Grasshoppers, like most other organisms, require sterols for the production of cellular membranes and, like other animals, as precursors to steroid hormones. However, grasshoppers, like other insects, cannot synthesize sterols de novo and must acquire these essential nutrients from their diets. Survival and performance of five grasshopper species, representing three phylogenetic lineages and with different host preferences, were compared on artificial diets that varied in sterol type. Sterols used were: cholesterol (Δ⁵) sitosterol (Δ⁵) stigmasterol (Δ⁵ʼ²²) spinasterol (Δ⁷ʼ²²) and lathosterol (Δ⁷); these are the most common plant sterols. For all species studied, none survived to the adult stage on stigmasterol, spinasterol or lathosterol. Performance on the cholesterol and sitosterol diets were similar for most species. This study suggests that grasshoppers, as a group, are extremely limited with respect to the sterols they can use. Grasshoppers, however, are highly polyphagous insects, often feeding on many different host-plants. Some of these host-plants contain unsuitable sterols. The effect of sterol mixtures on survival and performance was examined by rearing the grasshopper, Schistocerca americana, on artificial diets containing sitosterol (suitable) and spinasterol (unsuitable) presented either alone or in combination. Results indicated that spinasterol, even in the presence of high levels of a sitosterol, could prevent development to the adult stage. Biochemical analysis of grasshopper tissues revealed that dietary sterols with Δ⁷ and/or Δ²²-configurations could not be metabolized to cholesterol. Unmetabolized dietary sterols tended to accumulate in the midgut tissues. Grasshoppers are known to regulate feeding in response to some nutrients in relation to need. To determine if grasshoppers could regulate intake with respect to different sterols, a series of behavioral assays was performed. The grasshopper S. americana was observed feeding on artificial diets with different sterols, in some cases up to seven hours. Results suggest that S. americana developed aversion responses to diets that contained unsuitable dietary sterols (Δ⁷ and/or Δ²²) Additional experiments suggested that the aversion response developed through a combination of post-ingestive feedbacks and associative learning. Taken together, these results suggest that sterol metabolic limitations may partially determine host-plant affiliations in grasshoppers.

The insect neuropeptide eclosion hormone (EH) interacts in a positive feedback loop with two other hormones to trigger ecdysis behavior in the hawkmoth, Manduca sexta. There is also evidence that EH may act at other targets to coordinate physiological changes that must occur with ecdysis. Recently, the use of an antibody that recognizes the second messenger cyclic guanosine monophosphate (cGMP) in fixed tissue has allowed us to identify a novel target for EH in the proximal region of the abdominal transverse nerves of Manduca. Here we show that a significant increase in cGMP can be measured in vivo in the transverse nerves at a time when EH is normally released in the animal. Furthermore, in vitro experiments revealed that the ability of the transverse nerve to respond to EH is abruptly lost after the pupal ecdysis. Finally, it was found that up to 40% of the cGMP produced by the STNR in response to EH is exported out of the cells in probenecid-sensitive manner, and there is a significant increase in the hemolymph concentration of cGMP at the time of pupal ecdysis. Microscopic examination of transverse nerve sections revealed that the cGMP-positive filaments are the processes of intrinsic cells, and the area in which they reside was named the subtransverse nerve region (STNR). Ultrastructurally, the cells of the STNR fall into two groups: large-granule containing cells and ribosome rich cells. EH-stimulated cGMP immunoreactivity appears to be restricted to the ribosome rich cells. The discrete STNRs of the abdominal transverse nerves can be identified as early as the first instar and probably originates embryologically. After the time of wandering in the last instar, the cells of the STNR begin to divide and spread in a flat sheet between the transverse and dorsal nerves of each ganglion, and are positive for myosin by pupal stage 8. These STNR-derived cells appear to form the ventral diaphragm muscles of the adult. This is the first evidence in any insect that a reservoir of myoblasts exists in close proximity to the transverse nerves and the first evidence that myoblasts are a target for EH.

