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 / The hemlock woolly adelgid (HWA) is an invasive insect species from Asia that is the most important pest of eastern and Carolina hemlock trees in the eastern U. S. Laricobius osakensis is a small beetle from Japan that feeds only on HWA, and been released since 2012 in the eastern United States to control HWA populations. It is important to determine if L. osakensis populations were able to survive and spread in the eastern United States, and if the predator has an effect on HWA populations and the health of hemlock trees. In 2014, extreme cold temperatures in Virginia resulted in wide-scale death of HWA populations, depleting the food source for L. osakensis. Consequently, only 17 L. osakensis beetles were found on hemlock trees near release sites; however, the following year, 147 beetles were found. The ability to survive extreme cold temperatures is important for selecting a biological control agent for release in such regions. We can find out information about how tolerant a species is to cold temperatures by determining the supercooling point, the temperature at which it cannot stop itself from freezing. In this study we compared the supercooling points of a northern and southern population of L. osakensis, as well as another previously released and established biological control agent of HWA, Laricobius nigrinus. I found that there was no significant difference in supercooling point between the different types of Laricobius beetles suggesting that none of these species or populations appears to be anymore cold tolerant than the other for release in the colder regions of the U. S.

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 / <i>Laricobius nigrinus</i> is a beetle predator of hemlock woolly adelgid (HWA), an aphid like insect covered by wool. HWA is a serious pest of both eastern and Carolina hemlock, two important tree species found in Appalachia which provide species diversity and habitat to numerous animal and plant species. Damage to hemlock trees include death, dieback, and disease. We began releasing <i>L. nigrinus</i> in 2003 in the eastern United States to manage HWA. There are now over 900 documented releases of <i>L. nigrinus</i> from Maine to Georgia. Nine field sites were set up to assess the impact <i>L. nigrinus</i> is having on HWA populations throughout the geographic range of its releases. At many of these sites we found that <i>L. nigrinus</i> adults and larvae are significantly reducing HWA populations. A large proportion of the predators of HWA recovered at these sites are <i>L. nigrinus</i>, which shows that this species is primarily responsible for the observed predation. The second study conducted assessed for establishment of <i>L. nigrinus</i> at release sites around Virginia. <i>Laricobius nigrinus</i> was found at four of the fourteen sites sampled. Continued sampling is necessary to get a more accurate assessment of establishment since many of the sites were recovering from low HWA populations resulting from extreme cold temperatures in 2014 and 2015.

Eastern and Carolina hemlock in the eastern United States are experiencing high mortality due to the invasive non-native hemlock woolly adelgid (HWA). The most promising means of control of HWA is the importation of natural enemies from the native range of HWA for classical biological control. Prior to release, natural enemies must be tested for suitability as a control agent, including the ability to locate the target prey. Coleopteran predators, including Scymnus coniferarum and Laricobius osakensis are under consideration as a means of biological control of HWA. Laricobius nigrinus was released in hemlock forests in 2003. It was recently discovered to hybridize with the native Laricobius rubidus. Behavioral responses of these predators to HWA and host tree foliage were observed using a 4-chambered olfactometer, and genetic analysis was used to differentiate responses of L. nigrinus, L. rubidus, and hybrids. In the olfactometer, insects are allowed to amble about the arena and respond to volatile cues from each treatment. Host foliage with and without HWA was tested, as were various comparisons of eastern versus western foliage, host versus non-host foliage, and foliage containing HWA and a congeneric feeding beetle. Olfactometer bioassays demonstrated that foliage from hosts where prey is commonly found is preferable to foliage where prey is seldom found, and that the presence of HWA-induced volatile cues is the strongest driver of behavior, and trumps the presence of a competitor. There is evidence in the study that supports the reliability-detectability phenomenon common in parasitoid biological control agents. Hybrid individuals were found to behave similarly to released L. nigrinus, although in some cases intermediate behavioral traits were evident, with respect to the parental species. This study and others support the continued need for strict testing of potential biological control agents prior to release, as well as a strong impetus for the inclusion and implementation of genetic analysis as a standard component of agent evaluation.

Hemlock woolly adelgid, Adelges tsugae, native to Asia and the Pacific Northwest of North America (Pacific Northwest), has devastated eastern hemlock (Tsuga canadensis) in a major portion of its range in the eastern U.S. After many years and much effort directed towards rearing and releasing biological control agents to manage HWA, one of these agents, Laricobius nigrinus, native to the Pacific Northwest, is now well-established in sites from the southern to the mid-Atlantic states of the eastern U.S. However, there have yet to be studies of its efficacy in lowering A. tsugae densities, and there has been no noticeable drop in A. tsugae densities. Population models for A. tsugae have suggested that even upwards of 90% predation on eggs laid by the overwintering generation will have minimal effect in reducing the population densities of A. tsugae, if A. tsugae are at high density, due to compensatory density-dependent survival in the progrediens generation. Additionally, no studies showing insect predators are indeed what regulate A. tsugae in its native range exist. We established predator exclusion experiments, and recorded A. tsugae densities, mortality factors, and fecundity for multiple generations in both the native and invaded ranges. In the invaded range, we studied A. tsugae populations in sites with well-established populations of L. nigrinus to test its efficacy in reducing A. tsugae and tested model predictions of minimal difference in A. tsugae densities between treatments with and without predators. In the Pacific Northwest we tested the effect of insect predators and tree species, western (Tsuga heterophylla) and eastern hemlock, on populations of A. tsugae. In the invaded range we found that L. nigrinus predation was significantly higher in unbagged branches, however, model predictions were validated, and there was no effect of predation by treatment on the A. tsugae summer generation. In our plots in the Pacific Northwest we found that tree effects were not significant, but that summer-active predators were significantly lowering levels A. tsugae densities on unbagged branches. Our study demonstrates the importance of summer-active predators in reducing A. tsugae and suggest that summer- and winter-active predators are needed to suppress A. tsugae to innocuous densities.

