The rare and elusive wood-living beetle Tragosoma depsarium, once widespread all over Sweden, is associated with large, sun-exposed pine logs required for breeding. Due to modern forestry and fire suppression, this type of substrate has become so rare in the landscape that the beetles’ existence in Sweden is threatened. Recently, the female sex pheromone of this species was synthesised, providing a new method to monitor beetle populations using pheromone traps. Such a method was used in the current study to relate the presence-absence of T. depsarium with different landscape variables at different scales; 100, 500, 1000 and 2000 meters, respectively. The occurrence of T. depsarium in Östergötland County was associated with the amount of protected areas and clear-cuts at 500 and 1000 meters, respectively. Additionally, the amount of pine forest within 2000 m from the trap showed a weak, positive effect on the number of beetles. In the whole south-east boreo-nemoral zone of Sweden, a positive correlation between beetle occurrence and protected areas were found at 2000 m together with a negative correlation for volume pine wood within 100 m. Also, emergence holes on pine logs around each trap were strongly associated with beetle occurrences. To conclude, using pheromone traps were an easy way of detecting beetles. Although the number of caught T. depsarium varied greatly over time, the sampling period matched the flight period well. For long-term survival of this beetle, the forests must contain breeding substrates and be opened up through prescribed burning, selective cutting and active forestry.

Many species of caridean shrimp are protandrous hermaphrodites, maturing initially as males but developing into females as they age and grow. A unique sexual system was recently discovered in the peppermint shrimp, Lysmata wurdemanni. In this species, individuals are initially male, but become simultaneous functional hermaphrodites over time. As in most caridean shrimp, L. wurdemanni can mate as a male during the intermoult period, but can reproduce through female function for only a short period after moulting. Ecdysis does not occur en masse in this species, and thus the operational sex ratio found in populations of L. wurdemanni is extremely male-biased. Sexual selection theory suggests that these conditions will result in increased competition for access to mates. Evolutionary pressures should therefore have selected for mechanisms that permit individuals to quickly identify and locate potential mating partners. L. wurdemanni were exposed to chemical stimuli collected from recently moulted conspecifics of varying reproductive condition. Test animals were able to distinguish among the different conditions, and physically manipulated only the plastic nozzle used to pump solutions collected from shrimp with ovaries filled with vitellogenic oocytes. It was subsequently hypothesized that methyl farnesoate, a hormone associated with ovarian maturation in crustaceans, might be a key component of sex pheromones used by L. wurdemanni. However, a series of methyl farnesoate concentrations did not elicit responses, indicating this species does not use this hormone alone when determining reproductive condition. Reproductive behaviour in L. wurdemanni was observed to differ both before and after copulation, as well as with increasing population density. Intermoult individuals were more likely to approach, follow and remain in the vicinity of a near-moult shrimp before mating could occur, and under high density conditions. The near-moult shrimp approached conspecifics only under low density conditions, and performed rapid escape behaviours only after copulation had occurred. The unusual occurrence of epibiota upon L. wurdemanni was described, and the location, size and age of barnacles quantified. The effect of salinity upon grooming activities was tested. Results indicated that carapace grooming was depressed at low salinities, and could account for the considerable epibiota found in this region.

The use of traditional tools for the discovery and characterization of biological systems has resulted in a wealth of biological knowledge. Unfortunately, only a small portion of the biological world is well-understood to date, and the study of the rest remains a daunting task. This work involves using time-varying stimuli in order to more rapidly interrogate and characterize signaling pathways. The time-dependent stimulation of a signaling pathway can be used in conjunction with a model of the pathway to efficiently evaluate and test hypotheses. We are developing this technology using the yeast pheromone signal transduction pathway as a model system. The time-varying stimuli will be applied to the yeast cells via a novel microfluidic device, and the pathway output will be measured via various fluorescent reporters. The output of the pathway can then be compared to the output from a computational model of the pathway in order to test hypotheses and constrain our knowledge of the pathway. Initial work shows that a computational model can be used to identify stimuli time-courses that increase the parameter sensitivity, meaning that corresponding experiments could potentially be much more informative. / Poster presented at the 2005 ICSB meeting, held at Harvard Medical School in Boston, MA.