Aphis gossypii Glover (cotton-melon aphid) is a major pest of agriculture worldwide. Cucumis melo L. (melon) possesses monogenic resistance to this aphid, and is a good model for the study of aphid resistance mechanisms in plants. This dissertation presents analyses of the effects of the resistance gene on A. gossypii, and of the gene's effects on biochemical and molecular-genetic properties of melon plants. Nearly isogenic lines (NILs) of melon, either resistant or susceptible to A. gossypii, were compared for their influence on aphid life history traits and feeding behavior. The resistance trait delayed development, increased mortality, and markedly decreased reproduction of aphids confined to leaves of resistant plants. Aphids on resistant plants salivated into phloem sieve elements significantly longer, and were less likely to begin sap ingestion after salivation, suggesting that the resistance factor acts within phloem sieve elements. Biochemical properties of callose synthase were compared between NILs to test the hypothesis that callose deposition plays a role in the resistance mechanism. No differences were detected between resistant and susceptible melon genotypes with respect to callose synthase subunit abundance or in vitro enzyme activity. Sixty-four F₃ families from a melon mapping population were tested for aphid resistance to place the resistance locus on a genetic map of the melon genome. Four molecular markers were found to be linked to the aphid resistance phenotype. The name Agr ( Aphis gossypii resistance) is proposed for this locus. The closest flanking markers were positioned at 4.3 and 7.0 cM from Agr. Evidence suggests Agr might be a member of the nucleotide binding site-leucine-rich repeat (NBS-LRR) family of plant resistance genes, which are known to cluster in plant genomes. Melon genomic DNA sequences homologous to this gene family were isolated to test the hypothesis that Agr is an NBS-LRR homolog. Two of these sequences were tested for genetic linkage to Agr in a population of F₂ plants segregating for the resistance trait. DNA gel blot analysis determined that one sequence, NBS-2, is approximately 2.7 cM distant from Agr, which suggests Agr resides in a cluster of NBS-LRR homologs and could be a member of this gene family.

Insect and rodent samples were collected from suspected VSV-NJ enzootic areas over 2 consecutive field seasons (1996-1997) and from southern Arizona only during 1998. Insect samples were screened for arboviruses, and rodent sera were tested for the presence of VSV-NJ and VSV-IN neutralizing antibodies. Vesicular stomatitis virus New Jersey serotype was isolated from a pool of Culicoides sp. collected in 1997 near Belen, New Mexico. All rodent sera were negative for specific VSV-NJ and VSV-IN antibodies. Genetic analysis of the hypervariable region of the phosphoprotein gene demonstrated that the 1997 Belen VSV-NJ isolate was more closely related to viruses isolated from livestock during the 1982-83 western U.S. epizootic than to other VSV-NJ isolates. This suggests that VSV-NJ may be enzootic in the western U.S. Simulium vittatum was shown to be a competent vector of VSV-NJ. Virus-infected females were allowed to feed on laboratory mice and on deer mice. All laboratory mice seroconverted by day 21 post-exposure. Neutralizing antibody titers increased from an average of 1:4 at baseline to >1:1,024 on day 21. An age-related effect on viral pathogenesis was evident in Peromyscus maniculatus following VSV-NJ exposure by black fly bite. Lethal encephalomyelitis was evident in all 6-week-old mice, but in only one 6-month-old mouse. Peromyscus maniculatus did not meet the standard definition of a reservoir host for VSV-NJ because a viremia, was not detected. Nonetheless, P. maniculatus may play a role in virus maintenance since non-infected black flies became infected while co-feeding with infected black flies on the same non-viremic host. These results represent the first example of a western U.S. insect species becoming infected with VSV-NJ by feeding on a host. Simulium vinatum and S. notatum were shown to be competent laboratory vectors of VSV-IN. Saliva from experimentally infected Simulium vittatum and S. notatum was collected and tested for the presence of infectious virus. Virus was detected in the saliva of both species following oral infection. Independent experiments were conducted to determine if transovarial transmission of VSV-NJ and VSV-IN occurs in black flies. Transovarial transmission was not detected. Transstadial transmission of both virus serotypes was detected.

