Northeastern Naturalist Vol. 23, No. 1 K.L. Mooneyham, S.M. Salom, and L.T. Kok 2016 141 2016 NORTHEASTERN NATURALIST 23(1):141–150 Release and Colonization of Laricobius osakensis (Coleoptera: Derodontidae), a Predator of the Hemlock Woolly Adelgid, Adelges tsugae Katlin L. Mooneyham1,*, Scott M. Salom1, and Loke T. Kok1 Abstract - Biological control of Adelges tsugae (Hemiptera: Adelgidae; Hemlock Woolly Adelgid [HWA]) is an essential component of a management plan for this non-native pest of Tsuga spp. native to eastern N. America. The predatory beetle Laricobius osakensis Montgomery and Shiyake (Coleoptera: Derodontidae) shows potential as a biological control agent because of its coevolved life-cycle with HWA in its native habitat and its voracious appetite for HWA. In the first releases ever made for this species, adult beetles were introduced at 4 sites in the eastern United States with 2 long-term objectives: (1) suppressing HWA populations, and (2) developing field insectaries for re-distribution of predators in the future. Our immediate objectives were to determine the predator’s survival and colonization in eastern Tsuga forests following their introduction. In addition, L. osakensis eggs were set out at the 4 release sites. Results from the first year post-release of adult beetles indicated that reproduction occurred at these sites and that beetles survived the summer aestivation period and emerged the following fall. Second-year data were impacted by high HWA mortality due to periods of extreme low winter temperatures. No beetles have yet been collected from any of the 4 egg-release locations. Introduction Adelges tsugae Annand (Hemiptera: Adelgidae; Hemlock Woolly Adelgid [HWA]) is a non-native insect pest that feeds on Tsuga canadensis (L.) Carrière (Eastern Hemlock) and Tsuga caroliniana Engelm. (Carolina Hemlock) (Havill et al. 2011, McClure 1987). This strain of HWA was introduced from Japan (Havill et al. 2006), most likely from imported nursery stock, and was first recorded in Richmond, VA, in the early 1950s (Havill et al. 2011, Souto et al. 1996). In its native range, Asia and the west coast of North America, HWA is not considered a pest species on hemlocks. Natural resistance of other Tsuga spp., as well as thriving predator populations, keep this species from reaching the damaging levels seen in the eastern United States (McClure and Cheah 1999). The spread of HWA throughout the eastern United States is driven by a variety of sources including wind and movement by humans and animals, and appears limited by extreme cold winter temperatures (Evans and Gregoire 2007, McClure 1990). HWA causes injury to hemlocks by feeding on twigs, at the base where the needles attach (McClure 1987). It is active in the winter and feeds where it is able to access the tree’s stored photosynthate and reserves, depleting the necessary nutrients for that tree to create new growth in the spring (Young et al. 1995). Hemlock 1Department of Entomology, Virginia Tech, Blacksburg, VA 24061. *Corresponding author - katlinm@vt.edu. Manuscript Editor: David Orwig Northeastern Naturalist 142 K.L. Mooneyham, S.M. Salom, and L.T. Kok 2016 Vol. 23, No. 1 death is slow and prolonged due to HWA feeding and the cyclical populations of the adelgids as they grow and feed intensively and then decline along with hemlock health (McClure 1991). These insects do not discriminate between size and age of hemlocks, and thus all trees are susceptible. Damage appears on the trees as bud mortality, foliage discoloration, and overall loss of needles (USDA 2008). Loss of hemlocks in the forest changes the forest canopy structure, species composition, and forest soil temperatures (Jenkins et al. 1999, Orwig and Foster 1998). Establishment of a classical biological control program became a priority in the effort to develop means of suppressing HWA (Mausel et al. 2010, Onken and Reardon 2011). Classical biological control is the importation of natural enemies from the native range of the pest species where they control the insect below damaging levels (Clausen 1951, Dahlsten and Mills 1999, Franz 1961, Hall and Ehler 1979). HWA is ideal for biological control because of its immobile lifestyle and easy access for predation due to its persistence on trees for extended periods of time. Predators of HWA from both Asia and the United States have been evaluated since the mid-1990s (Onken and Reardon 2011, Wallace and Hain 2000). Initially, 3 predators were seriously evaluated for their potential as control agents: Scymnus sinuanodulus Yu & Yao (Coleoptera: Coccinellidade), Sasajiscymnus tsugae Sasaji (Coleoptera: Coccinellidade), and Laricobius nigrinus Fender (Coleoptera: Derodontidae) (Mausel and Salom 2013). Members of the genus Laricobius have been studied for their potential as biological control agents since 1997 because they are known to be adelgid specialists (Vieira et al. 2011, Zilahi-Balogh et al. 2002). While L. nigrinus has clearly been the most successful of the 3 original predators, in terms of establishment and spread (Mausel et al. 2010), only 2 site-specific studies have been able to assess the short-term impacts of the predator (Mausel et al. 2008, Mayfield et al. 2015). In a continued effort to find and utilize the most effective biological control agent available, Laricobius osakensis Montgomery and Shiyake (Coleoptera: Derodontidae), native to the same location where the HWA that invaded the eastern United States originated, is one such predator currently being evaluated (Havill et al. 2006, Montgomery et al. 2011). First found on Tsuga sieboldii from a sampling effort that took place in the Kansai region of Japan in 2005 (Montgomery et al. 2011), this predator has evolved with this strain of HWA. It is promising as a biological control agent because of its increased predation and reproduction compared with L. nigrinus (Vieira et al. 2012). Laboratory studies have shown that L.osakensis does not impact L. nigrinus or the native L. rubidus, a predator of Pineus strobi (Hartig) (Hemiptera: Adelgidae; Pine Bark Adelgid), through limited predation on one another and because all 3 are compatible for predation on HWA (Story et al. 2012). Additionally, while L. nigrinus and L. rubidus can successfully hybridize, L. osakensis cannot produce viable eggs from mating with either (Fischer et al. 2015). Both L. osakensis and HWA undergo an aestivation period at the same time and have co-evolved life cycles as is expected with a predator that has a narrow host-range (Vieira et al. 2013). This predator has a univoltine lifecycle, active from November to April in its native range in Japan. Additionally, laboratory studies Northeastern Naturalist Vol. 23, No. 1 K.L. Mooneyham, S.M. Salom, and L.T. Kok 2016 143 show that L. osakensis can only complete development on HWA, and is therefore not a threat to other species native to eastern forests (Vieira et al. 2011). In 2010, this species was approved for removal from quarantine by the USDA Animal and Plant Health Inspection Service (APHIS) and subsequently was allowed to be released (Lamb et al. 2011). In this study, we considered both the number and life stage of L. osakensis necessary to optimize release and colonization success. The methodology for field release of L. osakensis is adapted from the protocol developed for L. nigrinus adults by Mausel et al. (2010). All introductions of L. nigrinus were adult releases, except in Georgia, where they have also been set out at the egg stage. The potential benefit to releasing eggs is that it greatly reduces the effort needed to rear these beetles. Given the similarities between climate in the southern Appalachians and that of its native range in Japan, the primary objectives of this research were to determine how well L. osakensis survives and colonizes in eastern forests. Additionally, we hope to create a protocol for other forest-health professionals to use when releasing these beetles on their own. Methods Field site description Release locations for L. osakensis occurred on federal, state, and private lands. Sites suitable for releases had the following attributes: >10 ha of forest primarily composed of Tsuga spp., trees with high live-crown ratios, light to moderate HWA infestation, and >2 km away from any previous L. nigrinus release locations. Trees receiving L. osakensis beetles were clustered around a central HWA infestation if one could be located. We selected individual release trees on the basis that they were light to moderately infested with HWA, had minimal crown decline, and had foliage accessible from the ground for beat-sheet sampling. For each tree, we affixed an aluminum tag at breast height for identification and attached flagging for visibility within the stand. In the fall of 2012, we released 500 adults at both the Little Meadows Hunt Club (Giles County, VA) and Carnifex Ferry Battlefield State Park (Summersville, WV). To be able to compare colonization success of different release densities, we set out 80, 170, and 250 beetles on 3 separate trees, respectively, within each site. During the fall of 2013, we released 2000 beetles at both the