James C. Sellmer, William D. Morewood, Patricia Neiner and Kelli Hoover
Pennsylvania State University, Depts. Of Horticulture and Entomology, University Park, PA

Asian longhorned beetle (Anoplophora glabripennis) is a devastating wood-boring pest from China that has been found infesting urban shade trees in North America (New York, Chicago, and Jersey City in the U.S. and Toronto, Canada) and in Europe (Braunau, Austria). If allowed to establish throughout the urban areas of the United States, it is estimated that 1.2 billion urban trees may be lost with a compensatory value of $669 billion. In recent years a large amount of research on this pest has focused on understanding the biology of the species, improving detection techniques, and testing immediate eradication strategies in known infested areas within the U.S. Presently, eradication efforts still rely on visual detection using bucket trucks and tree climbers surveying for beetles and evidence of damage, followed by removal of infested trees. Most behavioral and biological research has been limited to laboratory conditions or in the field in China. A picture of adult preference and larval performance among commonly planted landscape trees has begun to emerge after several years of testing. To date we have completed screening of 16 species of trees representing 13 genera and 10 families including the Betulaceae, Aceraceae, Oleaceae, Fagaceae, Fabaceae, Rosaceae, Sapindaceae, Platanaceae, Tiliaceae, Ulmaceae. This presentation will discuss the results of greenhouse-based ALB oviposition preference and larval performance trials conducted at Penn State University on commonly planted landscape trees.

Keywords: Anoplophora glabripennis, susceptibility, oviposition, larval development, resistance

The Asian longhorned beetle (ALB) (Anoplophora glabripennis) is an invasive and destructive wood-boring insect that continues to threaten America's urban and suburban forests. The federal government, New York State, and Illinois have spent more than $2.4 billion on efforts to eradicate this pest (Stewart 2002), involving the destruction of almost 8000 infested trees in six areas of New York, five areas of Illinois, and one area of New Jersey (USDA Forest Service 2004). Losses of up to 1.2 billion urban shade trees, with a compensatory value of $669 billion, have been estimated if ALB becomes established and spreads across the United States (Nowak et al. 2001).

Present eradication strategies includes replanting infested areas with tree species thought to be non-hosts after removal of infested trees (Haack et al. 1997); the host range of ALB is not well known but appears to be very broad. Furthermore, the host range is expanding as the beetle invades new areas and encounters new potential host species (Nowak et al. 2001).

We have been investigating the potential for a wide variety of tree species to act as hosts for ALB, concentrating on species that are cultivated commercially for planting in urban and suburban settings. To date we have evaluated 16 tree species representing 13 genera (Acer, Betula, Carpinus, Celtis, Crataegus, Fraxinus, Gleditsia, Koelreuteria, Platanus, Pyrus, Quercus, Tilia, and Zelkova) and 10 families including the Betulaceae, Aceraceae, Oleaceae, Fagaceae, Fabaceae, Rosaceae, Sapindaceae, Platanaceae, Tiliaceae, Ulmaceae (Ludwig et al. 2002, Morewood et al. 2003, 2004a, 2004b). We have further plans to evaluate 12 species representing 12 genera (Alnus, Cladrastis, Corylus, Ginkgo, Gymnocladus, Liquidambar, Liriodendron, Malus, Pinus, Quercus, Sorbus, and Tsuga) and eight families (Betulaceae, Ginkgoaceae, Magnoliaceae, Fabaceae, Hamamelidaceae, Rosaceae, Pinaceae, and Fagaceae). The objective of this presentation is to highlight the results from screening experiments conducted within an APHIS certified quarantine greenhouse facility at The Pennsylvania State University. Our research efforts have focused on host suitability for oviposition and larval development using living trees under greenhouse conditions (Ludwig et al. 2002).

Some highlights of our results are as follows. Adult beetles showed a strong preference for sugar maple (Acer saccharum), well known as a preferred host in the field, confirming that our experimental system would produce results comparable to field conditions. Northern red oak (Quercus rubra) was accepted by adults for feeding and oviposition and we have since determined that larvae can complete development to the adult stage in this tree species. This result was both unexpected and important because no species of oak had previously been reported as a host for ALB and oaks have been used to replant infested areas. Golden-rain tree (Koelreuteria paniculata) was very attractive to adult beetles – at least as attractive as sugar maple in a direct comparison – but larval survival was relatively poor in this tree species. Honeylocust (Gleditsia triacanthos) was virtually ignored by adult beetles, even when the beetles had no other tree species available. Most beetles fed very little on honeylocust and the females failed to develop any eggs. Even females that had previously fed on Norway maple and developed eggs refused to lay eggs in honeylocust trees.

