Essential host plant cues in the grapevine moth

Host plant odours attract gravid insect females for oviposition. The identification of these plant volatile compounds is essential for our understanding of plant–insect relationships and contributes to plant breeding for improved resistance against insects. Chemical analysis of grape headspace and subsequent behavioural studies in the wind tunnel show that host finding in grapevine moth Lobesia botrana is encoded by a ratio-specific blend of three ubiquitous plant volatiles. The odour signal that attracts mated females to grape consists of the terpenoids (E)-β-caryophyllene, (E)-β-farnesene and (E)-4,8-dimethyl-1,3,7-nonatriene. These compounds represent only a fraction of the volatiles released by grapes, and they are widespread compounds known throughout the plant kingdom. Specificity may be achieved by the blend ratio, which was 100:78:9 in grape headspace. This blend elicited anemotactic behaviour in moths at remarkably small amounts. Females were attracted at release rates of only a few nanograms per minute, at levels nearly as low as those known for the attraction of male moths to the female sex pheromones.     

Attractants from Bartlett pear for codling moth, Cydia pomonella (L.), larvae

The alkyl ethyl and methyl esters of (2E,4Z)-2,4-decadienoic acid found in head-space samples of ripe Bartlett pear (Pyrus communis L.) stimulated a response from neonate larvae of the codling moth (CM), Cydia pomonella (L.), in both static-air Petri-plate and in upwind Y-tube and straight-tube olfactometer bioassays. In comparison with the known CM neonate attractant, (E,E)-!-farnesene, ethyl (2E,4Z)-2,4-decadienoate was attractive at 10-fold and 1,000-fold lower threshold dosages in the Petri-plate and in the Y-tube bioassays, respectively. Methyl (2E,4Z)-2,4-decadienoate was attractive to CM neonates in these bioassays at much higher doses than ethyl (2E,4Z)-2,4-decadienoate. Other principal head-space volatiles from ripe pear fruit and pear leaves, including butyl acetate, hexyl acetate, (Z)-3-hexenyl acetate, and (E)-#-ocimene, were not attractive to CM neonates. The potential uses of these pear kairomones for monitoring and control of CM in walnuts and apple are discussed.     

Insecticide resistance may enhance the response to a host-plant volatile kairomone for the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis> (L.)

The behavioral and electroantennographic responses of Cydia pomonella (L.) to the ripe pear volatile ethyl (2E,4Z)-2,4-decadienoate (Et-E,Z-DD), were compared in insecticide-susceptible and -resistant populations originating from southern France. A dose–response relationship to this kairomonal attractant was established for antennal activity and did not reveal differences between susceptible and resistant strains. Conversely, males of the laboratory strains expressing metabolic [cytochrome P450-dependent mixed-function oxidases (mfo)] or physiological (kdr-type mutation of the sodium-channel gene) resistance mechanisms exhibited a significantly higher response to Et-E,Z-DD than those of the susceptible strain in a wind tunnel experiment. No response of the females to this kairomone could be obtained in our wind-tunnel conditions. In apple orchards, mfo-resistant male moths were captured at significantly higher rates in kairomone-baited traps than in traps baited with the sex pheromone of C. pomonella. Such a differential phenomenon was not verified for the kdr-resistant insects, which exhibited a similar response to both the sex pheromone and the kairomonal attractant in apple orchards. Considering the widespread distribution of metabolic resistance in European populations of C. pomonella and the enhanced behavioral response to Et-E,Z-DD in resistant moths, the development of control measures based on this kairomonal compound would be of great interest for the management of insecticide resistance in this species.     

A pear-derived kairomone with pheromonal potency that attracts male and female codling moth, Cydia pomonella (L.)

Ethyl (2E, 4Z)-2,4-decadienoate, a pear-derived volatile, is a species-specific, durable, and highly potent attractant to the codling moth (CM), Cydia pomonella (L.), a serious pest of walnuts, apples, and pears worldwide. This kairomone attracts both CM males and virgin and mated females. It is highly attractive to CM in both walnut and apple orchard contexts, but has shown limited effectiveness in a pear orchard context. Rubber septa lures loaded with ethyl (2E, 4Z)-2,4-decadienoate remained attractive for several months under field conditions. At the same low microgram load rates on septa, the combined gender capture of CM in kairomone-baited traps was similar to the capture rate of males in traps baited with codlemone, the major sex pheromone component. The particular attribute of attracting CM females renders this kairomone a novel tool for monitoring population flight and mating-ovipositional status, and potentially a major new weapon for directly controlling CM populations.     