Clinch Mountain Wildlife Management Area (Saltville, Russell County, VA) and the George Washington National Forest (Goshen, VA). We set out 250 beetles on each of 8 trees at both sites. All field introductions of beetles during 2012–2013 occurred in November, corresponding to when the sistens generation breaks diapause. Differences in numbers released per site in 2012 compared to 2013 were due largely to the availability of beetles from the Virginia Tech Insectary. Predators released The L. osakensis beetles used for field release were lab-reared adults from beetles collected in northern Japan from HWA-infested Tsuga sieboldii Carr. (Southern Northeastern Naturalist 144 K.L. Mooneyham, S.M. Salom, and L.T. Kok 2016 Vol. 23, No. 1 Japanese Hemlock). Laboratory rearing of these beetles took place at the Virginia Tech Insectary, utilizing methods established for L. nigrinus (Lamb et al. 2005). In the fall, after adults emerged from the soil, they were held in 3.8-L containers with up to 50 beetles on HWA-infested Eastern Hemlock bouquets. Laricobius osakensis beetles were given fresh HWA every two weeks up until the point where they were released in the field. Adults were held in environmental chambers at 2–10 °C, depending on day length, and at 70–90% relative humidity. Prior to release, we checked the beetles to ensure none were missing appendages, showed abnormal behavior, or exhibited low vigor. Release methods We qualitatively assessed tree health prior to release using 5 measures: percent live-crown ratio, crown density, live tips, live branches, and percent new foliage (Table 1). We determined percent live-crown ratio by examining the ratio of a hemlock’s live crown in relation to that of its total height, crown density by examining the percent live branches and foliage that blocks light from coming through the crown, and percent new foliage, live branches, and live tips by visually estimating what percent of branches had live foliage and what percent of those branches contained new growth. In addition to the tree health measures, we determined the average number of adelgids per branch tip by picking 10 branches 20 cm in length at random and counting the number of live HWA on each branch (Table 1). Prior to release, bouquets of HWA-infested hemlock were prepared at the Virginia Tech Insectary. The bouquets were made up of 10–15 twigs (10 cm in length) and held in ventilated escape-proof polyethylene containers (950 mL, Rex Tech Corp., Kent, OH), which were sealed with parafilm. Each container contained approximately 50 beetles which we hand-carried to release sites in a cooler and kept at an ambient temperature, approximately 18 °C, and shaded before release within 24 h of packaging. We placed the hemlock branches from the insectary that held the predators on the release tree on branches that we had flagged because they contained at least 20 HWA/15 cm. All predator releases occurred on clear days, with little to no wind, and mild, cool temperatures. We also set out L. osakensis eggs at each of the 4 field sites, but at different locations from where adult releases took place, separated by a minimum of 1 km. All egg introductions took place in March following adult releases that occurred the previous November. The objective was to compare the success of colonization Table 1. Mean health-index values for site trees and mean number of adelgids per branch for each of the 4 release sites. All values given are means ± s.d. No. of adelgid given is per 20-cm branch. % live-crown % live % tips % new % crown No. of Site Year ratio branches alive foliage density adelgid Little Meadows, VA 2012 94 ± 2 88 ± 8 82 ± 13 73 ± 16 77 ± 6 16 ± 3 Carnifex, WV 2012 76 ± 10 82 ± 8 83 ± 11 88 ± 3 73 ± 16 15 ± 1 Goshen, VA 2013 81 ± 12 79 ± 11 83 ± 9 85 ± 5 74 ± 7 17 ± 2 Saltville, VA 2013 72 ± 15 81 ± 6 89 ± 5 83 ± 4 76 ± 10 15 ± 1 Northeastern Naturalist Vol. 23, No. 1 K.L. Mooneyham, S.M. Salom, and L.T. Kok 2016 145 from egg releases with colonization using the adult-release method. At each site, we placed an estimated 1000 eggs on HWA-infested trees that had met the same criteria as the infested trees where adult releases occurred. All sites had 10 HWAinfested hemlocks selected for egg release. We attached ~5 HWA-infested branches from the insectary (20–30 cm in length), each branch laden with approximately 5 L. osakensis eggs inside HWA ovisacs, to flagged branches on infested release trees for a total of 100 eggs per tree. Beat-sheet sampling for predators We conducted beat-sheet sampling for L. osakensis once monthly from November