Research Methodology
Susceptibility to attack is evaluated by offering adult beetles living trees housed in large walk-in insect cages within the quarantine greenhouse. Nursery liners of various landscape tree species are planted in 20-gallon containers and grown in a pot-in-pot nursery established on the University Park campus of Penn State. Trees are moved into the greenhouse as necessary to allow for acclimation to greenhouse conditions. For initial screening, the beetles are offered a choice of four different tree species, with two of each species placed in each of the four walk-in insect cages. Male-female pairs of beetles are marked for individual identification and then released into the cages containing the trees. The location and behavior of each beetle are recorded three or four times daily and the beetles are removed after 30 days. At this time the trees are examined for feeding damage and oviposition sites, and the trees are held in the greenhouse for a further 90 days to allow for egg hatch and larval establishment. Then the trees are dissected and the numbers of larvae and body mass of living larvae are recorded. Relative preferences of adult beetles are further defined by offering the beetles different combinations of tree species or more limited choices (e.g., only two tree species). Tree species that adult beetles neglect or in which no larvae are found are further tested for unsuitability by offering adults the relevant tree species in no-choice situations and/or through artificial insertion of larvae (Ludwig et al. 2002).

Summary of Screening Results
During the past three years the following trees (Figure 1) have been screened for adult oviposition preference and larval survival. Based on the oviposition and larval development results we have developed a hierarchical table of preference and suitability of common landscape trees for ALB attack and larval development (Table 1).

Figure 1. Compilation of oviposition preference, larval number, and larval weight data from ALB adult screening and larval development evaluation trials for common landscape trees tested to date. Within each four species screening experiment, the means followed by the same letter for oviposition, larval number, or larval weight are not significantly different (P>0.05).

Table 1. Relative ranking of tested trees for suitability as a host for Asian Longhorned beetle based on number of oviposition sites, average survival of larvae after 90 days, and larval weight after 90 days. Rankings are given as High, Moderate, Low, and Very Low.

Extended Research Activities
Several follow-up experiments have been designed to further clarify results from the previous screening experiments. Based on the previous screenings four curious trends were identified including: suitability of red oak to ALB oviposition and larval development; the high preference of ALB females for golden-rain tree as an oviposition host equal to that of sugar maple combined with a low level of larval survival; limited preference and survival of ALB adults and larvae to honeylocust; and the apparent high death rate of ALB adult and larvae feeding on Callery pear. The following clarifying experiments have been conducted:

Larval development in red oak:
We have found through screening experiments that ALB will lay eggs on and larvae can develop in red oak, at least up to 90 days. This is of great concern because it was believed that oaks were not hosts. A larval development experiment was initiated by inserting larvae into red oak to determine whether the larvae could complete development. Four adults emerged recently from red oak; all are female, all are quite small (0.45 ± 0.08 g), and all still alive after four weeks. These results put into question the use of red oak in replanting activities.

Evaluation of golden-rain tree:
A strategy proposed for monitoring the infested areas of Chicago and New York involves the use of “sentinel” trees, i.e., trees that the beetle will attack and that can be checked periodically for signs that beetles are in the area. Based on screening experiments golden-rain tree appears to be a good candidate because it is highly attractive to ALB, but many of the larvae die in the tree due to the profuse amount of sap released by the tree in response to wounding. To determine the suitability of golden-rain tree we conducted a direct oviposition preference trial comparison with sugar maple. When offered a choice of equal numbers of golden-rain trees and sugar maples interspersed within a cage, the beetles were observed more frequently on the sugar maples than on the golden-rain trees. However, the proportion of observations in which the beetles were actively feeding was the same for the two tree species and females actually laid more eggs in golden-rain tree than in sugar maple. To further test suitability a separate larval development experiment was initiated to determine whether ALB larvae could complete development in golden-rain tree. Three adults have emerged from golden-rain tree, two (one male and one female) from a dead tree and one (male) from a live tree. These adults are also small (0.36, 0.32, and 0.58 g, respectively; the latter approaching normal size) but two of the three (the female and the larger male) died in less than 2 weeks.

