Laricobius nigrinus is an insect predator introduced into the eastern United States for the biological control of hemlock woolly adelgid, Adelges tsugae. Laricobius nigrinus dispersal characteristics and predation on A. tsugae were evaluated by monitoring its immature life stages that were distinguished from larvae of the native, L. rubidus using a portion of the cytochrome oxidase subunit one gene. Conventional polymerase chain reaction (PCR) followed by restriction fragment length polymorphism produced DNA fragment sizes that were unique to each species. Real-time PCR used a TaqMan assay with two species-specific nucleotide sequence probes labeled with fluorescence molecule VIC or FAM that indicated the DNA template was L. nigrinus or L. rubidus, respectively. Laricobius nigrinus beetles released in the lower hemlock crown (< 7 m) dispersed to the upper crown (>15 m) for oviposition. Four years post-release, its rate of spread into the surrounding forest was 75 m/yr and the number of L. nigrinus detected on hemlock trees was positively correlated with the density of A. tsugae, regardless of proximity to the release area. Adelges tsugae survival rate was lower on release trees than on geographically separated control trees for three years at a L. nigrinus release site in each Pennsylvania and North Carolina. Yet, the density of A. tsugae remained greater on release trees than control trees. In the eastern United States, the average density of A. tsugae was 3.8 times greater than the upper 95% confidence interval in the native range of L. nigrinus and A. tsugae, as observed in Seattle, WA. This contributed to the average ratio of predator to prey remaining 4.6 times lower than the 95% confidence interval observed in WA. Five to seven years post-release, hemlock health of release and geographically separated control trees declined at similar rates, though the average A. tsugae infestation level was 35% greater on release trees at the time of L. nigrinus release. The full efficacy of L. nigrinus predation was likely underestimated because this study assessed experimental release sites. Interactive roles of biotic and abiotic pressures on A. tsugae should continue to be evaluated at L. nigrinus introduction sites. / Ph. D.

The hemlock woolly adelgid (HWA), Adelges tsugae Annand (Homoptera: Adelgidae), is forest pest introduced to eastern North America in the early 1950's. Although this pest occurs on both landscape and nursery stock as well as in natural stands of hemlock forest, pesticides are only practical and effective in urban settings. Ecological and economical considerations prevent utilization of chemical treatment in the forest setting, thus biological control is viewed as the most promising option for slowing the spread of HWA. It is essential for researchers to be able to accurately assess the population levels of biocontrol agents after release into the environment. No method currently exists for sampling HWA predators. This project was designed to determine whether two species of predators are able to utilize olfactory cues from eastern hemlock and/or HWA in host-finding. If predators use olfactory cues, we may develop an attractive sythnetic blend of compounds to draw them to a trap, thereby simplifying the sampling and improving its accuracy. To address this question we executed three experiments. The first involved examination of the antennae of the predators for the presence and abundance of olfactory sensilla. The second experiment was designed to detect a behavioral response by the predators following exposure to host volatile compounds. The final experiment involved identifying compounds emitted from eastern hemlock, and the affect of HWA-feeding on volatile emissions. Laricobius nigrinus Fender (Coleoptera: Derodontidae) antennae are densely populated with sensilla, several of which are potentially olfactory in function. In addition, we observed a behavioral response to olfactory cues which included altered flight behavior. However, the behavior was not clearly attraction. Pseudoscymnus tsugae Sasaji and McClure (Coleoptera: Coccinellidae) has few sensilla on a very short antennae and only one type of sensilla possesses wall pores suggestive of an olfactory function. In addition, we did not observe a significant behavioral response to host-volatiles. It seems unlikely that this species uses olfaction in long-range host location. We identified 10 monoterpenes that were consistently expressed in the hemlock volatile profile and were unable to isolate volatile emissions from HWA. There is an increased monoterpene release rate from HWA-infested hemlock foliage as compared to uninfested foliage apparently driven indirectly by HWA through a reduction in new growth at branch tips. In addition there was a slight but statistically significant change in the percent composition of the individual compounds. We see potential in developing a more efficient sampling procedure for L. nigrinus through utilization of olfactory cues. More biological assays must be conducted to determine whether an attractive blend exists and electrophysiological assays are required to isolate physiologically active compounds. However, our data suggest that P. tsugae is not likely to be reliant on olfaction in long-range host location. / Master of Science