to May, 2012–2014, using canvas beat sheets (71 cm2, BioQuip, Rancho Dominguez, CA). We examined branches in the lower canopy of both the release tree and the surrounding non-release hemlocks within 20 m and recorded any predators discovered. We placed beat sheets under the branches with high HWA populations and then tapped the branches ~10 times with a stick. We identified, recorded, and returned unharmed to the tree from which they fell all L. osakensis. We sampled only on days when the temperature was above 0 °C, when the adult Laricobius spp. are active (Zilahi-Balogh et al. 2003), and when it was not windy, raining, or snowing. When sampling at each release site, we recorded the number of adults, release tree, site conditions, and sample date. Branch-clipping sampling procedure for larvae Mausel et al. (2010) determined that field colonization of Laricobius was more accurately assessed by sampling for larvae rather than adults. To use this method for L. osakensis, we clipped branches once a year at each site, on each release tree, in March or April, depending on when HWA oviposition was at its peak. We used Eastern Redbud (Cercis canadensis L.) flowering as a phenological indicator to guide the start of larvae sampling (Mausel et al. 2010). Using hand pruning shears, we cut heavily infested branches on the assumption that they would most likely have L. osakensis larvae within the ovisacs. Branches from all areas within reach on each release tree were cut into 20–30 cm lengths, loosely placed in individually labeled plastic bags (26.8 x 27.3 cm), and stored in a cooler within a covered vehicle. We then transported the samples to the Virginia Tech Insectary where each sample was put in a rearing funnel (Salom et al. 2012). After the L. osakensis larvae developed through the 4 instars, they dropped as pre-pupae into mason jars attached to the funnel. We inspected these funnels daily for larval drop. We counted and recorded the larvae according to release tree and site, then moved them to soil containers to undergo pupation and aestivation. We placed dead larvae found during the funnel checks in a vial containing 99% ethanol for later molecular analysis, since Laricobius larvae are morphologically indistinguishable among species. We used the mitochondrial cytochrome oxidase I (COI) gene for species identification (Davis et al. 2011). Partial cytochrome oxidase subunit I (COI) was amplified for 21 Laricobius larvae recovered over the 2012–2013 field season. We performed amplification of the COI gene according to protocols described by Davis et al. (2011) and Fischer et al. (2014). Northeastern Naturalist 146 K.L. Mooneyham, S.M. Salom, and L.T. Kok 2016 Vol. 23, No. 1 Results Annual field survey and Laricobius osakensis collection The total number of adult beetles recovered at Little Meadows during the first year (2012–2013) was 15, while the total number of adults at Carnifex was 4. We collected no beetles at either site in March (Fig. 1A). The beetles recovered this first season were all lab-reared adults from generation P1 released the previous November. The sampling of these adults was to ensure that they were still active and present during the first field season. During the 2013–2014 field season, we recovered 2 adult F1 L. osakensis at Little Meadows and 5 at Carnifex (Fig. 1B). During the first year, we recovered 209 larvae from Little Meadows and 8 from Carnifex. Of the larvae recovered from the Little Meadows site, the majority (148) was from the tree where 250 beetles were released. This trend did not hold up, as the second greatest amount of larvae drop (48) came from the tree where the least amount of adults was released (80). The release tree where 170 adults were released had the least amount of larval drop (13). The Carnifex site followed the same trend. The 250-adult release tree had 5 larvae drop, the 80-adult release tree had 2, and the 170-adult tree had 1. Molecular analysis on a subsample of these larvae found 14 of the 21 larvae collected to be L. osakensis and 7 to be the native L. rubidus. We clipped branches in the 2014 and 2015 field seasons to monitor F2 larvae from the F1 adults, but no larvae dropped during these field seasons. The total number of P1 adult beetles recovered at the Saltville field site the first year (2013–2014) was 7, while the total number recovered at Goshen was 1 (Fig. 1B). Branches were clipped to monitor larval drop, however no larvae were ever recovered from either of these field sites during spring of 2014 . We released eggs at Little Meadows and Carnifex in 