Evaluation of honeylocust:
Three pairs of ALB adults that were maturation fed on honeylocust twigs prior to being placed in cages with honeylocust trees as a no-choice experiment for oviposition were maintained on those trees for 25 days (by replacing them as they died off). After 25 days only 8 oviposition sites were found on only one of the four trees and none contained an egg. Furthermore, all 8 females used in the experiment were dissected at the end and none contained any eggs. Three pairs of beetles that had been given sugar maple twigs for maturation feeding were then released on the same trees and monitored until they all died. All three males were dead after one week and all three females were dead after two weeks. At that time an additional 6 oviposition sites were found (3 on the tree that had 8 previously and 3 on another tree) but none contained an egg. The three females were dissected and found to contain 0, 2, and 5 eggs. Larvae have been inserted into honeylocust trees to determine whether larval development and survival is possible. Those trees have not been destructively harvested to evaluate larval survival at this time.

Evaluation of Callery pear:
Experiments were conducted to confirm the apparent resistance of callery pear to ALB larval establishment. Because of the very limited extent of oviposition on callery pear trees, larval inserts were used to assess larval survival in Callery pear compared to sugar maple. Sugar maple is known to support ALB larval development in the field (Smith et al. 2002). In the first experiment, young larvae that had previously become established on artificial diet after being extracted from oviposition logs were inserted into either Callery pear or sugar maple trees as described by Ludwig et al. (2002). Only 6% (n = 18) of the larvae inserted into Callery pear were still alive after four weeks, compared to 78% (n = 18) of the larvae inserted into sugar maple, a highly significant difference that is consistent with our observation that no larvae survived after 90 days in the Callery pear trees from the oviposition preference experiment. In the second experiment, first-instar ALB larvae were transferred directly from the Norway maple oviposition logs in which they had eclosed from the egg stage to artificial diet that was prepared either according to our standard recipe (Dubois et al. 2002) or with powdered Callery pear bark and wood in place of the cellulose component. Only 14% (n = 28) of the larvae on the diet containing Callery pear were still alive after two weeks, compared to 69% (n = 29) on the standard diet, again a highly significant difference, indicating that this antibiosis is a result of the constitutive chemical composition of Callery pear bark and/or wood, rather than an induced response of living trees.

We have initiated fractionation of Pyrus wood and bark to identify the compound(s) in Callery pear that may be involved in the premature death of both ALB larvae and adults. We have obtained evidence that the biologically active compound(s) in Callery pear can be chemically extracted without losing biological activity. The trunk of a Callery pear tree was dried and ground to a powder with a Wiley mill, and then processed overnight using acetone in a Soxhlet extractor. The crude (i.e., no further fractionation) acetone extract was suspended in dimethyl sulfoxide (DMSO) and then incorporated into artificial diet. Larvae of Anoplophora glabripennis were extracted from oviposition logs and placed on standard artificial diet for 7-10 days, to eliminate any mortality resulting from the extraction process, and then transferred to one of four experimental diets, including both positive and negative control treatments. After 3.5 weeks, larval survival did not differ between standard artificial diet and one containing DMSO (P > 0.25), indicating that the DMSO itself had no detrimental effect that might confound the results. Similarly, larval survival did not differ between artificial diets containing the crude chemical extract or dried shredded callery pear cambial tissues (P > 0.10), indicating that the chemical extract had biological activity comparable to the actual plant tissues. This new information strongly supports the probability of successfully extracting and identifying the active compound(s) involved in adult and larval death.

An obvious pattern of oviposition preference and larval development suitability exists for ALB relative to commonly planted landscape trees in the United States. The host range remains quite broad with highly, moderately, and limited preference trees for oviposition. Ultimately this makes field scouting and tree replacement difficult in infested areas; however, the availability of a preference list also enhances field scouting efficiency.

Unanticipated outcomes have also been found, most notably the suitability of red oak as a host for oviposition and its ability to support larval development through to adult emergence. This reduces red oak’s usability as a replant tree, especially young trees with thin bark similar to those employed in these screening trials. From a positive standpoint, two trees (Pyrus calleryana ‘Aristocrat’ and Gleditsia triacanthos var. inermis ‘Halka’) have been identified to have limited suitability as a host for oviposition, adult feeding, and larval survival. These two trees appear to be strong candidates for replanting. In addition, these findings generate several questions regarding the chemistry behind their unsuitability, the extent to which the traits are found beyond these specific cultivars to include the species, the genera, and the families, and finally how readily the chemical(s) or traits can be isolated and employed in management of other arthropod pests.

Special Thanks:
J. Frank Schmidt & Son Co.
Carlton Plants, LLC
Pennsylvania Department of Agriculture
Horticultural Research Institute
Pennsylvania Landscape and Nursery Association
Pennsylvania State University, College of Agricultural Sciences
Alphawood Foundation-Chicago
International Society of Arboriculture

Literature cited: