Hemlock woolly adelgid (Adelges tsugae Annand) is causing defoliation and mortality of eastern hemlock (Tsuga canadensis (L.) Carrière) in the eastern United States. The objectives of this study were to quantify changes in tree-ring width and wood anatomy for trees that survived adelgid infestation, and to contrast dendroclimatic relationships across a latitudinal gradient. Six sites spanning the current range of hemlock woolly adelgid (HWA) infestation were selected. At each site, 23 infested eastern hemlocks were cored and two trees were felled at the Virginia site and thin-sectioned using a sliding microtome for analysis of wood anatomy. Tree cores were cross-dated and ring widths were measured. For each site, t-tests were used to determine if there was a difference in radial growth pre- and post-HWA arrival. To compare differences in pre- and post-HWA cell properties, t-tests were used. For dendroclimatic analysis, Pearson correlation coefficients were calculated between radial growth and monthly climate variables. Three sites showed significant suppression in radial growth after HWA arrival and latewood produced post- HWA arrival had significantly smaller cells with reduced cell-wall thickness than latewood produced before HWA arrival. This indicates that HWA can reduce a tree's photosynthate production. The relationship between hemlock growth and climate also varied with latitude and site, with trees growing further south or on shallower soils being more sensitive to moisture levels. This sensitivity to drought can also partially explain the variation in hemlock response to adelgid feeding, as trees affected by moisture stress tend to be more sensitive to insect attack. / Master of Science

Tsuga canadensis (L.) Carr. (eastern hemlock), a long lived and shade tolerant coniferous tree species native to eastern North America, is currently threatened by the hemlock woolly adelgid (Adelges tsugae Annand, HWA). This exotic, invasive insect poses a serious threat to T. canadensis stands throughout their native range. The loss of this unique tree species is often coupled with numerous ecological consequences. HWA-induced mortality has exceeded 95% in some forest types, and will likely continue to decimate T. canadensis populations in the coming years. Shortly after HWA was found in Great Smoky Mountains National Park (GSMNP), long-term monitoring plots were established across a gradient of T. canadensis associated forest communities to monitor HWA impacts and stand decline. Our objectives were to (1) determine how T. canadensis populations have changed in conjunction with HWA infestation, and (2) document how HWA has affected vegetative community dynamics following five to six years of infestation in GSMNP. We assessed T. canadensis associated overstory communities and determined that crown density, top dieback, and T. canadensis stem density has significantly declined over the observational period. Furthermore, we found that midstory T. canadensis trees were experiencing greater rates of decline than those of the overstory. Even with these losses, overstory and midstory community composition did not significantly change from 2003 to 2008/09. Many studies have documented rapid pulses of understory vegetative productivity in conjunction with T. canadensis defoliation, which often allows increased amounts of solar radiation to reach the forest floor. Although we observed significant T. canadensis mortality at most plots sampled, understory species composition did not significantly change following HWA infestation. We attribute this lack of species response to the overwhelming abundance of Rhododendron maximum in the mid/understory, which restricts increased light from reaching the forest floor despite the formation of small overstory gaps. However, we found that understory composition significantly varied among ecological groupings, and differences were largely associated with soil properties and elevation. Overall, results of this project suggest that T. canadensis may largely disappear from southern Appalachian forests resulting from infestations of HWA. Although community composition of T. canadensis associated stands has not yet been altered, changes are likely to occur as duration of infestation progresses.

The hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Sternorrhyncha: Adelgidae) is a small invasive insect that frequently causes hemlock (Tsugae spp.) mortality in the eastern United States. Studies have shown that once healthy hemlocks become infested by the adelgid, nutrients are depleted from the tree, leading to both tree decline and a reduction of the adelgid population. Since A. tsugae is dependent on hemlock for nutrients, feeding on trees in poor health may affect the insect's ability to obtain necessary nutrients and consequently affect their population and physiological health. A cluster analysis, based on quantitative and qualitative tree health measurements, grouped sample trees into categories of lightly and moderately impacted trees. The A. tsugae population health on each tree was determined by measuring insect density, survival from aestivation, and peak fecundity. A. tsugae physiological health was determined similarly by measuring insect biomass, total carbon, carbohydrate, total nitrogen, and amino nitrogen. A. tsugae from moderately impacted trees exhibited significantly greater fecundity; however, A. tsugae from lightly impacted hemlocks contained significantly greater levels of carbohydrates, total nitrogen, and amino nitrogen. All A. tsugae physiological parameters increased significantly over time as the insects matured and reproduced regardless of tree health classification. While the results of the physiological analysis generally support our hypothesis that A. tsugae on lightly impacted trees are healthier than those on moderately impacted trees, this was not reflected in the population fitness measurements of the insects. Further examination of A. tsugae egg health may elucidate this apparent contradiction. / Master of Science in Life Sciences

The hemlock woolly adelgid (HWA), Adelges tsugae Annand, (Hemiptera: Adelgidae) is an invasive species in the eastern United States and Canada. This scale-like insect feeds on the xylem ray parenchyma cells of eastern hemlock, Tsuga canadensis (L.) Carrière, (Pinales: Pinaceae) and Carolina hemlock, Tsuga caroliniana Englem., resulting in multiple physical and physiological symptoms, all of which commonly leads to tree mortality within 4 to 10 years. Currently, HWA has spread throughout most of the eastern hemlock range and all of the range of Carolina hemlock and threatens the forest ecosystems they serve. In its introduced range, there are no natural enemies specialized to feed on HWA and suppress HWA populations below damaging levels. In the urban environment and at some public locations, the use of chemicals, such as imidacloprid, have been used to temporarily protect hemlocks, however; this does not provide a permanent solution. In the 1990's a classical biological control program for HWA was created with the goal to suppress HWA populations below damaging levels to reduce tree mortality, and to reduce the negative effects associated with the loss of either hemlock species. Presently, four predators have become the main focus of this program: Laricobius nigrinus Fender, Laricobius osakensis Shiyake and Montgomery (Coleoptera: Derodontidae), Leucotaraxis argenticollis (Zetterstedt), and Leucotaraxis piniperda (Malloch) (Diptera: Chamaemyiidae). Throughout the eastern US, La. nigrinus has established populations and expanded its distribution beyond where it has been released. Although it has successfully established and continues to spread, its effect on eastern hemlock health has been unknown. A 1-year study, carried out in 2021, revealed that La. nigrinus predation on the sistens generation not only reduced the sistens generation, but also had a similar net photosynthetic rate to the negative control on 1-2 year old shoot growth. In addition, significantly more shoots were produced for treatment branches with the highest La. nigrinus density. This also led to a significantly reduced aestivating sistens density compared to the no predator treatment. However, predation was insufficient to reduce HWA populations below the suggested damaging threshold of eastern hemlock (< 4 HWA/cm), suggesting that additional predators would be needed. Recently, the western strain of Le. argenticollis had been released in the eastern US. However, its phenology in the eastern US was unknown. A 2-year study, from 2021-2023, revealed that Le. argenticollis eggs and larvae were present when both generations of HWA adults with eggs and HWA nymphs were present. In addition, Le. argenticollis was capable of completing development in NY and VA, showing promise that this species could establish populations in the eastern US. Timing the adult fly releases into cages at the time when sistens were producing eggs resulted in Le. argenticollis phenology to synchronize well with HWA phenology at both locations, thus providing additional support for its release in the eastern US. Another 2-year study conducted in 2021-2022, at five locations (MD, NC, PA, TN, and VA), sought to determine how the predation of La. nigrinus and Le. piniperda, alone and together, would impact the sistens and progrediens generations, as well as how their predation would affect the aestivating sistens density and new shoot growth production. From this study, La. osakensis was found as the dominant Laricobius species present at the TN site, and similarly to La. nigrinus, reduced intact ovisac density in. This provided a small preview in La. osakensis capabilities and how this predator may be impacting HWA populations at locations where it has established. In order to assess Le. piniperda's impact on the progrediens generation, mesh cages were applied to treatment branches at each site. The study found that Le. piniperda could reproduce at all field sites and