We examined the effects of two fungicides (copper+mancozeb and sulfur) and five reduced-risk insecticides (chlorantraniliprole, cyantraniliprole, lambda-cyhalothrin, novaluron, and spinetoram) on Hippodamia convergens (Guerin-Meneville) (Col.: Coccinellidae), an important natural enemy in western United States orchards. Acute toxicity of pesticides was tested via three different exposure routes: oral, residual, and topical. Lambda-cyhalothrin caused significant mortality to adults and larvae via all exposure routes (57-100%). Cyantraniliprole was toxic to adults and larvae when orally exposed, but it was less toxic for other exposure routes. Cyantraniliprole, spinetoram, and novaluron were moderately toxic to larvae when topically exposed (40-63% mortality), but not to adults (<4% mortality). Chlorantraniliprole, copper+mancozeb, and sulfur demonstrated low toxicity (<30% mortality) to adults and larvae, regardless of exposure route. Lethal and sublethal effects were tested by treating insects topically in the inactive stages (egg, pupa) and simultaneously through oral, residual, and topical exposure in the active stages (larva, adult). Larvae were vulnerable to all pesticides except copper+mancozeb, and lambda-cyhalothrin also reduced survivorship for pupae and eggs (3-19%). Exposure to novaluron lowered survivorship for treated eggs (14%) and fertility of treated females (9%). Fecundity was increased by exposure to copper+mancozeb. Corrected acute adult mortality alone was sufficient to detect the harmful effects of chlorantraniliprole and lambda-cyhalothrin, but combined effects of corrected adult mortality and daily fertility were needed to highlight the harmful effect of novaluron. Estimates of the intrinsic rate of increase indicated substantial delays in the population recovery of H. convergens following exposure to chlorantraniliprole, cyantraniliprole, novaluron, and sulfur, and local extinction for lambda-cyhalothrin. The effects of a combination pesticide (chlorantraniliprole and lambda-cyhalothrin) were tested on larvae and adults exposed to residues aged 0, 1, 2, 4, 8, 16, and 38 d in a laboratory or field setting. For field-aged residues, both life stages were highly susceptible to 0 and 1d residue (> 80%), but acute mortality declined for residues aged 16 and 38 d adults (< 53%). For laboratory-aged residues, both life stages had high acute mortality rates (> 80%) regardless of residue age. The rate of decay of the pesticide impact on adults (0.059) was greater than for larvae (0.024) for the field-aged residues, and greater than for adults exposed to laboratory-aged residues (a = 0.004). The results indicate that even reduced-risk insecticides may have harmful effects that vary by pesticide, life stage, and exposure route. Pesticide risk assessments can be improved by using lethal and sublethal data to estimate changes in population growth, and by incorporating both laboratory and field-based bioassays to better understand how pesticides impact natural enemies in the field.

In this dissertation I expand upon our knowledge in regards to the utility of population genetic approaches to be used for the study of the evolution of introduced biological control agents and their target pests. If biological control methods are to provide sustainable pest management services then more long-term studies will be necessary, and these studies should also include the use of population genetic approaches. For existing biological control programs, post-release population genetic studies could be initiated using museum voucher specimens for baseline data. In Chapter 2, I explored what factors influence our ability to extract usable genomic material from dried museum specimens, and whether we could use non-destructive techniques for parasitic hymenoptera. I found that the age of the specimen was the most important determinant for the amplification of PCR products, with nuclear loci having a higher probability of amplification from older specimens than mitochondrial loci. With these sequence results I was able to differentiate voucher specimens of different strains of the biological control agent Trioxys pallidus and I was able to confirm the identification of an unknown parasitoid reared from the invasive light brown apple moth.

For population genetic surveys to be conducted more frequently in biological control programs, some of the barriers to developing molecular markers that are variable enough for these types of surveys need to be overcome. One barrier is the time required to develop polymorphic microsatellite markers. Therefore, in Chapter 3, I developed a novel bioinformatics pipeline that searches through next-generation sequence (NGS) data and uses the raw sequencing reads to identify polymorphic loci. Using this approach I was able to rapidly develop microsatellite markers for two of my study species (T. pallidus and Chromaphis juglandicola). For both species more than 60% of the target markers amplified and were found to be polymorphic, compared to previous approaches where the success rates were much lower (published studies often show rates between 1 and 20%).

I then examined evolutionary factors that may affect the sustainability of two classical biological control programs; 1) the biological control of walnut aphids, and 2) the biological control of invasive knotweeds. The walnut aphid biological control program is a textbook successful biological control program, but has shown recent evidence of localized breakdowns, whereas the biological control program for invasive knotweeds is currently under review in the United States and Canada. In Chapter 4, I explored whether hybridization between introduced “strains” of T. pallidus is responsible for recent breakdowns in this control program. In that study I found low levels of hybridization – thus it is unlikely hybridization is playing an important role in these breakdowns – as well as evidence that two of the strains may actually be cryptic species; one being a specialist and another being a generalist. In Chapter 5, I explored whether the geographic mosaic theory of coevolution might help explain these localized breakdowns. In that chapter I found evidence for a geographic mosaic in the walnut aphid biological control program, and commented on how components of the geographic mosaic theory of coevolution can help us predict what systems we might expect localized breakdowns to occur in. In Chapter 6, I explored whether endosymbionts might play a role in shaping the host-associations of two strains of the candidate biological control agent for invasive knotweeds. I found that while strains of the psyllid Aphalara itadori showed no barriers to hybridization of their nuclear genomes, there were curious patterns of horizontal transmission of their primary endosymbiont. I also found that one haplotype of the secondary endosymbiont Sodalis sp. dramatically changed in frequency during the hybrid crosses reared on giant knotweed. When compared with previous studies of this species, the results I observe suggest that endosymbionts may play an important role in the differing fitness levels of these two strains.

In conclusion, population genetic approaches provide valuable tools for the study of post-release dynamics in biological control settings. While biological control programs promise to be useful study systems for evolutionary interactions, post-release studies will allow for that promise to come to fruition. In my future research endeavors I would like to continue to monitor the effects of hybridization and the frequency of geographic mosaics of coevolution in biological control settings. In addition, I would like to conduct post-release population genetic studies of both previous successful introductions and programs that resulted in failures. I believe these post-release studies will allow us to better determine what evolutionary factors affect the sustainability of biological control services and will allow for better management practices.

Background: Illumina sequencing with its high number of reads and low per base pair cost is an attractive technology for development of molecular resources for non-model organisms. While many software packages have been developed to identify short tandem repeats (STRs) from next-generation sequencing data, these methods do not inform the investigator as to whether or not candidate loci are polymorphic in their target populations.

Results: We provide a python program iMSAT that uses the polymorphism data obtained from mapping individual Illumina sequence reads onto a reference genome to identify polymorphic STRs. Using this approach, we identified 9,119 candidate polymorphic STRs for use with the parasitoid wasp Trioxys pallidus and 2,378 candidate polymorphic STRs for use with the aphid Chromaphis juglandicola. For both organisms we selected 20 candidate tri-nucleotide STRs for validation. Using fluorescent-labeled oligonucleotide primers, we genotyped 91 female T. pallidus collected in nine localities and 46 female C. juglandicola collected in 4 localities and found 15 of the examined markers to be polymorphic for T. pallidus and 12 of the examined markers to be polymorphic for C. juglandicola.

Conclusions: We present a novel approach that uses standard Illumina barcoding primers and a single Illumina HiSeq run to target polymorphic STR fragments to develop and test STR markers. We validate this approach using the parasitoid wasp T. pallidus and its aphid host C. juglandicola. This approach, which would also be compatible with 454 Sequencing, allowed us to quickly identify markers with known variability. Accordingly, our method constitutes a significant improvement over existing STR identification software packages.

This dissertation focuses on three different aspects of the biotic and abiotic effects on pest management and community structure in agricultural systems, at a local level for a specific insect pest, at a landscape level for an insect community and in mesocosm experiments to examine the effects of predation on indirect interactions. Agricultural crops and pastures cover approximately 38% of the available land surface globally, and the intensity of agricultural production has continually increased over the past 300 years. The intensification of management practices in agricultural ecosystems, from pesticide applications to simplified rotations, have had wide-ranging consequences, from a rise in secondary pests to negative effects on native species and the efficacy of biological control from generalist natural enemy assemblages.

The walnut husk fly, Rhagoletis completa (Diptera: Tephritidae), is a secondary pest that has become more problematic in California walnut orchards with the decline in use of organophosphates, and a seasonal emergence pattern that is not well understood. To more effectively manage R. completa the factors that affect the timing of adult emergence were examined using both field data and mechanistic laboratory studies. A phenology model was developed for R. completa using a large historic data set to more accurately predict the timing of the flight period and optimize management decisions. The relationship between cumulative trap catch and degree-day accumulation for R. completa in California was modeled to predict thermal requirements for the start and mid-point of the flight period. Latitude, walnut cultivar leaf-out time, orchard age and year all contributed significantly to explain deviations from model predictions. The negative relationship between year and accumulated degree days at the start of the flight period corroborates grower observations and concerns that R. completa is emerging earlier in the season than it was 18 years ago.

A series of laboratory studies were conducted to further examine the effects of incubation temperature, pre-chill and chill durations, latitude, cultivar and size on the post-diapause development of R. completa puparia. The thermal requirement and developmental thresholds for adult emergence were estimated. Patterns based on sex were also examined in relation to puparial weight and post-diapause development. Percent adult emergence declined at both higher and lower incubation temperatures. Chill and pre-chill duration affected the thermal requirement, survival and synchronization of emergence for R. completa, with climate change implications for the future. Latitude and plant host species also affect the thermal requirement. The effects of these environmental factors on the post-diapause development of R. completa are discussed in relation to observations from other Rhagoletis species.

Insect pests and natural enemies respond to land-use intensity at varying scales. As agricultural production has intensified on the local scale over the last century, landscapes have also become increasingly homogenized. The effects of land-use intensity at both landscape and local scales on pests, generalist predators and sorghum yield were studied in Uruguay. Greater landscape intensity increased densities of pest species and provided evidence that they may interact indirectly through apparent commensalism leading to a decrease in the abundance of the generalist coccinellid community. In contrast to many field studies that have evaluated effects of local intensity on organic agriculture, in this study a multi-year rotational system was compared to continuous agriculture. Crop-grazing rotational systems resulted in lower coccinellid species richness, winter crop cultivation caused a higher proportion of non-native coccinellid individuals, and insecticide-use increased pest density. The use of path analysis provided a robust approach to examine the effects of land-use intensification on insect communities in a lesser known multi-year management system and an under-studied geographic region.

Generalist predators are increasingly recognized as important contributors to the reliability of conservation biological control. In addition, indirect interactions between prey species sharing a common predator are increasingly recognized as drivers of community dynamics. Consideration of how indirect interactions between prey species affect biological control potential is critical. Cage experiments with Acyrthosiphon pisum and Aphis gossypii were performed with varying levels of predation by adult Hippodamia convergens as well as varying levels of habitat complexity. Across predation pressure the early growth of A. gossypii appears beneficial to the net change in abundance of A. pisum over the course of the experiment, however the early growth of A. pisum provides no explanatory power for the net change in abundance of A. gossypii. Both increasing predation pressure and decreasing habitat complexity appear to change the sign and strength of indirect interactions between A. gossypii and A. pisum as predicted by ecological theory.

The negative impacts of agricultural intensification are wide-reaching and insect communities respond to the changes in their environment at multiple levels. Sustainable pest control strategies must consider long-term data sets, whenever possible, take into account environmental, local and landscape variables and evaluate patterns in the context of community ecology.

Invasive species are recognized as one of the main drivers of global environmental change. The majority of invasive species escape from their coevolved natural enemies (predators, parasitoids and parasites), but in some cases can either act as a transport vector for coevolved parasites, or encounter biotic resistance from resident competitors or enemies, in a new region. When resident enemies are unable to suppress an invasive species, specialist natural enemies from the native range can be imported and implemented to reduce its abundance in a novel environment (classical biological control). As top-down effects of natural enemies can interact with bottom-up effects from host plants, it is of increasing interest to understand how multitrophic interactions influence the suppression of invasive species. The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), provides a unique opportunity to examine mechanisms for the suppression of an exotic species and in the context of novel multitrophic interactions. Native to Australia, E. postvittana is a highly polyphagous leafroller that was confirmed to be present in coastal California in 2006. For my dissertation research I have focused on several different aspects of the trophic interactions among E. postvittana and its natural enemies.

Firstly, I investigated the occurrence and fitness consequences of infanticide for Goniozus jacintae Farrugia, a coevolved gregarious larval ectoparasitoid of E. postvittana. Bethylid parasitoids, such as G. jacintae, have long been recognized for their aggressive adult behavior and their use of infanticide to kill the offspring of competing females. In this laboratory study I investigated the clutch size and handling time of G. jacintae, compared its life history performance between primary and secondary (laid after infanticide events) broods, and estimated lipid and protein concentrations in the hemolymph of non-parasitized and parasitized hosts. I found that secondary clutches were significantly larger than primary clutches in ovicide treatments and also experienced greater brood survivorship. Lipid concentrations were consistently higher in the hemolymph of parasitized hosts and protein concentrations were also higher until egg hatch when parasitoid larvae began to consume the resources available. This study was the first to provide evidence that improved nutritional quality could be an important benefit of infanticide for an insect parasitoid, allowing for larger clutch size and improved brood survivorship among secondary broods.

Subsequently, I discovered a microsporidian pathogen infecting field populations of E. postvittana in California and I investigated both its identity and pathogenicity. Using ultrastructure of different spore stages in the life cycle and phylogenetic analysis of sequences from selected genetic markers (ITS, SSU and RPB1), I was able to confirm that the microsporidian was a member of the Nosema fumiferanae species complex (N. fumiferanae postvittana subsp. n.). I was also able to confirm that this microsporidian can be vertically transmitted and that it has significant negative effects on the life-history performance of E. postvittana under laboratory conditions. To further explore the potential of this microsporidian infection to provide biotic resistance to the invasiveness of E. postvittana, I further examined its pathogenicity in the context of dose-response relationships and the latent period of infection in the laboratory, and quantified pathogen prevalence and intensity in the field using quantitative real-time PCR (qPCR) for five populations in the San Francisco Bay Area of California. In the laboratory, the median lethal dose (LD50) was estimated to be 1.8 x 104 spores, the mean latent period for infection with 103 spores was 12.67 days, and compared to healthy larvae, those infected with up to 105 spores showed a reduction in intrinsic rate of natural increase from 0.18 to 0.008. From the field sampling I detected N. fumiferanae postvittana in all five populations with an overall prevalence of 5% and a mean microsporidian intensity of 226 spores. Although the laboratory results demonstrated the potential for host suppression, the field sampling indicated that the prevalence and intensity were too low to account for the continued decline in population densities of E. postvittana in coastal California.

Finally, I investigated the role of multitrophic interactions among E. postvittana, its host plants, and its resident enemies in California. In a common garden experiment in the field, parasitism rates of egg masses and larvae by resident parasitoids in the field were found to vary among host plant species, with a higher probability of egg parasitism on taller plants and a higher probability of larval parasitism on shorter plants. In the laboratory, parasitoid search time for an egg mass varied among plant species, but longer search times did not necessarily correspond to lower rates of egg parasitism in the field. When controlling for plant species, the probability of a parasitoid contacting an egg mass decreased with increasing trichome density. I also found significant effects of plant diet on the fitness of both healthy and microsporidian-infected E. postvittana larvae under laboratory conditions, with evidence of synergistic effects between diet and infection for some host plant species. Overall, I demonstrated significant variation in the extent of enemy-free space for E. postvittana based on plant species, but not plant origin.

My dissertation highlights the importance of biotic resistance in buffering a resident community against an exotic invader and demonstrates that this resistance is often dependent on complex multitrophic interactions.

With the increase of globalization, the introduction of exotic species into new regions has become a worldwide threat for biodiversity and agricultural production. However, invasiveness of alien species depends on the extent to which abiotic and biotic factors affect the impact of exotic species in a new region. The Light Brown Apple Moth (LBAM), Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), is a leafroller native to southeastern Australia that was discovered in California in 2006. At the time of its discovery, little was known about how abiotic and biotic factors might limit its distribution and impact in California and North America. I therefore measured high and low temperature tolerance of LBAM in laboratory studies and found that it exhibited only moderate tolerance of extreme temperatures, which may limit its potential distribution in California and North America. I also monitored LBAM populations in the field and measured parasitism rates over a four year study period to determine the influence of of biotic resistance and to provide baseline population data for use in management decisions. I found that LBAM populations were cyclic or decreasing, had strongly overlapping generations and produced three generations annually in San Francisco and and four in Santa Cruz. The parasitoid assemblage of LBAM in California consisted of many species and parasitism rates were unusually high, but provided no evidence of an ability to regulate LBAM populations. I also found that low density LBAM populations did not exhibit demographic Allee effects, but instead populations of all sizes exhibited strong negative density dependence.

I also studied life history parameters of Meteorus ictericus, the most abundant parasitoid of LBAM in California, and found that it has a number of traits that could account for its dominance in the parasitoid assemblage. It was able to attack and develop in a wide range of host larval instars, had a preference for late larval instars, a female only lifestyle, a low generation time ratio in relation to LBAM, and similar temperature maxima and minima for development. However, M. ictericus exhibited an unusually low lifetime fecundity, which could pose an important constraint on its potential to suppress LBAM populations.

Overall, the results of these studies have provided valuable insights that can be used to better understand the potential geographic distribution of LBAM, and to better inform management decisions. In addition, I suggest that the high level of resistance from resident parasitoids on LBAM in California, in combination with other pest management strategies, could prevent the widespread losses from agricultural crops that were originally anticipated in the United States.