The effect of larval and early adult experience on behavioural plasticity of the aphid parasitoid <Emphasis Type="Italic">Aphidius ervi</Emphasis> (Hymenoptera,Braconidae, Aphidiinae)

The relevance of the integration of preimaginal and eclosion experiences on the subsequent habitat preferences and mate finding by the adult has been rarely tested in holometabolous insects. In this work, the effect of larval and early adult experiences on the behavioural responses of adult males of the aphid parasitoid, Aphidius ervi, towards volatiles from the host–plant complex (HPC) and from conspecific females were evaluated. Two experience factors were considered: host diet (normal diet=ND; artificial diet=AD), and eclosion, i.e. extraction or non-extraction of the parasitoid larva from the parasitised aphid (extracted=EX; non-extracted=NE). Thus, four treatments were set up: ND/NE, ND/EX, AD/NE and AD/EX. Glass Y-tube olfactometers were used to investigate the responses of adult A. ervi males to the odour sources used. Males from the ND/NE treatment showed a shorter latency to the first choice of olfactometer arms, displayed a marked preference towards the HPC olfactometer arm, and spent more time in the HPC arm than males from the other treatments. Only the interaction of host diet and eclosion experiences proved to be relevant in explaining the differences in latency to first choice, time spent in olfactometers arms, and behaviours displayed in the olfactometer arms. These results show the importance of the integration of larval and eclosion experiences in the development of stereotyped responses of the adults. This process may involve memory retention from the preimaginal and emergence period, but further research is needed to disentangle the contribution of each stage. The response to conspecific females was much less affected by the treatments in relation to first arm choice and times in olfactometer arms, suggesting a pheromone-mediated behaviour, even though a prompter and more intense wing fanning courtship behaviour was registered in the ND/NE males compared to males from the AD/NE treatment. These results show that sexual behaviours are less affected by early experiences than behaviours related with finding a HPC for foraging or oviposition. Taken together, our results demonstrate for first time that larval and eclosion experiences affect in a differential way the early responses of the adult towards environment-derived cues and mate related cues.     

Nocturnal herbivore-induced plant volatiles attract the generalist predatory earwig <Emphasis Type="Italic">Doru luteipes</Emphasis> Scudder

Numerous studies have demonstrated that entomophagous arthropods use herbivore-induced plant volatile (HIPV) blends to search for their prey or host. However, no study has yet focused on the response of nocturnal predators to volatile blends emitted by prey damaged plants. We investigated the olfactory behavioral responses of the night-active generalist predatory earwig Doru luteipes Scudder (Dermaptera: Forficulidae) to diurnal and nocturnal volatile blends emitted by maize plants (Zea mays) attacked by either a stem borer (Diatraea saccharalis) or a leaf-chewing caterpillar (Spodoptera frugiperda), both suitable lepidopteran prey. Additionally, we examined whether the earwig preferred odors emitted from short- or long-term damaged maize. We first determined the earwig diel foraging rhythm and confirmed that D. luteipes is a nocturnal predator. Olfactometer assays showed that during the day, although the earwigs were walking actively, they did not discriminate the volatiles of undamaged maize plants from those of herbivore damaged maize plants. In contrast, at night, earwigs preferred volatiles emitted by maize plants attacked by D. saccharalis or S. frugiperda over undamaged plants and short- over long-term damaged maize. Our GC-MS analysis revealed that short-term damaged nocturnal plant volatile blends were comprised mainly of fatty acid derivatives (i.e., green leaf volatiles), while the long-term damaged plant volatile blend contained mostly terpenoids. We also observed distinct volatile blend composition emitted by maize damaged by the different caterpillars. Our results showed that D. luteipes innately uses nocturnal herbivore-induced plant volatiles to search for prey. Moreover, the attraction of the earwig to short-term damaged plants is likely mediated by fatty acid derivatives.     