The insecticide imidacloprid, has been found to be highly effective in suppressing hemlock woolly adelgid, Adelges tsugae. Laricobius nigrinus is a predatory beetle released as a biological control of adelgids in the eastern U.S. This project was designed to develop a pest management strategy that utilizes both tactics concurrently within the same site. It will assess the efficiency of this strategy in reducing HWA populations and improving the health of hemlock forests. The project was started in 2010 and data were collected annually through 2016. The project spanned three sites in three different states (KY, WV, and TN). Results show that tree health has generally declined across all sites for each year. HWA population index values are highly variable and are more strongly influenced by the occurrence of low winter temperatures than by treatment effect. Cross-correlation analysis of tree health and HWA population, revealed characteristics of their temporal relationship. In two of the three sites, tree health lagged up to three years behind changes in HWA population, and HWA populations lagged approximately one year behind changes in tree health. L. nigrinus did not establish at any site as of 2016. The lack of sustained recovery of the beetle may be attributable to the occurrence of extremely cold temperatures during the winters of 2014 and 2015 which produced subsequent crashes in the HWA population at two of the three sites. In TN, the L. nigrinus population may have never established due to a decline in the HWA population shortly after release. / Master of Science in Life Sciences / The insecticide imidacloprid, has been found to be highly effective in suppressing hemlock woolly adelgid, <i>Adelges tsugae. Laricobius nigrinus</i> is a predatory beetle released as a biological control of adelgids in the eastern U.S. This project was designed to develop a pest management strategy that utilizes both tactics concurrently within the same site. It will assess the efficiency of this strategy in reducing HWA populations and improving the health of hemlock forests. The project was started in 2010 and data were collected annually through 2016. The project spanned three sites in three different states (KY, WV, and TN). Results show that tree health has generally declined across all sites for each year. HWA population index values are highly variable and are more strongly influenced by the occurrence of low winter temperatures than by treatment effect. Cross-correlation analysis of tree health and HWA population, revealed characteristics of their temporal relationship. In two of the three sites, tree health lagged up to three years behind changes in HWA population, and HWA populations lagged approximately one year behind changes in tree health. <i>L. nigrinus</i> did not establish at any site as of 2016. The lack of sustained recovery of the beetle may be attributable to the occurrence of extremely cold temperatures during the winters of 2014 and 2015 which produced subsequent crashes in the HWA population at two of the three sites. In TN, the <i>L. nigrinus</i> population may have never established due to a decline in the HWA population shortly after release.

Insects form close partnerships, or symbioses, with bacteria. These partnerships allow the insects to use resources that would be unavailable to them otherwise. Certain insects, hemipterans, are able to feed on nutrient-poor plant-sap because these bacteria supplement their diets. While this association is beneficial for both the insect and bacteria, it also comes with consequences: the genomes of bacterial symbionts typically undergo extreme degradation, becoming small and lacking many genes necessary for typical bacterial functioning. In the Hemiptera, aphids, mealybugs, cicadas, true bugs,and their relatives, these bacterial partnerships tend to be stable over millions of years. However, adelgids (Aphidoidea: Adelgidae) are highly unusual in that their symbiotic bacteria have been frequently replaced. These replacements offer a unique opportunity to explore the effects of symbiont role and age on symbiont genome degradation. My dissertation uses the pattern of adelgid symbiont gains and losses to understand the process of symbiont replacements and co-symbiont gain. I accomplished this by sequencing and annotating the genomes of adelgid symbionts from across the family, first focusing on the symbionts from a pest species, then expanding to representatives from across the family, and finally conducting an in-depth exploration of how the genomes of a symbiont found in two branches of the adelgids varies between species. Through this work I demonstrate that adelgid symbionts are nutritional providers, they have a unique pattern for distributing the work of providing nutrients between the symbiont pairs, and that a symbiont’s precedence, whether it was there first or whether it joined another symbiont, has an impact on genome degradation.

Eastern hemlock (Tsuga canadensis) is in decline because of infestation by the invasive hemlock woolly adelgid (Adelges tsugae; ‘HWA’) and, to a lesser extent, the elongate hemlock scale (Fiorinia externa; ‘EHS’). Many conifers respond to insect herbivory by inducing oleoresin-based defenses, however it is unknown whether eastern hemlock is capable of this inducible response. We conducted a plantation setting study of artificially infested saplings to determine if feeding by HWA or EHS induces changes in the tree’s volatile chemistry. The induced changes in volatiles we found were unlike the terpenoid-based defenses of related conifers. Only HWA feeding elevated methyl salicylate, a plant signal for systemic acquired resistance, and benzyl alcohol, a known antimicrobial and aphid deterrent. The influence of environmental conditions and tree life-stage on hemlock volatile chemistry, potentially important factors for wild hemlock populations, is unknown. We investigated whether mature and immature forest trees respond to HWA infestation with the same patterns of volatile production as plantation saplings in full sun and amended soils. HWA induced volatile changes comparable to those of plantation saplings, with many-fold benzenoid increases and no terpenoid-based resinosis. Nearly all volatiles were substantially more abundant in forest than plantation trees, suggesting the effect of site conditions should be addressed in investigations of mechanisms operative in HWA-resistant biotypes of eastern hemlock. Our findings represent the first important step toward understanding the influence of inducible phytochemical responses on hemlock susceptibility to exotic pests, and highlight the possibility of salicylic acid-dependent biosynthetic activity in this gymnosperm system.

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