2013 and Goshen and Saltville in 2014. Beat-sheet sampling from the release trees the following fall after release did not result in the recovery of any adult L. osakensis beetles from any of the sites. Figure 1. Number of adult Laricobius osakensis recovered in Virginia and West Virginia during the 2012–2013 field season (A) and the 2013–2014 field season (B) by release site ( LM = Little Meadows Hunt Club in Giles County, VA: CAR = Carnifex Ferry Battlefield State Park in Summersville, WV; SALT = Clinch Mountain Wildlife Management Area in Saltville,VA; and GOSH = George Washington National Forest in Goshen, VA). Northeastern Naturalist Vol. 23, No. 1 K.L. Mooneyham, S.M. Salom, and L.T. Kok 2016 147 Discussion Beat-sheet sampling to monitor the colonization of L. osakensis yielded positive results during the first field season at each site. We recovered adult P1 beetles throughout the field season after release, as well as larvae showing that the released beetles were reproducing in the field. It is known that beat-sheet sampling is difficult for small, hard to find insects and that non-recovery of these beetles does not confirm their absence (Mausel et al. 2010, Venette et al. 2002). A critical issue with the recovery of adult beetles was the extreme winter temperatures experienced during the 2014 field season. Winter temperatures reached as low as -25 °C in certain regions of Virginia and caused considerable adelgid mortality (T. McAvoy, Virginia Tech University, Blacksburg, VA, 2014, unpubl. data). We brought branches infested with HWA back from the Carnifex site in January 2014 and found 99% HWA mortality at this site. This high incidence of mortality might explain the lack of recovery for both L. osakensis adults and larvae. The loss of prey and cold weather together likely caused the decline in L. osakensis adults recovered from December to May of 2014. After December, there was a decrease in recovery numbers, which corresponded to the extreme cold winter temperatures. Evidence suggests that beat-sheet sampling is less effective in the winter than in the fall and spring (Zilahi-Balogh et al. 2003). The weather conditions also explained the lack of larvae recovered during this time. The low recovery of adult beetles could be the result of these beetles being intolerant of the freezing temperatures experienced during the 2014 field season. Testing must be conducted to determine the supercooling point of L. osakensis. The other contributing factor is the lack of prey because the only way to sample for predators i s to find their prey. The egg-release results from March of both 2013 and 2014 are still inconclusive, as only 1 adult has been recovered thus far from all 4 sites. While it is much easier to rear the beetles just through the egg stage versus through adulthood, the lack of recovery suggests more sampling time is necessary to determine if this is a reasonable alternate method for releasing L. osakensis compared with releasing lab-reared adults. Continued fieldwork is needed to assess establishment of L. osakensis over time at each of the field sites. The ultimate goal is to create successful field insectary sites where adult L. osakensis beetles can be collected and redistributed for release at other impacted locations. Establishing these sites can take many years, as has been the case with L. nigrinus (Mausel et al. 2010). This effort is designed to relieve rearing pressure from insectaries. Colonization occurred at the first 2 release sites, yet is hard to fully quantify due to the onset of consecutive harsh winters. These difficult environmental conditions killed prey, which likely delayed establishment of these predators. It appears that L. osakensis is suited for the eastern United States because reproduction occurred naturally in the field as evidenced by the larvae recovered at both initial release sites. Continued sampling is necessary to assess L. osakensis colonization in the eastern United States. Measuring their success in establishing viable populations as well as their impact on the ecosystem is critical in determining their potential for suppressing HWA in the eastern United States. Northeastern Naturalist 148 K.L. Mooneyham, S.M. Salom, and L.T. Kok 2016 Vol. 23, No. 1 The lack of recovery of L. osakensis in 2014 is not specific to this species of Laricobius. For L. nigrinus, the numbers collected were directly related to the number of HWA present (Mausel et al. 2008), we believe this helps explain our observations with L. osakensis. 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