Characteristics of volatile compounds removal in biogas slurry of pig manure by ozone oxidation and organic solvents extraction

Biogas slurry is not suitable for liquid fertilizer due to its high amounts of volatile materials being of complicated composition and peculiar smell. In order to remove volatiles from biogas slurry efficiently, the dynamic headspace and gas chromatography-mass spectrometry were used to clear the composition of volatiles. Nitrogen stripping and superfluous ozone were also used to remove volatiles from biogas slurry. The results showed that there were 21 kinds of volatile compounds in the biogas slurry, including sulfur compounds, organic amines, benzene, halogen generation of hydrocarbons and alkanes, some of which had strong peculiar smell. The volatile compounds in biogas slurry can be removed with the rate of 53.0% by nitrogen stripping and with rate of 81.7% by the oxidization and stripping of the superfluous ozone. On this basis, the removal rate of the volatile compounds reached 99.2% by chloroform and n-hexane extraction, and almost all of odor was eliminated. The contents of some dissolved organic compounds decreased obviously and however main plant nutrients had no significant change in the biogas slurry after being treated.     

Male scent-marking pheromone of <Emphasis Type="Italic">Bombus ardens ardens</Emphasis> (Hymenoptera; Apidae) attracts both conspecific queens and males

To explore the role of the volatiles emitted from male labial gland (LG) of the bumblebee Bombus ardens ardens, we investigated the responses of virgin queens and males to volatiles using a gas chromatography–electroantennographic detector (GC–EAD) system and Y-tube olfactometer. GC–EAD analysis revealed that citronellol, the main compound detected in the male LG, caused clear electrophysiological responses in the antennae of B. a. ardens virgin queens and males although two minor compounds elicited antennal responses when applied in a high concentration. Behavioral tests using a Y-tube olfactometer showed that queens and males were significantly attracted to both LG extracts and citronellol more than to the solvent alone. This is the first study to demonstrate that citronellol as a major compound of male scent-marking pheromone in B. a. ardens functions as a sex attractant for queens. The results also suggest that this compound has another function as a trail marker used by males.     

Evidence for a male-produced pheromone in <Emphasis Type="Italic">Tetropium fuscum</Emphasis> (F.) and <Emphasis Type="Italic">Tetropium cinnamopterum</Emphasis> (Kirby) (Coleoptera: Cerambycidae)

(E)-6,10-dimethyl-5,9-undecadien-2-ol (geranyl acetol), termed here fuscol, was identified as a male-produced pheromone emitted by Tetropium fuscum (F.) and Tetropium cinnamopterum Kirby. In field experiments, traps baited with synthetic fuscol alone were not significantly attractive, but the combination of fuscol plus host volatiles (a synthetic blend of monoterpenes plus ethanol) attracted significantly more male and female T. fuscum and female T. cinnamopterum than did host volatiles alone. This is the first homoterpenoid alcohol to be described in the Cerambycidae, and the first pheromone reported from the sub-family Spondylidinae.     

Anatomical localization and stereoisomeric composition of <Emphasis Type="Italic">Tribolium castaneum</Emphasis> aggregation pheromones

We report that the abdominal epidermis and associated tissues are the predominant sources of male-produced pheromones in the red flour beetle, Tribolium castaneum and, for the first time, describe the stereoisomeric composition of the natural blend of isomers of the aggregation pheromone 4,8-dimethyldecanal (DMD) in this important pest species. Quantitative analyses via gas chromatography–mass spectrometry showed that the average amount of DMD released daily by single feeding males of T. castaneum was 878 ± 72 ng (SE). Analysis of different body parts identified the abdominal epidermis as the major source of aggregation pheromone; the thorax was a minor source, while no DMD was detectable in the head. No internal organs or obvious male-specific glands were associated with pheromone deposition. Complete separation of all four stereoisomers of DMD was achieved following oxidation to the corresponding acid, derivatization with (1R, 2R)- and (1S, 2S)-2-(anthracene-2,3-dicarboximido)cyclohexanol to diastereomeric esters, and their separation on reversed-phase high-performance liquid chromatography at −54°C. Analysis of the hexane eluate from Porapak-Q-collected volatiles from feeding males revealed the presence of all four isomers (4R,8R)/(4R,8S)/(4S,8R)/(4S,8S) at a ratio of approximately 4:4:1:1. A walking orientation bioassay in a wind tunnel with various blends of the four synthetic isomers further indicated that the attractive potency of the reconstituted natural blend of 4:4:1:1 was equivalent to that of the natural pheromone and greater than that of the 1:1 blend of (4R,8R)/(4R,8S) used in commercial lures.     

