Host preference between saltcedar (Tamarix spp.) and native non-target Frankenia spp. within the Diorhabda elongata species complex (Coleoptera: Chrysomelidae)

Biological Control 51 (2009) 337–345

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Perspective

Host preference between saltcedar (Tamarix spp.) and native non-target Frankenia spp. within the Diorhabda elongata species complex (Coleoptera: Chrysomelidae) John C. Herr a,*, Raymond I. Carruthers a, Daniel W. Bean b, C. Jack DeLoach c, Javid Kashefi d a

USDA-ARS Western Regional Research Center, Exotic and Invasive Weeds Research Unit, Albany, CA 94710, USA Colorado Department of Agriculture, Biological Pest Control, Palisade Insectary, Palisade, CO 81526, USA c USDA-ARS Grassland, Soil and Water Research Laboratory, Temple, TX 76502, USA d USDA-ARS European Biological Control Laboratory, Thessaloniki 54622, Greece b

a r t i c l e

i n f o

Article history: Received 14 February 2009 Accepted 28 July 2009 Available online 3 August 2009 Keywords: Biological control of weeds Host range Host specificity testing Non-target impacts Diorhabda elongata Tamarix Frankenia

a b s t r a c t Since its release in 2001 for the biological control of saltcedar (Tamarix spp., Tamaricaceae), the leaf beetle Diorhabda elongata (Brullé) from China and Kazakhstan, has become successfully established in many locations in the western United States. However, it failed to establish in the southern and western portions of the saltcedar infestation, creating the need to test additional populations of the beetle from other areas within its region of origin. The host specificity of seven Eurasian populations of D. elongata was evaluated by testing larval development and adult ovipositional preference on a variety of non-target agricultural, ornamental and native plants, with emphasis placed on native Frankenia spp. (Frankeniaceae), which were shown to be laboratory hosts in previous tests. No larvae survived on any of the non-target test plants except for Frankenia spp., where survival to the adult stage ranged between 15% and 92%, and was often not significantly different from survival on Tamarix controls. Adult Diorhabda from Crete laid significantly more eggs on Tamarix ramosissima Ledebour than on Frankenia spp. in a multiple-choice oviposition test but showed very little discrimination between Tamarix and Frankenia species in a no-choice test. In paired-choice tests, all seven Diorhabda populations laid significantly more eggs on T. ramosissima than Frankenia salina (Molina) I.M. Johnston. However, the percentage of total eggs laid on F. salina ranged from 0.8% to 15.7%, suggesting that some utilization of this native plant might occur in the field, despite the presence of a preferred host plant. Significant differences were found between some Diorhabda populations in the percent of total eggs laid on F. salina, indicating a variable degree of risk to these non-target plants. