The hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae) is a small, non-native insect that feeds on hemlock (Tsuga spp.) species native to the eastern United States. One biological control agent, Laricobius osakensis, shows promise due to its voracious feeding on and synchronous life cycle with HWA. This beetle species is originally from Japan and has undergone all the necessary testing while in quarantine, gaining permission for field release in 2010. Field releases began in the fall of 2012 and continued the following fall. Reproduction occurred at the first year's field sites with beetles recovered at both sites the following fall. During the second year of field work, beetle recovery was low due to winter temperatures that caused high HWA mortality. All of the L. osakensis beetles released in the field are first reared in an Insectary on Virginia Tech's campus. To improve health of these beetles during rearing, a series of chemical amendments were added to the water baths where HWA infested cut hemlock branches were placed. HWA samples were taken from each treatment and analyzed for carbon, nitrogen and total carbohydrates and beetles emergence was recorded in relation to prescribed treatment. None of the treatments appeared as the best for improving HWA health, but there were differences between the sampling months within the two years. These differences in month correlate with the biological processes occurring within HWA at these times such as reproduction and preparation for aestivation. No treatment helped increase beetle emergence. / Master of Science in Life Sciences

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

Hemlock woolly adelgid, Adelges tsugae Annand, is an invasive pest threatening eastern (Tsuga canadensis (L.) Carrière) and Carolina hemlock (T. caroliniana Englem.) forests in the eastern US. A new predator, Laricobius osakensis Montgomery and Shiyake, has been found in association with A. tsugae in Japan. Laricobius osakensis was evaluated in a series of pre-release studies to assess its potential as a biological control agent for A. tsugae. Host-range studies indicated that L. osakensis is a specific predator that feeds predominantly and reproduces only on A. tsugae. The functional response "prey consumption changes in response to changes in prey density" was similar for both L. osakensis and Laricobius nigrinus Fender adults. However, L. osakensis had a higher numerical response"changes in oviposition in response to changes in prey density"than L. nigrinus. Laricobius osakensis larvae had a higher functional response than L. nigrinus larvae. Laricobius osakensis\' higher numerical and functional response indicates that this species can potentially be more effective than L. nigrinus. In the evaluation of L. osakensis in sleeve cages in the field from December to April high rates of adult survival, feeding, and reproduction were found. A pair of predators in a cage killed on average five adelgids/day. Peak oviposition occurred in March and April. Larvae from eggs placed in the cages reached maturity in 28-50 days, depending on the season, and only 6.7 % died before reaching maturity. Laricobius osakensis was able to survive, feed, develop, and reproduce in USDA cold-hardiness zones 5b and 6a of southwest Virginia. Behavior of L. osakensis and L. nigrinus was qualitatively similar but varied quantitatively. Laricobius osakensis was more active and had a lower association with T. canadensis. Interactions between species were minimal and not detrimental to either. Intrasexual copulation attempts were observed between males and to a lesser extent between females; however, intrasexual interactions were less frequent than intersexual interactions between the two species. Otherwise activity, including oviposition, was not altered by the presence of the other species. These studies indicate that L. osakensis has the potential to be a valuable addition to the natural enemies complex against A. tsugae. / Ph. D.

Hybridization was evaluated among three Laricobius spp. involved in the biological control of hemlock woolly adelgid (Adelges tsugae Annand). Following lab mating studies, there was no evidence that Laricobius osakensis Montgomery and Yu could produce hybrid progeny with either Laricobius nigrinus Fender or Laricobius rubidus LeConte. Interaction between L. osakensis and L. nigrinus did not result in a lower production of progeny as a result of fitness costs associated with interspecific mating attempts. Laricobius nigrinus and L. rubidus hybrids were produced in the lab and collected in the field. Hybrid progeny showed very little evidence of decreased fitness. For example, there was no significant difference in the number of days it took for hybrids and pure parental species to develop from egg hatch to the prepupal stage, there was no difference among hybrids and pure parental species in the head capsule widths and larval lengths for the first through third instar, and there was evidence of an F2 generation from field collected specimens. Hybrids produced in the lab had intermediate shaped genital paramere angles compared with parental species, and had elytra coloration similar to that of L. rubidus. Hybrids showed no host preference in the lab, but a preference for Adelges tsugae in the field. Of 12 site factors examined, only the number of years that L. nigrinus was present at the site was found to be associated with percent hybrids. Contamination of the L. osakensis colony with Laricobius naganoensis Leschen resulted in the need to develop molecular methods to differentiate L. osakensis from L. naganoensis. Three restriction enzymes were found that correctly differentiated the two species. / Ph. D.