Alcoholism in cockchafers: orientation of male Melolontha melolontha towards green leaf alcohols

Chemical orientation of the European cockchafer, Melolontha melolontha L., a serious pest in agriculture and horticulture, was investigated by field tests and electrophysiological experiments using plant volatiles. In total, 16 typical plant volatiles were shown to elicit electrophysiological responses in male cockchafers. Funnel trap field bioassays revealed that green leaf alcohols (i.e. (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol and 1-hexanol) attracted males, whereas the corresponding aldehydes and acetates were behaviourally inactive. Furthermore, male cockchafers were attracted by volatiles from mechanically damaged leaves of Fagus sylvatica L., Quercus robur L. and Carpinus betulus L. However, volatiles emitted by damaged leaves of F. sylvatica attracted significantly more males than those from the other host plants. Odour from intact F. sylvatica leaves was not attractive to M. melolontha males. Females were not attracted by any of the tested volatile sources. The results suggest that plant volatiles play a similar role as a sexual kairomone in mate finding of M. melolontha, as has been shown for the forest cockchafer, Melolontha hippocastani F. Nevertheless, both species show remarkable differences in their reaction to green leaf alcohols.     

Olfactive detection of fig wasps as prey by the ant<Emphasis Type="Italic"> Crematogaster scutellaris</Emphasis> (Formicidae; Myrmicinae)

In the species-specific and obligate mutualism between the fig (Ficus carica) and its pollinator (the fig wasps Blastophaga psenes), a third participant, the ant Crematogaster scutellaris, is a predator of the wasps. Here, we ask how ant workers can rapidly localise such prey, whose availability is limited in time and space. Using a Y-tube olfactometer, we tested ant response to odours emitted by different types of figs (receptive female, ripe female or male figs) and by fig wasps (pollinators or non-pollinators). We demonstrate that ants were significantly attracted only to odours emitted by pollinators, either alone or associated with odours of male figs (releasing wasps). Detection of prey odour by ants is an important trait that can explain their observed high rate of predation on pollinators, and could have important implications on the stability of the fig/fig wasp mutualism.     

Pre-pupation behaviour of the aphid parasitoid <Emphasis Type="Italic">Aphidius ervi</Emphasis> (Haliday) and its consequences for pre-imaginal learning

Olfactory learning may occur at different stages of insect ontogeny. In parasitoid wasps, it has been mostly shown at adult emergence, whilst it remains controversial at pre-imaginal stages. We followed larval growth of the parasitoid wasp, Aphidius ervi Haliday, inside the host aphid, Acyrthosiphom pisum Harris, and characterised in detail the behaviour of third instar larvae. We found that just before cocoon spinning begins, the third instar larva bites a hole through the ventral side of the mummified aphid exoskeleton. We then evaluated whether this period of exposure to the external environment represented a sensitive stage for olfactory learning. In our first experiment, the third instar larvae were allowed to spin their cocoon on the host plant (Vicia faba L.) surface or on a plastic plate covering the portion of the host plant exposed to the ventral opening. Recently emerged adults of the first group showed a preference for plant volatiles in a glass Y-olfactometer, whereas no preference was found in adults of the second group. In a second experiment, during the period in which the aphid carcass remains open or is being sealed by cocoon spinning, third instar larvae were exposed for 24 h to either vanilla odours or water vapours as control. In this experiment, half of the parasitoid larvae were later excised from the mummy to avoid further exposure to vanilla. Adult parasitoids exposed to vanilla during the larval ventral opening of the mummy showed a significant preference for vanilla odours in the olfactometer, regardless of excision from the mummy. The larval behaviour described and the results of the manipulations performed are discussed as evidences for the acquisition of olfactory memory during the larval stage and its persistence through metamorphosis.     

Irresistible bouquet of death—how are burying beetles (Coleoptera: Silphidae: Nicrophorus) attracted by carcasses

Chemical composition of volatiles emitted from fresh mouse carcasses (laboratory mice, Mus musculus) was studied using solid sample injection technique (solid-phase micro-extraction), two-dimensional gas chromatography with time of flight mass spectrometric detection and gas chromatography with electroantennographic detection. Electroantennography (EAG) and laboratory olfactometric behavioural observations were used to study the antennal sensitivity to identified infochemicals and their attractiveness for burying beetles Nicrophorus vespillo and Nicrophorus vespilloides (Silphidae: Nicrophorinae). Chemical analysis showed that immediately after death, emitted volatiles did not differ from those emitted by a living organism. However, in the course of time, sulphur-containing chemicals, specifically methanethiol, methyl thiolacetate, dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide appear. EAG measurements revealed antennal sensitivity to these compounds. Behavioural tests in laboratory olfactometer showed that dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide are highly attractive to both studied species. The data suggest that sulphur-containing chemicals are involved in mediating the fresh carcass attractiveness for N. vespillo and N. vespilloides.     

Macroglomeruli for fruit odors change blend preference in Drosophila

The olfactory circuitry of Drosophila melanogaster is becoming increasingly clear. However, how olfactory processing translates into appropriate behavioral responses is still poorly understood. Using a sibling species approach, we tested how a perturbation in the olfactory circuitry affects odor preference. In a previous study, we found that the sibling species of D. melanogaster, the specialist D. sechellia, overrepresents a sensillum, ab3, the A neuron of which is sensitive to hexanoate esters, characteristic of the species’ sole host, the Morinda citrifolia fruit. Concordantly, the corresponding glomerulus, DM2, is enlarged. In this study, we found that the ab3B neuron, the expansion of which was previously assumed to be pleiotropic and of no ecological significance, is in fact tuned to another morinda fruit volatile, 2-heptanone (HP). Axons of this neuron type arborize in a second enlarged glomerulus. In behavioral experiments we tested how this has affected the fly’s odor preference. We demonstrate that D. sechellia has a reversed preference for the key ligands of these macroglomeruli, especially at high concentrations. Whereas D. melanogaster was repelled by high concentrations of these odors, D. sechellia was highly attracted. This was the case for odors presented singly, but more notably for blends thereof. Our study indicates that relatively simple changes, such as a shift in sensillar abundance, and concordant shifts in glomerular size, can distort the resulting olfactory code, and can lead to saltatory shifts in odor preference. D. sechellia has exploited this to align its olfactory preference with its ecological niche.     

Mastrus ridibundus parasitoids eavesdrop on cocoon-spinning codling moth, Cydia pomonella, larvae

Cocoon-spinning larvae of the codling moth, Cydia pomonella L. (Lepidoptera: Olethreutidae) employ a pheromone that attracts or arrests conspecifics seeking pupation sites. Such intraspecific communication signals are important cues for illicit receivers such as parasitoids to exploit. We tested the hypothesis that the prepupal C. pomonella parasitoid Mastrus ridibundus Gravenhorst (Hymenoptera: Ichneumonidae) exploits the larval aggregation pheromone to locate host prepupae. In laboratory olfactometer experiments, female M. ridibundus were attracted to 3-day-old cocoons containing C. pomonella larvae or prepupae. Older cocoons containing C. pomonella pupae, or larvae and prepupae excised from cocoons, were not attractive. In gas chromatographic-electroantennographic detection (GC-EAD) analyses of bioactive Porapak Q extract of cocoon-derived airborne semiochemicals, ten compounds elicited responses from female M. ridibundus antennae. Comparative GC-mass spectrometry of authentic standards and cocoon-volatiles determined that these compounds were 3-carene, myrcene, heptanal, octanal, nonanal, decanal, (E)-2-octenal, (E)-2-nonenal, sulcatone, and geranylacetone. A synthetic 11-component blend consisting of these ten EAD-active compounds plus EAD-inactive (+)-limonene (the most abundant cocoon-derived volatile) was as effective as Porapak Q cocoon extract in attracting both female M. ridibundus and C. pomonella larvae seeking pupation sites. Only three components could be deleted from the 11-component blend without diminishing its attractiveness to M. ridibundus, which underlines the complexity of information received and processed during foraging for hosts. Mastrus ridibundus obviously eavesdrop on the pheromonal communication signals of C. pomonella larvae that reliably indicate host presence.

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A contact sex pheromone component of the emerald ash borer Agrilus planipennis Fairmaire (Coleoptera: Buprestidae)

Analyses of the elytral hydrocarbons from male and female emerald ash borer, Agrilus planipennis Fairmaire, that were freshly emerged vs. sexually mature (>10 days old) revealed a female-specific compound, 9-methyl-pentacosane (9-Me-C₂₅), only present in sexually mature females. This material was synthesized by the Wittig reaction of 2-decanone with (n-hexadecyl)-triphenylphosphonium bromide followed by catalytic reduction to yield racemic 9-Me C₂₅, which matched the natural compound by gas chromatography/mass spectrometry (retention time and EI mass spectrum). In field bioassays with freeze-killed sexually mature A. planipennis females, feral males spent significantly more time in contact and attempting copulation with unwashed females than with females that had been washed in n-hexane to remove the cuticular lipids. Hexane-washed females to which 9-Me-C₂₅ had been reapplied elicited similar contact time and percentage of time attempting copulation as unwashed females, indicating that 9-methyl-pentacosane is a contact sex pheromone component of A. planipennis. This is the first contact sex pheromone identified in the Buprestidae.     

