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The distribution and abundance of six bumblebee species (Bombus) and their associated flowers were studies for 12 weeks in 26 discrete subalpine meadows over the summer of 1981. Principal component analysis of flower densities identified phenological patterns of blooming and differential affinities of plants for moisture as the major contributors to flower variation among the meadows. A positive relationship between bumblebee species diversity and meadow area in the early summer and mark-recapture experiments suggested that queens are attracted to large meadows for nest establishment. After colony initiation meadow floristics govern Bombus species diversity and abundance. Results showed that: (1) mid- to late-summer bumblebee diversity, the temporal occurrence of new queens, and the densities of two species were correlated with the density of nectar producing flowers; (2) flower composition in meadows where local extinctions of at least one Bombus species occurred differed from those where all species persisted; and (3) the number of species reaching reproductive fruition was independent of bumblebee species composition. These results underscore the importance of nectar resources in Bombus colony and guild development. Multiple regression analyses were used to relate the effects of meadow size, elevation, and floristics to observed densities of Bombus flavifrons and B. rufocinctus. In mid- to late summer a significant portion of the variation in densities not explained by these variables was accounted for by densities of congenerics. In meadows where all Bombus were removed except B. flavifrons or B. rufocinctus significant positive density compensation was observed for the latter, but not the former. These results are suggestive of relationships among bumblebee densities, intensity of competition, colony ontogeny, colony reproduction, and local floristics that question both the (1) simplistic views of Bombus guild organization based on the paradigm of tongue length, corolla depth, and character displacement; and (2) the stochastic view that flowering phenologies and developmental time lags thwart resource-based guild organization.

This dissertation is a study in how heme facilitates biology using two heme proteins as examples. I write about my mechanistic studies on Cimex nitrophorin and preliminary studies on Ecdysone inducible protein 75, respectively. Nitrophorins are salivary heme proteins used by bloodfeeding insects to deliver NO to the victim, leading to vasodilation and antihemostasis. The bedbug nitrophorin cNP, a thiolate heme protein accomplishes this via an unusual heme-assisted S-nitrosation reaction, requiring proximal ligand cleavage. This dissertation explores this mechanism through mutational, crystallographic and transient kinetic approaches. I present the detailed investigation of the two NO binding events, one at the heme and the other at the proximal cysteine. The heme nitrosyl shows marked pH dependence arising out of the apparent protonation of the proximal cysteine ligand, a feature crucial to cNP function. The structures and spectroscopy of cNP mutant proteins reveal the SNO modification to be regulatory in nature. Laser flash photolysis measurements and the structures of mutant proteins reveal the negative influence of steric hindrance on SNO stability.Studies of insect embryogenesis and metamorphosis reveal the regulatory role of the hormone ecdysone via its target, the ecdysone receptor. Ecdysone triggers expression of several nuclear receptors in a time and tissue dependant fashion, which in turn carry out gene regulation. Ecdysone inducible protein 75 (E75), a nuclear receptor and an early ecdysone responsive gene product, regulates a subset of the developmental activities attributed to ecdysone. We are investigating E75 from Aedes aegypti to uncover its role in ecdysone signaling in mosquitoes. I have expressed and partially purified the full length protein using the baculovirus driven expression in SF9 cells, and purified to homogeneity the heme binding domain resolubilized from inclusion bodies obtained by expression in E. coli. Preliminary characterization of the proteins using UV-visible spectroscopy indicates that E75 has a b type heme with a low spin six-coordinate ferric iron. In the E75 heme binding domain, the heme exhibits an unstable ferrous state and only binds NO and CO at high non-physiological levels. These data place into doubt the suggested roles for E75 as a gas regulated transcription regulator.

The next generation of Bollgard® cotton was evaluated for agronomic and insecticidal efficacy under central Arizona growing conditions. Two novel lines were compared with their recurrent parents, DP50 and DP50B. There were no seasonlong differences observed among the varieties in most plant development and insect parameters. However, DP50 had significantly lower emergence than the other lines tested (possibly related to seed quality). The lower plant population may have been responsible for greater whitefly abundance observed on two dates mid-season. During early-season ratings of secondary “pests” (15 DAP) (scaled on damage and/or presence), the two test lines received lower ratings for thrips and flea beetle when compared with DP50, DP50B and DP50Bu (untreated for Lepidoptera). However, these difference are likely as a result of the difference in seed treatments that the two test lines received (Gaucho®) and the others did not. This seed treatment does have known activity against thrips and beetle pests. In mid-season, the two test lines received lower ratings for beet armyworm when compared to DP50, DP50B and DP50Bu (although, not significantly different from DP50B or DP50Bu). Efficacy against pink bollworm (PBW) was assessed one time at the end of the season (we were limited to this time, so as to not affect yield), and DP50 was the only variety in which PBW exit holes were observed and PBW larvae collected. However, the low Lepidoptera pressure experienced during the season limited assessments of the two novel lines’ efficacy toward PBW. There was no significant difference in yield (bale/A) among the varieties. Although, one of the test lines had a lower lint turnout than each other variety. The two novel Bollgard lines performed well under our growing conditions, but continued evaluations will be necessary under more conditions and more insect pressures before “varietal” performance and gene efficacy can be assessed adequately.

Green bolls (100/field) were sampled from the uppermost internodes within adjacent fields of Bt (Deltapine 33B) and non-Bt refuge (Hyperformer HS 44 or Deltapine 20) experiencing severe pink bollworm pressure late in the growing season. Evidence of 3rd instar or larger pink bollworm larvae survival was higher in susceptible bolls sampled from transgenic Bt cotton late in the 1998 growing season, compared to that observed late in the 1997 growing season.

Lygus became the number one pest of cotton in 1998 with statewide losses of over $16 million in spite of individual costs to the grower of over $55/A for control. Selective technologies for whitefly and pink bollworm control reduce the number of broad spectrum sprays that incidentally control Lygus. Control of Lygus depends mainly on just two related chemical classes of insecticides, organophosphates and carbamates. Over reliance on such a limited diversity of chemical controls increases the risk of resistance. Further, FQPA threatens the future availability of many of our main stay chemical controls. The study reported here sought to investigate the commercial suitability of a new compound, Regent®, for the control of Lygus. This novel mode of action represents one of the few potential new tools under development for Lygus management. Under a federal Emergency Use Permit (EUP), Regent was tested against two standards of Lygus control (Orthene® and Vydate®) and an untreated check. In a test of unusually high Lygus densities, Regent provided excellent control of small (instars 1–3) and large (instars 4–5) Lygus nymphs and may provide marginally better control of adults than current standards. None of the tested agents provided quick control or knockdown of adults. Rather, adult levels were reduced over time, most likely as a result of prevention of the development of new adults via nymphal control. All three materials protected cotton producing yields significantly higher than the check. The Orthene treatment had the highest yield, though not significantly higher than the Regent treatment which was effectively sprayed one less time than the other compounds.

The three keys to whitefly management are sampling, effective chemical use, and avoidance. This study examines factors relevant to the latter two keys in the context of Arizona’s cotton pest spectrum. Insect growth regulators (IGRs) are central to Arizona’s success in whitefly management. The basic usage guidelines developed for the IGRs—initial treatment timing, prescribed intervals between successive uses, and one use each seasonal limits—are all valuable in the development of a sustainable use pattern. Re-treatment timing guidelines for the second IGR has been the subject of investigation for the past two years. However, whitefly pressure in 1998 was strikingly different and lower than in any other post-introduction year. Re-treatment was unnecessary and thus could not be evaluated this year. Lygus, on the other hand, were at damaging levels early in plant development and for a protracted period. Future successes in whitefly management should consider the whole pest spectrum and depend on integrating chemical controls for all sprayed pests. While our primary focus is to optimize management of whiteflies in the context of other pests, this study examined the impact of Lygus controls on whitefly population dynamics and cotton production. Three sprays were required to control Lygus populations in this study. These sprays were atypically non-disruptive to whitefly population dynamics, and instead, helped to suppress low-level populations of whiteflies even further. This lack of disruption may have been due in part to the reduced abundance and role of natural enemies in this study. Lygus sprays did protect yields with a 3-fold advantage over untreated plots. Furthermore, there were a series of negative consequences of poor Lygus control. Plants tended to be more vegetative and more difficult to defoliate. Lower lint turnouts were documented for the Lygus-untreated areas. Sources of this additional loss were identified and included increased gin trash and larger seed size in Lygus-untreated areas. The lint also had significantly more sticking points as measured by manual thermodetector. While all cotton was determined to be non-sticky, this increased contamination may have been also related to the higher trash levels. Because of the differences in outcome in 1997 and 1998 in terms of Lygus spray effects on whiteflies, it is even more imperative that we further test whitefly management systems under near commercial conditions. A better understanding of the relationship between the control programs for these two major pests will help guide decisions on remedial inputs. This study also serves as an annual, replicated, and systematic accounting of whitefly population dynamics and control requirements useful for making historical comparisons across years. Inferences may be drawn about what are and are not the underlying causes of the unusual population dynamics observed in 1998.

Starting in 1993, we developed a field-based protocol for bioassaying sweetpotato whiteflies (SWF) for susceptibility to buprofezin (Applaud®). Since then, we have monitored Arizona SWF populations (up to 5 regions) for susceptibility to Applaud in four out of the last six seasons. We observed no appreciable decrease in susceptibility. Instead, we have observed an increase in susceptibility of present day whiteflies when compared to populations bioassayed in 1993 and 1996, before any Applaud use in the U.S.. This result, however, is likely related to various procedural changes in the bioassay methodology. Nevertheless, our current estimates of whitefly susceptibility are similar to those obtained from various unexposed populations from around the world and to populations we bioassayed in 1997. Differences between our LC50 estimates and those of some other researchers can probably be explained by various procedural differences: 1) method of Applaud application, 2) whitefly stage collected and sources of leaf foliage, and 3) bioassay environmental conditions. Our results also showed each year that Applaud susceptibility does not decline after Applaud application(s) based on commercial paired field comparisons and replicated small and large plot evaluations. In fact, susceptibilities actually marginally increased after an Applaud application. This fact does not alter the recommendation for Arizona to limit Applaud use to one time per crop season, but does provide hope for the development of a sustainable use pattern even if usage continues on non-cotton hosts (i.e., on melons and vegetables under Section 18). Given the tremendous value of this mode of action, however, commodity groups should work together wherever possible to coordinate the usage of this and other valuable compounds so that whitefly generations are not successively exposed to this product.