Faculty of Science School of Animal,Plant and Enviromental Studies 0204322k thibbitts@tamu.edu / I investigated three mechanisms (endurance rivalry, contest competition, and mate choice) of sexual selection and the influence of multiple signals on intrasexual and intersexual encounters in the common barking gecko (Ptenopus garrulus). Aspects of the ecology of barking geckos were also studied to facilitate the investigation of sexual selection. Barking geckos exhibited sexual size dimorphism in relation to head size, with males having wider heads. No differences in diet or size of prey ingested were observed between the sexes, indicating that niche divergence was not occurring. Therefore, the difference in head width was best explained by sexual selection (male contest competition). Barking gecko diet was dominated by termites by number and volume. The peak reproductive season was in October for both sexes. I used activity patterns to determine if males emerged before females from winter dormancy, a key assumption of the protandry-based mating system model. Activity patterns were significantly different between males and females. Males were active in higher numbers early in the breeding season. Male and female activity patterns along with evidence that male territories were established before female emergence, testicular recrudescence likely coincides with male emergence, and larger males have larger territories and better reproductive success, suggest that barking geckos have a protandry-based polygynous mating system. I also tested for clustering of geckos on the landscape to determine if barking geckos lek. Clustering was found to occur in some instances, but barking geckos did not iii meet the criteria for a ‘classical’ lek species because males use calling sites containing resources (a burrow) that are also used by females. Lizards frequently rely on chemical cues to detect the presence of a conspecific. Male lizards in particular, may chemically sample potential refuges to avoid rivals. Barking geckos were equally likely to use an artificial refuge scented by another male compared to a control, indicating that males do not use scent when selecting refuges. I assessed the role of two signals, one acoustic (dominant call frequency) and one visual (yellow throat patch), in advertising residency and aggressive behavior in barking geckos. Larger males defended the largest home ranges and home ranges were maintained through calling, which is negatively correlated with body size. Body size also predicted some behavioural responses to field-playback trials. Small males retreated from the playback and large males were found to be aggressive towards the playback. Small relative throat patch size was also correlated with aggression and charging the playback. Finally, call frequency was correlated with the behaviour of charging the playback. I suggest that the frequencies of barking gecko calls constitute a long-range signal of body size, used by males for remote rival assessment and to advertise home range boundaries. I also assessed the role of multiple signals (acoustic and visual) in reproductive success and I studied the effect of one mechanism of sexual selection, endurance iv rivalry, on reproductive success. Activity levels were similar for males which bred compared to those that did not breed, suggesting that endurance rivalry is not a significant mechanism of sexual selection in this population. Body size was the best predictor of reproductive success, suggesting that call frequency functions as a long range signal of body size used by females to assess potential mates.

Exploiting the chemical ecology of an insect can unveil novel strategies for its pest management. Though much has been learned about the chemical ecology of Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), a major pest of solanaceous crops in the U.S., there has been little use of this knowledge in pest management. To better understand host plant selection by CPB, field and laboratory-choice experiments were conducted in Virginia. In laboratory studies, CPB preferred potato over both tomato and eggplant foliage and eggplant over tomato foliage. However, field studies using counts of live beetles on untreated paired plants and counts of dead beetles on insecticide-treated plants revealed no significant preference for potato over eggplant. Additional studies showed that the presence of adult male CPB on foliage greatly impacted host plant selection, with significantly more adults being attracted to eggplant with male beetles than any other treatment combination. Adult CPB have been shown to be attracted to (S)-3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol [(S)-CPB I], a male-produced aggregation pheromone. Field studies were conducted to determine if the opposite enantiomer of the pheromone, (R)-CPB I had an effect on CPB in the field. Results revealed no differences in counts of all CPB life stages between untreated potato plots with and without rows inundated with (R)-CPB I lures. In addition, the relative attraction of CPB adults to various racemic forms of the (S)- and (R)-enantiomers was also investigated and showed that racemic blends that were less than 97%(S) were not attractive to CPB adults. Combinations of the (S)-CPB I pheromone with synthetic plant volatiles consisting of (Z)-3-hexenyl acetate, (+)-linalool, and methyl salicylate were investigated in a trap crop strategy in potatoes, but failed to reduce CPB numbers in untreated middle rows of potatoes. Combinations of the (S)-CPB I pheromone with synthetic plant volatiles were also used in a novel CPB trap designed to catch colonizing adults in the field. Although the traps caught CPB adults, no differences were observed in traps baited with and without the attractant. Metaflumizone, a novel semicarbazone insecticide, was recently shown to be highly efficacious on CPB. Laboratory studies found the combination of metaflumizone and a low concentration (0.39 ppm) of the pyrethroid esfenvalerate was slightly synergistic on CPB adults and early (1st-2nd) instar larvae. Field trials combining a low rate of esfenvalerate and metaflumizone at one tenth the field rate controlled beetles as well as the full rate of metaflumizone. / Ph. D.

Field studies were conducted to evaluate the effect of mating disruption on the mating success of the gypsy moth, Lymantria dispar (L), in low-density populations. The gypsy moth is an insect pest of hardwood forests in many regions of the world. The discovery of the sex pheromone disparlure (cis-7,8-epoxy-2-methyloctadecane) produced by females marked the start of a new era in the control and management of gypsy moth populations. Sex pheromones, like disparlure, have been used for detecting new populations, monitoring the spread of populations and for population control based on the disruption of mating communication. Although mating disruption has been used against populations of insect pests in agricultural and forest systems, considerable information about the use of this method for managing gypsy moths is still lacking. Studies, therefore, were designed and carried out specifically to improve current understanding of the mechanism of mating success, to evaluate existing techniques for mating disruption, and to develop methods that would improve the application of pheromone used for mating disruption so as to reduce the costs associated with the use of this management tactic. The first study was conducted to compare the mating success and mortality of gypsy moth females in low-density populations in Virginia and Wisconsin because of differences, which have been observed in the population dynamics and the impact of management strategies between these two states. The results suggested that the higher rate of population spread in Wisconsin might be due to the increased mating success of females compared with Virginia, which may be due in part to increased long-distance dispersal of males and increased longevity of females. The effect of artificial pheromone applied at various doses and formulations on mating success in low-density gypsy moth populations also was studied. Dose-response relationships were obtained for pheromone doses ranging from 0.15 to 75 g a.i./ha. The doses of 37.5 and 15 g a.i./ha of pheromone were shown to effectively disrupt mating and, therefore, have been recommended for operational use. The results also showed that the disruption of mating and attraction of males to pheromone-baited traps as a result of application of pheromone formulated in plastic flakes (Disrupt® II, Hercon Environmental, Emigsville, PA) was stronger and lasted longer than for the pheromone formulated as microcapsule (3M Canada Co., London, Ontario, Canada) and in liquid (Shin-Etsu Chemical Co. Ltd, Tokyo, Japan). Another study was carried out to improve the use and efficacy of the pheromone for mating disruption by reducing the amount of pheromone that was sprayed and the flight distance during aerial application. This study showed that in mountainous landscapes the effect of disparlure along the valley between mountains could be observed at a larger distance (633 ± 63 m) from the treated area than across the valley (104 ± 22m). In a relatively flat area, the effective distance for mating disruption was similar to the effective distance across the valley in a mountainous area (67 ± 17m). These dispersal characteristics of the pheromone provided further evidence that it could be used effectively in mating disruption treatments. Finally, a portable Electroantennogram (EAG) device was evaluated for its ability to detect disparlure sprayed for mating disruption in gypsy moth populations. The study found no relationship between the dose of artificial airborne pheromone and response of gypsy moth antenna as measured by the voltage ratio. The inability to detect differences between airborne pheromone concentrations in the plots treated for mating disruption might have been due to high variability among antennae and also by the inability of the EAG device to detect the low concentration of airborne pheromone used operationally for mating disruption. Further studies are required to improve the sensitivity of the portable EAG device before it can be recommended for use in the field. / Ph. D.

The populations of aphidophagous insects in low-spray and conventional programs were surveyed in Virginia apple orchards. Two sampling methods were utilized;aphid colony collection and beating tray collection. The low-spray blocks employed the use of mating disruption for control of the key direct pests, codling moth, Cydia pomonella (Linnaeus), and leafrollers. The abundance and diversity of aphid predators in low-spray mating disruption programs and conventionally controlled apple orchards was compared. Both sampling methods suggested that aphid predators were more plentiful in the mating disruption blocks. Several mating disruption blocks were found to accumulate significantly more predator-days and diversity than the matched control blocks. The reduction of insecticide input into the low-spray blocks may have allowed higher populations of aphidophagous insects to occur. Aphid populations in mating disruption blocks sometimes declined faster and had less resurgence than those in the conventionally controlled blocks. The tufted apple bud moth (TABM), Platynota idaeusalis (Walker), populations in four apple orchards in Winchester, Virginia, were compared for resistance to azinphosmethyl. These orchards were suspected by the growers to have resistant populations of TABM. Pheromone traps with insecticide incorporated into the adhesive were used to collect and test the moths. The results were not analyzed due to the low number of moths collected and high variability in mortality. / Master of Science