that the percentage of disturbed progrediens was significantly higher for treatments with Le. piniperda present at all field sites, except at PA in 2022. However, the affect of both of these predators on the aestivating sistens density and eastern hemlock health was inconclusive. In addition, the percentage of new shoot growth was highly variable among treatments at all field sites, and there was no treatment effect on the mean length of new shoot growth produced at all field sites. The results of this study suggests that in order to fully understand the affects of these predators on HWA populations and on hemlock tree health, longer term studies should be conducted. However, this study does provide some insight on the capabilities of these predators in the short term and supports the continued release and redistribution of these predators. / Doctor of Philosophy / The hemlock woolly adelgid (HWA), is an invasive species in the eastern United States and in Canada. This small scale-like insect was introduced likely on nursery stock material from southern Japan and was first discovered in the eastern US in 1951 in Richmond, Virginia. Two generations of HWA occur each year known as the sistens and progrediens; both reproduce without males. This insect feeds on eastern hemlock and Carolina hemlock, and since its discovery has spread through most of their range. While this insect feeds, it weakens the tree, eventually causing tree death. In eastern North America, eastern hemlock is one of the major tree species in the forest and provides a unique habitat for various mammals, birds, fish, and invertebrate species. To protect this habitat, multiple management strategies have been developed. The two main management strategies that are in use are the use of pesticides and the release of predators of HWA. Pesticides have been useful in the urban environment and in parks and forests, but they only last for a short time and can lead to negative affects to the environment. Since the 1990's, the release of insect predators originally found in the native range of HWA, has occurred with the goal of predator establishment, to lower HWA populations, and to lower hemlock tree death in the eastern US. Four small insect predators have been the main focus of this program: Laricobius nigrinus, Laricobius osakensis, Leucotaraxis argenticollis, and Leucotaraxis piniperda. So far La. nigrinus and La. osakensis have populations at multiple locations throughout the eastern US, and a low number of sites have been recently found for Le. argenticollis and Le. piniperda. Past studies have indicated that La. nigrinus has a significant impact to the sistens generation of HWA, but its affect to eastern hemlock health has been unclear. A 1-yr study conducted in VA in 2021, released La. nigrinus adults into mesh cages, with two different population sizes of beetles, to determine if La. nigrinus predation on HWA populations lead to an indirect effect on eastern hemlock health at the branch level. Results confirmed that La. nigrinus had a significant impact to the sistens generation of HWA. Photosynthesis of branches with La. nigrinus, in June, was similar to the photosynthesis of the negative control branches, which started with a lower number of sistens present on the branch at the beginning of the study. Suggesting that branches with La. nigrinus may be healthier compared to branches without La. nigrinus. Laricobius nigrinus predation also significantly reduced the number of aestivating sistens on new shoot growth, but not below damaging levels (< 4 HWA/cm). This suggested that additional predators may be needed to target the progrediens generation of HWA so that HWA populations do not return to damaging levels after La. nigrinus predation. To learn more about the potential for Le. argenticollis to survive and reproduce in the eastern US, a 2-yr study was conducted in 2021 – 2023, to determine when different life stages of Le. argenticollis could occur in NY and VA. Results revealed that Le. argenticollis was able to reproduce in the mesh cages and complete its life cycle. In addition, Le. argenticollis eggs and larvae were present at the time when HWA adults were producing eggs and when HWA nymphs were present for both generations of HWA. These results provide insight on Le. argenticollis life cycle and shows that Le. argenticollis has a good chance in surviving throughout the eastern US. A 2-yr study at five locations in the eastern US (MD, NC, PA, TN, and VA), tried to combine two predators, La. nigrinus and Le. piniperda, to determine if their combined impact would be sufficient to reduce HWA populations and affect eastern hemlock tree health. Three assessments were conducted each year to determine the impacts of La. nigrinus and Le. piniperda on HWA populations and on eastern hemlock shoot growth production as a representation of tree health. Results confirmed that La. nigrinus significantly reduced the sistens generation at all locations, except at TN. At the TN site, La. osakensis was found to be the main predator of the sistens generation and had a similar affect on the sistens generation as La. nigrinus. Leucotaraxis piniperda was able to reproduce at all locations and also had a significant affect on progrediens ovisacs at four of the locations in 2021 (MD, NC, PA, and VA) and at three of the locations in 2022 (MD, NC, and VA). However, the results on the number of aestivating sistens on new shoot growth and eastern hemlock health were inconclusive. Therefore, it is unclear as to how the presence of both of these predators' affect eastern hemlock health. A long term study may be needed in order to understand the relationship between these predators and their indirect affect to eastern hemlock health.

Prey-mediated effects of imidacloprid were evaluated for Laricobius nigrinus Fender and Sasajiscymnus tsugae Sasaji and McClure after feeding on hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). Two methods were evaluated for detecting imidacloprid in hemlock tissues: a commercially available enzyme linked immunoassay (ELISA) kit and a high performance thin-layer chromatography technique for detecting and quantifying imidacloprid residues in hemlock wood and needle tissues. ELISA is advantageous because of its cost, sensitivity, and ease of use. However, matrix effects in the form of false positives and overestimated imidacloprid concentrations were evident in hemlock wood and needle tissue extracts. Matrix effects could be reduced by dilution with water, effectively raising the lower detection range of the kit from 0.2 to 200 ppb. High performance thin-layer chromatography was accurate, quick, easy to use, and matrix effects were not evident. However, the technique is sensitive in the lower ppm range and tissue samples from field-treated hemlocks are often in the ppb range, making this technique less desirable than more sensitive analytical methods. Lethal and sublethal effects on both predators were evident after eastern hemlock branches infested with HWA were spiked with imidacloprid in the laboratory. HWA mortality increased with dosage and time, and its 30 d LC50 was determined to be 242 ppb. Both predator species exhibited reduced survivorship and fitness parameters after feeding on HWA from the treated branches. In a topical application bio-assay, 6 d imidacloprid LD50 values for L. nigrinus and S. tsugae were 2.43 and 1.82 µg/g, respectively. Imidacloprid and its major metabolites in hemlock tissues were analyzed by liquid chromatography-tandem mass spectrometry. Imidacloprid recovery from beetle cadavers was correlated with beetle mortality from feeding on treated hemlock branches. Olefin was the primary imidacloprid metabolite recovered from hemlock wood tissues. When predators fed on HWA from field-treated trees, impacts on survivorship and fitness were variable. In 2005, significantly higher proportions of both species of beetles were affected by feeding on control branches compared with treated branches. In 2006, beetles feeding on HWA from some of the trees treated in the field exhibited longer fliptimes compared with beetles feeding on controls, although beetle mortality was not significant among treatements. In the field, imidacloprid controlled HWA populations 1-3 years post-treatment. Hemlock health improved in the highest dosage group, with significantly greater lengths of new shoots compared with shoots from control trees. Eastern hemlock trees primarily metabolized imidacloprid into the olefin metabolite, which can have increased insecticidal toxicity compared with imidacloprid. Imidacloprid was detected in beetle cadavers after feeding on HWA from treated branches, suggesting that prey-mediated impacts of systemic imidacloprid are possible on nontarget predators. However, because of HWA's sensitivity to imidacloprid, in field situations predators are more likely to be affected by reduced adelgid density and quality. / Ph. D.

The biology of Laricobius nigrinus Fender (Coleoptera: Derodontidae) and its potential for control of hemlock woolly adelgid (HWA), Adelges tsugae Annand (Homoptera: Adelgidae) in the eastern United States were investigated. Laricobius nigrinus completed development on HWA in laboratory studies. There are four larval instars. Mean larval consumption was 225.9 and 252.3 HWA eggs at 12 and 18 °C, respectively. Post-aestivation activity period was 36.6 and 30.8 weeks for males and females at 13 °C, respectively. Mean lifetime fecundity was 100.8 eggs over a mean ovipositional period of 13.2 weeks. Laricobius nigrinus is host specific. In paired-choice and no-choice oviposition tests, L. nigrinus laid more eggs in HWA ovisacs than other test species. Laricobius nigrinus consumed more eggs of HWA than eggs of Adelges piceae (Ratzeburg) and Pineus strobi (Hartig) but not of Adelges abietis (L.). In larval development tests, L. nigrinus only completed development on HWA. A two-year field study conducted in British Columbia showed that: 1) the life cycle of HWA in British Columbia is similar to that previously reported in Virginia and Connecticut; 2) L. nigrinus adults undergo an aestival diapause that coincides with the diapause of the first instar HWA sistens. Adult activity beginning in autumn coincides with resumption of development of the sistens generation; 3) oviposition and subsequent larval development of L. nigrinus coincide with oviposition by HWA sistens adults. Temperature-dependent development was determined for egg, larval, pre-pupal and pupal stages of L. nigrinus at five constant temperatures (9, 12, 15, 18 and 21 °C). Development time was inversely proportional to temperature between 9 and 18 °C. Laricobius nigrinus did not complete development at 21 °C. Minimum developmental temperatures for eggs (6.5 °C), larvae (5.1 °C), pre-pupae (3.8 °C), and pupae (3.1 °C) were determined by extrapolation of linear regression equations to the x-intercept. Degree-days calculated for eggs, larvae, pre-pupae, pupae, and egg to adult were 54.6, 161.3, 196.1, 212.8, and 666.7, respectively. The laboratory derived degree-day model for predicting egg hatch was validated with field data collected in British Columbia. Observed median egg hatch at three of four sites over two years was within 15% of the predicted DD value (54.6). / Ph. D.

The predatory beetle, Laricobius nigrinus Fender (Coleoptera: Derodontidae), has been released in the eastern U.S. since 2003 for the management of hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). The establishment of L. nigrinus at release sites in Virginia was assessed in 2017 and 2018. Sampling was performed in both years to determine presence or absence L. nigrinus. Stand level HWA densities, tree health, predator-prey ratios, and Laricobius spp. identifications were also assessed at each site. Laricobius nigrinus established at 82% of sites and was the primary species recovered. HWA densities appeared to decline, and tree health appeared to improve in year two. Predator-prey ratios were lower than those indicated in the native range, however much is left to be understood about the dynamics of this system in its introduced range. A separate two-year study to assess the impact of L. nigrinus on HWA was initiated in 2014 (Phase One) at nine sites in the eastern U.S. Significant predation of HWA sistens ovisacs was demonstrated during this period, therefore, it was continued from 2016-2018 (Phase Two) to provide longer-term evaluations. Predator exclusion cages were used to monitor predator and prey populations. In Phase Two of the study, mean ovisac disturbance rates on no-cage branches were significantly greater than caged branches and were as high as 80%, suggesting that L. nigrinus can have a significant impact on the sistens generation. Microsatellite analysis of Laricobius larvae indicated that L. nigrinus was the primary species recovered at study sites. / Master of Science in Life Sciences / Hemlock woolly adelgid (HWA), an invasive insect native to Asia and western North America, is a significant threat to two native hemlock species in the eastern U.S. Since 2003, a predatory beetle, Laricobius nigrinus, has been released for management of HWA. In biological control programs such as this one, it is important to evaluate the ability of predators to establish and impact prey in areas where they are introduced, as this helps guide future management decisions. As such, a study was conducted to examine the ability of L. nigrinus to establish after being released at 26 locations in Virginia. In those investigations we found that L. nigrinus established at 82% of the sites. Although promising, longer-term studies are needed to understand if the predation of HWA by L. nigrinus helps improve the health of our native hemlocks. A separate two-phase study was carried out from 2014-2016 and 2016-2018 evaluating the impact of L. nigrinus on HWA at nine release sites in the eastern U.S. In Phase Two reported here, cages were used to exclude L. nigrinus on some HWA infested branches while on others, no cage was applied to allow free access to HWA. Comparisons between branches were made to determine the level of predation by L. nigrinus. These studies showed a significant impact by L. nigrinus on the winter generation of HWA with as many as 80% of those insects being attacked on study branches which indicates that this species has potential as an effective predator.

The hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae), is an invasive pest from Japan that is causing significant hemlock mortality in the eastern U.S. The most promising control method is biological control. Beetles in the genus Laricobius are adelgid specialists. In 2005, Laricobius osakensis, was discovered in Japan and in 2010 was approved for release in the eastern United States and there have now been more than 60 releases. In 2014, the polar vortex resulted in significant HWA mortality in the eastern U.S., depleting the food source for L. osakensis, which resulted in low field recoveries of them. In the 2015-2016 field season, there were 14 larvae recovered from the field, and the 2016-2017 field season yielded 90 larvae. A significant correlation was found between Laricobius beetles recovered and HWA density, between HWA density and plant hardiness zone, and a negative correlation between Laricobius beetles recovered and average tree health. Understanding more about the supercooling point of L. osakensis gave insight to its ability to survive subfreezing temperatures as occurred in 2014. Comparison of the supercooling point between the northern and southern populations of L. osakensis, and to the other released HWA predator, L. nigrinus, will allow us to determine which biological control agent is best suited for release in the coldest regions. The overall mean supercooling points of northern L. osakensis was -13.52 oC, southern L. osakensis was -13.42 oC, and L. nigrinus was -13.57 oC. There were no significant differences between species or populations. / Master of Science in Life Sciences

Management of hemlock woolly adelgid (HWA), Adelges tsugae (Hemiptera: Adelgidae), is currently being implemented through several different methods including the release of host-specific predators such as Laricobius nigrinus Fender (Coleoptera: Derodontidae). Releases of this predator began in 2003. In 2014, an assessment of the efficacy of L. nigrinus at release sites from New Jersey to Georgia. Field sites were chosen based on the presence of moderate to high densities of HWA, that L. nigrinus was released at least four years prior to the start of the study, and that the predator was determined to be established at that site. Three treatments were set up at each of the sites: no cage, closed exclusion cage, and open cage. Three assessments were taken during key points throughout the season in order to monitor both HWA and L. nigrinus populations. Larval predator were recovered from most of the sites in year one and in higher numbers in year two. Many sites at which L. nigrinus were recovered showed high predation rates of HWA in uncaged samples ranging from 1.5 to 47.3% in year one and 0 to 66% in year 2. A survey for L. nigrinus establishment at previous release sites in VA was conducted. These sites date back as far as 2003 and as recently as 2015. Beat sheeting and branch clippings were conducted to recover Laricobius spp. adults and larvae, respectively. The recovered insects were then identified to species through genetic analysis. A mix of the introduced L. nigrinus and the native L. rubidus LeConte (Coleoptera: Derodontidae) were recovered at some sites, and only L. rubidus were recovered from others. Higher numbers of Laricobius were recovered in spring of year two. Overall, tree health in Virginia decreased from spring 2015 to 2016. / Master of Science in Life Sciences

The hemlock woolly adelgid, an invasive non-native insect, is threatening eastern hemlock in Kentucky. This study examined three techniques to map the distribution of eastern hemlock using decision trees, remote sensing, and species distribution modeling. Accuracy assessments showed that eastern hemlock was best modeled using a decision tree without incorporating satellite radiance. Using the distribution from the optimal model, risk maps for susceptibility to hemlock woolly adelgid infestation were created using two species distribution models. Environmental variables related to dispersal were used to build the models and their contributions to the models assessed. The models showed similar spatial distributions of eastern hemlock at high risk of infestation.