In the mid-1980s, a new biotype of Bemisia tabaci was introduced into the southern U.S. causing extensive damage to agricultural crops throughout the region. An augmentative biological control project was initiated using a native parasitoid, Eretmocerus eremicus to determine its efficacy against B. tabaci infesting cotton in the desert areas of California and Arizona. A series of experiments were conducted in 1992-1995: release rate studies in cages and open fields; parasitoid dispersal within fields to determine movement rates after point release; and experiments to determine the parasitoid-host spatial relationship. Cage release rate studies, demonstrated that rates of parasitism could be increased 61 to 79% in the highest release treatments, with reductions in whitefly densities of 80 to 100% relative to control treatments. Cotton yields in the high release treatments peaked at 2.5 bales/ac and were 2.6 to 4.2 times greater than in control treatments. Effective release rates were estimated to be equivalent to 770,000 to 1.1 million parasitoids/ha. Field releases equivalent to 7.9 million parasitoids/ha resulted in a peak rate of parasitism of 42% but there were no statistical differences in parasitism, whitefly densities, or cotton yield; relative to no-release plots. High levels of whitefly immigration from surrounding crops, and parasitoid dispersal from release plots, diluted the effects of release. In a second field release rate study, releases equivalent to 2.0 to 3.0 million parasitoids/ha increased levels of the percentage of discovered leaves to greater than 80%. Parasitoid dispersal was analyzed with mark-recapture experiments and data were fit to a diffusion model. One female wasp flew 82 m in one day though the majority of wasps flew a few meters or less. Estimated diffusion rates and median dispersal distances were 0.40 to 0.71 m²/min and 2.4 to 4.4 m/(4 to 8) days respectively. Analysis of dispersal data suggested that releases on 20 m centers would provide effective coverage within a field. Density independent parasitism was common at the spatial scale of leaves and plants. There was positive density dependence for the percentage of discovered leaves suggesting that parasitoids aggregate to high density patches of whitefly but fail to achieve high levels of parasitization possibly due to egg limitation or mutual interference.

The macroglomerular complex (MGC) in the antennal lobe of the sphinx moth Manduca sexta is the first brain region for processing sex-pheromonal information. How is the MGC is functionally organized, and how are chemical and physical features of the pheromone encoded by projection neurons (PNs) innervating the MGC (MGC-PNs). For some MGC-PNs with arborizations in the toroid, one of the two major glomeruli of the MGC, bombykal (a key pheromone component) can evoke a mixed (inhibitory/excitatory/inhibitory) response similar to that evoked by the pheromone blend. Likewise, for some neurons with arborizations in the cumulus, C-15 (a mimic of the second key component) can evoke a similar mixed response. The maximal pulse frequency encoded by these component-specific neurons was not increased in the presence of the blend, but seemed to arise through the convergence of two parallel pathways, one excitatory and one inhibitory, both activated by the same olfactory stimulus. Convergence of different synaptic pathways allowed MGC-PNs to resolve intermittent stimuli and thus to relay the temporal structure of the pheromonal signal to higher brain centers. In a subset of MGC-PNs that was excited by antennal stimulation with either of the two components (bombykal-C-15 cells, blend neurons), the ability to encode intermittent stimuli was improved when stimulating with the blend. The temporal character of the responses was dependent on the ratio of the two key components in the blend. Component-specific MGC-PNs responded over a range of increasing pheromone concentration with stronger inhibitory and excitatory postsynaptic potentials and more impulses but the responses were not affected by changing the blend ratio. Two basic response patterns emerged when the ipsilateral antennal flagellum was stimulated at different zones along its proximo-distal axis while the activity of MGC-PNs was recorded. A subset of neurons with broad receptive fields was excited regardless of the zone of the antenna stimulated, whereas another subset responded selectively to stimulation of the basal region of the antenna. A diverse array of MGC-PNs forms a heterogeneous group of parallel output channels that encode features of the pheromone signal that the moth is likely to encounter in the natural stimulus situation.

The phylogenetic relationships of flies within the family Drosophilidae are studied in four clades placed at three different taxonomic levels. The goal of examining phylogenetic relationships of nested clades within Drosophilidae is to study patterns divergence at several points, giving a more informative view of evolution in this family of flies. Two studies presented here examine the phylogenetic relationships within the Drosophila saltans and Drosophila obscura species groups. Another examines the relationships among several species groups within the subgenus Sophophora. The final study examines the phylogenetic relationships among genera within the family Drosophilidae. The molecular data gathered for members of the Drosophila saltans species group are congruent with the conclusions of previous taxonomic studies. All five species subgroups are monophyletic with respect to one another. Relationships among the subgroups indicate that the neocordata and elliptica subgroups are basal in the saltans species group. The saltans and parasaltans subgroups are derived sister taxa and the sturtevanti subgroup occupies an intermediate position within the saltans species group. The results of the Drosophila obscura analyses indicate that all five of the subgroups are monophyletic with respect to one another. Furthermore, the New and Old World obscura species groups are monophyletic, a result which has been suggested by some data in the past. This study also suggests, albeit weakly, that the Afrotropical microlabis subgroup may be the sister taxon of the subobscura subgroup. The results of this study indicate that Sophophora is monophyletic with respect to the subgenus Drosophila. Within Sophophora, the obscura and saltans species groups are highly supported as monophyletic. The willistoni and melanogaster species groups, however, may not be monophyletic. This is possibly due to rapid speciation at the base of these two species groups. The relationships inferred through combined analysis of all available molecular and morphological data indicate that the genus Drosophila is not monophyletic. Several genera, including Engiscaptomyza, Hirtodrosophila, Mycodrosophila, Paramycodrosophila, Samoaia, Scaptomyza, Zaprionus and Zygothrica diverge within the boundaries of what is currently considered the "genus" Drosophila. Furthermore, the subgenus Drosophila is not monophyletic and can be tentatively divided into at least two, and probably more, monophyletic groups. These clades correspond, although not perfectly, with the virilis-repleta and immigrans-tripunctata radiations described in earlier taxonomic studies. Finally, the subgenus Sophophora, previously considered the sister clade of the subgenus Drosophila, is shown to be a lineage distinct from the genus Drosophila.

Heliolithine moth larvae are serious agricultural pests causing hundreds of millions of dollars in agricultural damage annually. Mating disruption, a behavioral method of control of adult male sexual response to female pheromones, has been successfully administered in the field for many years, yet the underlying mechanisms that account for mating disruption are poorly understood. Habituation, a form of nonassociative learning, has been implicated as one possible mechanism that effects male moth behavior during the application of mating disruption strategies. To substantiate the claim that habituation can play a role in mating disruption, data were collected on five of habituation's classic characteristics: (1) The existence of a negatively accelerated relationship between trials and response strength; (2) Spontaneous recovery of response strength; (3) A long-term reduction in male moth pheromonal response strength; (4) More rapid reduction of response strength and greater spontaneous recovery of response with less intense stimuli; (5) Learning associated with a highly specific stimuli will not generalize to exceedingly dissimilar stimuli. Results of this study clearly indicate that all five characteristics related to habituation were present. Specifically, males, repeatedly exposed to pheromones of conspecifics, display a decrease in response strength. Response reduction and spontaneous recovery were both found to be greater with less complex stimuli. Long-term habituation was successfully demonstrated for up to 96 hours post habituation training. Most importantly, it was found that males, habituated to one blend, demonstrated little habituation when exposed to a second blend. Additionally, it was found that a substantial amount of individual differences in responsiveness to habituation training exists between males of this species. The findings of this study indicate two major points. First, habituation can be used as part of an effective integrated pest control strategy. However, because there is a substantial amount of individual differences in habituation susceptibility, some male moths may experience greater reproductive success. Further, because the males did not generalize habituation across the two stimuli presented in this study, there is concern that if all known pheromone components of this species are not used in field applications, males will habituate to the applied pheromone but not individual females.

The olfactory system of the hawkmoth Manduca sexta is able to discriminate among odors and to mediate a behavioral response, such as feeding or oviposition in females. This dissertation research describes attempts to elucidate neural coding involved in the processing of odors within the first-order olfactory center of the brain, the antennal lobe (AL), in female moths. This study focused on a small and identifiable subset of projection neurons within the AL, which have their cell bodies grouped on the anterior surface of the AL (AC neurons). The subpopulation of about 14-20 neurons was characterized physiologically and morphologically. The anatomical features of AC neurons and their physiological tuning specificities to plant-associated odors were investigated by means of electrophysiological recording and staining techniques. All AC neurons identified were output neurons, each having a distinct uniglomerular dendritic branching pattern. The entire population of AC neurons, which sometimes exhibited a separation of its cell body group into two clusters (primary and satellite), projected into two small groups of glomeruli. One group was located dorsally and medially in the posterior part of the AL and probably consists of five glomeruli. The other group was situated dorsally and anteriorly and possibly consists of one uniquely identifiable glomerulus. Furthermore, the results indicate response specificity and a narrow receptive range of certain AC neurons in response to flowers of Solanaceous hostplants. Among the odors tested were nitrogen (N)-bearing compounds, monoterpenes, aromatics and green-leaf volatiles. The response profiles were divided into different response categories: "excited" (multiphasic), "inhibited", "mixed" and "no response to odors tested". The majority of AC neurons were spontaneously active and exhibited rhythmic and periodic bursting (burst frequency 1-3 Hz). Also, AC neurons showed low-amplitude, membrane oscillations. These oscillations, as well as the bursts, are not odor-induced. In summary, the data show that the organization of a subset of unique, ordinary AC glomeruli is spatially distinct and recognizable, and functionally significant for hostplant odor processing in the AL. The mechanisms underlying processing seem to include a rate code (encoding concentration) and possibly a complex spatial code (encoding quality).

Polyhydroxylated steroids with insect molting hormone activity were discovered in plants over thirty years ago. The major endogenous molting hormone of insects is believed to be 20-hydroxyecdysone (20E) and interestingly, it is also the most commonly encountered phytoecdysteroid (PE) in plants. Ecdysteroids control developmental programs in both immature and adult insects however, the role of PEs in plants has not been demonstrated. PEs are hypothesized to function as either plant hormones or plant defenses against phytophagous insects. Many toxic secondary metabolites are concentrated in apical meristems where herbivory would result in the greatest reduction in plant fitness. Similarly, the highest concentrations of 20E in spinach were associated with the stems and vasculature while old leaves and roots displayed low levels. In plants, concentrations of toxic or deterrent metabolites are often rapidly induced following attack. In spinach roots, both mechanical damage and insect herbivory resulted in rapid increases in 20E concentrations. The plant wound signal, jasmonic acid was strongly implicated in signaling this response. Known plant hormones and chemical defenses are regulated differently. Pulse chase studies with [2-¹⁴C] mevalonic acid demonstrated that de novo root 20E biosynthesis occurred during the induction and, once synthesized, 20E was stable for over one month. This result is does not support the plant hormone hypothesis, as plant hormones typically undergo rapid conjugation or catabolism. The induction of root 20E concentrations occurred without similar changes in related membrane phytosterols. Simply, pathway specificity was demonstrated as increased 20E accumulation was not part of an overall increase in steroids. To empirically examine the hypothesis that PEs function as plant defenses against insects, a series of experiments were designed with the fungus gnat Bradysia impatiens. Results indicated that root herbivory by larvae induced 20E levels in roots, larval preference for diets containing induced 20E levels was reduced, larval survivorship on 20E containing diets was lower, and plants with induced root 20E levels were better protected from attack. Together, these results support the plant defense hypothesis at both the physiological and ecological level.