single application and polymethacrylate beads applied in two applications, were compared in two separate tests conducted in 1993 and 1994. The beads were applied in two applications spaced 2 weeks apart because preliminary tests had indicated that they released pheromone too rapidly to maintain adequate emission rates throughout the period of male flight. In 1993, the flakes were applied at a rate of 50 g a.i./ha, and the beads were applied at a rate of 15 g a.i./ha for each application. In 1994, the flakes were applied at a rate of 75 g a.i./ha and the beads were applied at rates of 32.5 and 42.5 g a.i./ha for the two applications. Beads with larger average particle size were used in 1994 to prolong disparlure release. The treatments applied in 1993 resulted in >97% reduction in mating and >82% suppression of population growth in the following year. Because of a 1995 collapse of gypsy moth populations in the vicinity of the tests, reliable population growth data were not available for the treatments applied in 1994, but significant mating disruption did occur under both treatments. Based on measurements of residual disparlure after field aging, the flakes released 32 and 48% of their disparlure content during the 6 weeks of male moth flight in 1993 and 1994, respectively. The smaller beads used in 1993 released 75% of their disparlure content, and the larger beads used in 1994 released 52% of their disparlure content, during the 6 weeks of male flight. The biological efficacy data suggest that the bead and flake formulations, as applied in these tests, have similar effects on gypsy moth mating disruption and subsequent population growth. Based on the observed release rates from both 1993 and 1994, a single application of the beads would provide emission rates equal to or greater than those provided by the flakes when applied at an equal dose.     

Rapid identification of the Asian gypsy moth and its related species based on mitochondrial DNA

The gypsy moth—Lymantria dispar (Linnaeus)—is a worldwide forest defoliator and is of two types: the European gypsy moth and the Asian gypsy moth. Because of multiple invasions of the Asian gypsy moth, the North American Plant Protection Organization officially approved Regional Standards for Phytosanitary Measures No. 33. Accordingly, special quarantine measures have been implemented for 30 special focused ports in the epidemic areas of the Asian gypsy moth, including China, which has imposed great inconvenience on export trade. The Asian gypsy moth and its related species (i.e., Lymantria monocha and Lymantria xylina) intercepted at ports are usually at different life stages, making their identification difficult. Furthermore, Port quarantine requires speedy clearance. As such, it is difficult to identify the Asian gypsy moth and its related species only by their morphological characteristics in a speedy measure. Therefore, this study aimed to use molecular biology technology to rapidly identify the Asian gypsy moth and its related species based on the consistency of mitochondrial DNA in different life stages. We designed 10 pairs of specific primers from different fragments of the Asian gypsy moth and its related species, and their detection sensitivity met the need for rapid identification. In addition, we determined the optimal polymerase chain reaction amplification temperature of the 10 pairs of specific primers, including three pairs of specific primers for the Asian gypsy moth (L. dispar asiatic), four pairs of specific primers for the nun moth (L. monocha), and three pairs of specific primers for the casuarina moth (L. xylina). In conclusion, using our designed primers, direct rapid identification of the Asian gypsy moth and its related species is possible, and this advancement can help improve export trade in China.     

Stimulation of sex pheromone production by PBAN-like substance in the pine caterpillar moth, Dendrolimus punctatus (Lepidoptera: Lasiocampidae)

Sex pheromone production in the female pine caterpillar moth, Dendrolimus punctatus is controlled by a PBAN-like substance located in the head of female moth. Pheromone titer was significantly decreased by decapitation of female moth, and restored by injection of either Hez-PBAN or head extract prepared from male or female moth. Stimulation of pheromone production by head extract followed a dose-dependent pattern from 0.5 to at least 4 head equivalent. A gland in vitro assay was used to study the relationship between gland incubation time and pheromone production as well as calcium involvement in the stimulation of pheromone production by head extract. Maximum pheromone production was occurred at 60 min after pheromone gland was incubated with two equivalents of head extracts. In vitro experiments showed that the presence of calcium in the incubation medium was necessary for stimulation of pheromone production. The calcium ionophore, A 23187, alone stimulated pheromone production. The pheromone components (Z,E)-5,7-dodecadienol and its acetate and propionate were produced in these experiments but in addition to the aldehyde, (Z,E)-5,7-dodecadienal was also found. This indicates that females are capable of producing four oxygenated functional groups. The PBAN-like substance control of the pheromone biosynthetic pathway was investigated by monitoring the incorporation of the labeled precursor into both pheromone and pheromone intermediates.