Identification of cuticular lipids eliciting interspecific courtship in the German cockroach, Blattella germanica

The cuticular surface of sexually mature females of the German cockroach contains a sex pheromone that, upon contact with the male's antennae, elicits a characteristic species-specific courtship behavior. This female-specific pheromone is a blend of several long-chain methyl ketones, alcohols and aldehydes, all derived from prominent cuticular hydrocarbons found in all life stages of this cockroach. We found that contact with the antennae of 5 out of 20 assayed cockroach species elicited courtship behavior in German cockroach males. The heterospecific courtship-eliciting compounds were isolated by behaviorally guided fractionation of the active crude extracts and compared to the native sex pheromone components. We identified two active compounds from the cuticular extract of the Oriental cockroach, Blatta orientalis -- 11-methylheptacosan-2-one and 27-oxo-11-methylheptacosan-2-one; the former compound was confirmed by synthesis and proved to independently stimulate courtship in German cockroach males. These compounds share common features with, but are distinct from, any of the known contact sex pheromone components. This suggests that sex pheromone reception in the male German cockroach is unusually promiscuous, accepting a wide range of compounds that share certain features with its native pheromone, thus resulting in a broad spectrum of behavioral response to other species. We propose that several characteristics of their mating system -- chiefly, absence of closely related species in the anthropogenic environment, resulting in relaxation of selection on sexual communication, and a highly male-biased operational sex ratio -- have driven males to respond with extremely low thresholds to a wide spectrum of related compounds.     

Olfactory host finding, intermediate memory and its potential ecological adaptation in Nasonia vitripennis

Associative learning of host-associated chemical cues was studied in Nasonia vitripennis, a parasitoid of fly pupae in nests of hole-nesting birds. When females encountered a fly pupa and performed one sequence of host recognition behaviour including drilling the ovipositor into the host in the presence of the artificial odour furfurylheptanoate (FFH), they were afterwards arrested by FFH in olfactometer experiments. The response vanished after 4 days and could be blocked after 3 days by feeding wasps with ethacrynic acid prior and after the training. This indicates the formation of an intermediate form of memory by one host experience in N. vitripennis. Interestingly, the trained wasps avoided odours that were not present during the host encounter, although naive wasps did not react to these odours. This unique behaviour probably causes wasps to focus during host searching on those chemical cues they have experienced in the host environment. Studies in nests of hole-nesting birds revealed that about 30% of all nests contained only one fly pupa, and laboratory studies showed that N. vitripennis females are able to parasitise around 100 fly pupae in their life. It is discussed that under these conditions, the formation of a non-permanent intermediate memory for host-associated odours after one host encounter is adaptive to avoid costs involved with formation and maintenance of memory for misleading cues. The demonstration of associative olfactory learning in N. vitripennis, the first parasitoid species with sequenced genome, opens the gate to study molecular mechanisms of memory formation and its ecological adaptation in parasitoids.     

Sexual behavior,cannibalism, and mating plugs as sticky traps in the orb weaver spider <Emphasis Type="BoldItalic">Leucauge argyra</Emphasis> (Tetragnathidae)

Unpublished field observations in Leucauge argyra, a tropical orb weaver spider, suggest the occurrence of conspicuous mating plugs that could reduce or prevent remating attempts. Otherwise, the sexual behavior of this species remains unknown. The aims of this study were to describe the courtship behavior and copulation in L. argyra and investigate mating plug formation in this species. Fourteen virgin females and 12 plugged females were exposed to up to three males and checked for mating plug formation. Of the 12 virgins that copulated, nine produced plugs (five immediately after copulation), and the five plugged females that copulated produced another mating plug immediately after copulation. We did not detect the transfer of any male substance during copulation but observed a whitish liquid emerging from female genital ducts. Plug formation was positively associated with male twanging during courtship. One virgin and four plugged females cannibalized males. In seven trials with virgins and in three trials with plugged females, the male’s palp adhered to a substance that emerged from female genital ducts and spread on her genital plate. The male had to struggle energetically to free his glued palp; two of these males were cannibalized while trying to release their palps. Females seem to determine copulation duration by altering the timing of mating plug formation and through sexual cannibalism. This is the first case reported of a mating plug as a sticky trap for males.