Swede midge (Contarinia nasturtii, Diptera: Cecidomyiidae) is a small invasive fly that is currently threatening Brassica vegetable and oilseed production in the Northeastern U.S. and Canada. Larvae feed on plant meristems, resulting in deformed leaves, stems, and heads. Extremely low damage thresholds for heading Brassica vegetables, multiple overlapping generations, and lack of effective organic insecticide options present serious challenges for managing this pest. Pheromone mating disruption (PMD), which involves confusing male insects with unnaturally large doses of sex pheromones, is particularly promising for swede midge management because it prevents mating and subsequent oviposition. One major challenge to PMD for swede midge management is that the chiral female pheromone blend, a 1:2:0.02 blend of (2S, 9S)-diacetoxyundecane, (2S, 10S)-diacetoxyundecane and (S)-2-acetoxyundecane, is expensive to synthesize due to the structural complexity of the compounds. Here, we explored three ways to reduce the cost of swede midge PMD: the use of lower-cost racemic pheromones containing all possible stereoisomers, single-component blends, and the possibility of using timed pheromone dispensers by testing for diel patterns of midge reproductive behavior. Although we found that males were not attracted to blends containing the racemic stereoisomers of the main pheromone component, (2S, 10S)-diacetoxyundecane, racemic blends functioned equally as well as chiral blends in confusing males and altering female behavior in PMD systems. We observed 95% and 87% reductions in males caught in monitoring traps in three-component chiral and racemic PMD plots of broccoli, respectively. In addition to confusing males, we also found that females altered their reproductive behavior in response to both chiral and racemic pheromones. Females released pheromones more frequently when exposed to three-component chiral and racemic blends, and were less likely to mate afterward. Single-pheromone treatments containing either chiral or racemic 2,10-diacetoxyundecane neither confused males nor influenced female behavior. We identified a total of eight hours during the day and night when midges do not exhibit mate-seeking behavior, during which programmable PMD dispensers could be turned off to save pheromone inputs. We found that up to 81% of females released pheromones to attract males for mating in the early morning shortly after dawn. Most females emerged in the morning as well, releasing pheromones soon after eclosing. Because midges are receptive to mates shortly after emergence, they may mate at their emergence site. Overall, we found relatively high levels of crop damage in our pheromone-treated plots, likely due to the migration of mated females into our plots. If midges mate at emergence sites, rotation of Brassica vegetable crops may result in overwintered midges emerging in fields where host plants are not currently grown. Further research is needed to determine where midges mate in order to determine where to install PMD dispensers.