Competitive interactions among two specialist predators, <i>Laricobius nigrinus</i> Fender (Coleoptera: Derodontidae) and <i>Sasajiscymnus tsugae</i> Sasaji and McClure (Coleoptera: Coccinellidae), and a generalist predator, <i>Harmonia axyridis</i> Pallas (Coleoptera: Coccinellidae), of hemlock woolly adelgid were evaluated using laboratory, field and video studies. The two specialist predators are part of a biological control program for <i>A. tsugae</i>, and the potential for competition among these species and previously established generalist predators is unknown. In laboratory studies of predator groups in Petri dish assays, the only significant negative effects from competition occurred among conspecifics, resulting in reduced net egg production by <i>L. nigrinus</i> and <i>H. axyridis</i> and reduced feeding by <i>H. axyridis</i>. In contrast, heterospecific combinations showed non-interference. In longer duration field studies of predator groups, held in branch enclosures, predator survival and feeding were not significantly affected by additional predators. Net reproduction was again significantly reduced by conspecifics, while heterospecifics showed non-interference for all predator responses. All predators reduced the number of <i>A. tsugae</i> nymphs of the next generation relative to no-predator controls; however, <i>L. nigrinus</i> had much greater impact overall due to the large number of progeny produced. Video studies revealed that predator behavior varied qualitatively and quantitatively by species, and did not appear to be coordinated temporally or spatially. All species exhibited continuous activity patterns that were punctuated by longer periods of rest. The specialist predators were more selective of feeding and oviposition sites, and rested at more concealed locations than <i>H. axyridis</i>. Conspecifics significantly altered the time allocated to specific behaviors for <i>L. nigrinus</i> and <i>H. axyridis</i>, resulting in reduced predator effectiveness due to increased searching and decreased feeding and oviposition. All predator groups maintained a high degree of spatial separation relative to assay size, suggesting that chemical or tactile cues may be used to regulate their distributions. Overall, these studies suggest that the three predator species will be compatible in this system. Management implications include using multiple-predator species combinations over single-species for biological control of <i>A. tsugae</i> and implementing low-density releases to reduce the potential negative effects of intraspecific competition. / Ph. D.

Hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae), is an introduced pest injurious to hemlock trees (Tsuga spp.) in the eastern United States. HWA currently infests hemlock in over 50% of its geographic range and has the potential to spread throughout its entire range. Since HWA populations in the eastern United States are not regulated effectively by natural enemies (Wallace and Hain 2000), classical biological control is the most promising option for controlling this pest in the forest setting. This work evaluates Laricobius nigrinus Fender (Coleoptera: Derodontidae), a predator associated with HWA in western North America, is being evaluated as a potential biological control agent for HWA (Cheah et al. 2004). Observations suggest that natural enemies may help keep HWA populations below injurious levels in the western United States (Mausel, pers. comm.). Laboratory studies have revealed that L. nigrinus feed selectively on HWA and field studies in British Columbia, Canada have shown that its lifecycle is highly synchronous with HWA phenology. This predator is being further evaluated for its suitability as a biological control agent. In order to be a viable candidate, L. nigrinus must survive under natural field conditions in the eastern United States, reduce the density of HWA, and it must be amenable to mass rearing. The survival, reproductive capability and predator efficiency of L. nigrinus were assessed over 3 years in the field. In addition, the environmental factors regulating processes in the life cycle to develop and improve rearing procedures for this insect were identified. In the first year, adults survived from February - May, laid up to 41 eggs/beetle, and consumed approximately 4.3 adelgids per day. In the second season, L. nigrinus adults survived from November - April, laid up to 38 eggs/beetle and consumed approximately 4.5 adelgids per beetle throughout the study. In both seasons, adelgid populations were significantly lower on branches with predators than those without predators. This impact was demonstrated on both the winter and spring generation of HWA. The feasibility of caged field releases of L. nigrinus was determined in a third field study. An estimated 10, 000 L. nigrinus eggs were liberated in field cages in spring 2003. The density of adelgids in the subsequent (spring) generation of the adelgids was significantly lower on branches with larval activity than those with no predators. Despite extensive sampling, no F1 adults were observed, however F2 adults were recovered in the fall of 2004, 20 months after release. The L. nigrinus life stages incurring high mortality during rearing were identified and factors affecting survival in the feeding and non-feeding life stages were investigated. Studies on the survival and feeding of adults, length of ovipositional period, density per cage, and survival of larvae were conducted. The effect of type of pupation medium, moisture level, disturbance, soil sterilization, temperature, and photoperiod on survival of the non-feeding stages and time of emergence from aestivation was examined. The most noteworthy finding, regarding rearing, is that aestivation can be extended by storing adults at high temperatures and long daylength throughout the summer and decreasing the temperature and daylength in the fall. Based on the results of these studies, procedures for rearing L. nigrinus have been developed and it is currently being reared at two other institutions. To date, over 8, 000 adults produced at Virginia Tech have been released in 6 states and both F1 and F2 adults have been recovered from multiple locations. / Ph. D.

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.