Most Aphidinae species produce and use (E)-ß-farnesene (Eßf) as an alarm pheromone. This sesquiterpene is released by individuals under attack by a predator, and nearby aphids exhibit a variety of alarm behaviours. This PhD thesis aims to better understand how aphids manage their production and emission of alarm pheromone (Chapter IV). We also wanted, in a second step, to improve our knowledge on the roles that Eßf could play in the relationships that aphids have with their predators (Chapter V) and tending ants (Chapter VI), in order to better pinpoint the problem in this very tough context. The aphid predators have indeed a real advantage to be able to use the odorant cues emitted by their prey, to locate them and to select an adequate oviposition site. Ants establish with certain aphid species mutualistic relationships, which occurrence could be facilitated by the use of aphids odours. In Chapter IV, we have highlighted that aphid colonies non subjected to attack by predators release constantly small quantities of Eßf in their headspace, which means that this molecule could have additional roles than just acting as an alarm pheromone. In a second study, we demonstrated that the release of Eßf was not contagious, and therefore that a non stressed aphid receiving the alarm signal does not release additional Eßf. Since the production of alarm pheromone is likely to entail physiological cost, we tested and validated the hypothesis that aphids regulate their Eßf production according to their social environment. In Chapter V, we studied the ability of the hoverfly predator Episyrphus balteatus (Diptera, Syrphidae) to be used as biological control agent against aphids infesting tomato plants (Lycopersicon esculentum). After identifying the odours emitted by aphid infested plants, we have demonstrated that although this Diptera is able to perceive all the odours released by the system, it mainly uses Eßf to select its oviposition site. However, the E. balteatus larvae are not adapted to the architecture of tomato plants. We also showed that the Asian ladybeetle Harmonia axyridis (Coleoptera, Coccinellidae) olfaction was adapted to the perception of Eßf and that this beetle is also attracted by this sesquiterpene. Finally, in Chapter VI, we characterized the benefits accruing to aphid populations that have established mutualistic relationships with Lasius niger (Hymenoptera, Formicidae), and have demonstrated the role of Eßf and honeydew, respectively in locating aphid colonies and in the persistence of the mutualism. La plupart des espèces de pucerons appartenant à la sous-famille des Aphidinae produisent et utilisent le (E)-ß-farnésène (Eßf) comme phéromone dalarme. Ce sesquiterpène est relargué par les individus stressés par lattaque dun prédateur et cause chez les individus qui le perçoivent un comportement dalerte. La présente thèse de doctorat a pour objectif de comprendre comment les pucerons gèrent la production et émission de phéromones dalarme (Chapitre IV). Nous voulions aussi, dans une seconde étape, améliorer les connaissances sur les rôles potentiels que cet Eßf peut jouer au sein des relations que les pucerons entretiennent avec leurs prédateurs (Chapitre V) et avec les fourmis (Chapitre VI) Les prédateurs de pucerons retirent en effet un réel avantage à pouvoir saider des odeurs émises par leurs proies pour les localiser et pour sélectionner un site doviposition adéquat. Les fourmis, quant à elles, établissent des relations de mutualisme avec certaines espèces de pucerons. Les rencontres entre fourmis et pucerons pourraient être facilitées par lutilisation des odeurs de pucerons. Les résultats obtenus peuvent être résumés de la manière suivante: Dans le chapitre IV, nous avons mis en évidence que les colonies de pucerons non soumises à lattaque de prédateurs relarguent constamment de faibles quantités dEßf, ce qui permet dassumer que cette molécule puisse avoir dautres fonctions que celle de phéromone dalarme. Dans une deuxième étude, nous avons démontré que lémission du Eßf nétait pas contagieuse, et donc quun puceron non stressé percevant le signal dalarme német pas à son tour de le Eßf. Puisque la production de phéromone dalarme a inévitablement un coût physiologique, nous avons testé et validé lhypothèse selon laquelle les pucerons régulent leur production de Eßf en fonction de leur environnement social. Dans le chapitre V, nous avons étudié la possibilité dutiliser le syrphe prédateur Episyrphus balteatus (Diptera, Syrphidae) en lutte biologique contre les pucerons infestant les plants de tomate (Lycopersicon esculentum). Après avoir identifié les odeurs émises par les plants infestés, nous avons démontré que, si ce Diptère est capable de percevoir lensemble des odeurs émises par ce système tritrophique, il utilise principalement lEßf pour sélectionner son site doviposition. Cependant, les larves dE. balteatus ne sont pas adaptées à larchitecture des plants de tomate. Nous avons également montré que la coccinelle asiatique Harmonia axyridis (Coleoptera, Coccinellidae) possède le matériel olfactif nécessaire à la perception du Eßf et quelle est aussi attirée par ce sesquiterpène. Enfin, dans le chapitre VI, nous avons caractérisé les bénéfices retirés par les populations de pucerons ayant établi des relations de mutualisme avec Lasius niger (Hymenoptera, Formicidae), et avons démontré le rôle du Eßf et du miellat, respectivement dans la localisation des colonies de pucerons et dans la persistance du mutualisme.

Les pucerons sont considérés comme des ravageurs majeurs, dune part en raison de leur action directe sur le végétal, mais également en tant que vecteurs de nombreux virus phytopathogènes. A lheure actuelle, la lutte contre certaines espèces de pucerons, au moyen dinsecticides de synthèse, devient extrêmement difficile en raison de lapparition de populations résistantes. Le développement de nouveaux produits antiparasitaires, visant spécifiquement les pucerons, devient, par conséquent, hautement souhaitable. Dans ce contexte, nous avons choisi de nous intéresser aux enzymes de la famille des « short-chain » isoprényle diphosphate synthases (scIPPS). Ces enzymes constituent une classe de prényltransférases impliquées dans le métabolisme des isoprénoïdes. Cette classe comprend la géranyle diphosphate synthase, la farnésyle diphosphate synthase et la géranylgéranyle diphosphate synthase qui respectivement synthétise le géranyle diphosphate (GPP, C10), le farnésyle diphosphate (FPP, C15), et le géranylgéranyle diphosphate (GGPP, C20). Nous avons cloné chez le puceron un nouveau type de scIPPS. Contrairement à d'autres scIPPS connues, lenzyme recombinante du puceron affiche une activité bifonctionnelle ; cette dernière étant capable de synthétiser à la fois le GPP, le précurseur commun des monoterpènes, mais également le FPP, le précurseur des sesquiterpènes. Chez les pucerons, ces deux précurseurs sont supposés jouer un rôle clé dans la biosynthèse de l'hormone juvénile et des phéromones d'alarme et sexuelle. Dans le but de fournir une explication structurelle à la bifonctionnalité observée chez la GPP/FPP synthase de puceron, nous avons entrepris de résoudre sa structure tridimensionnelle en se basant sur les structures cristallographiques du poulet. Plusieurs mutants ont été ensuite conçus et caractérisés.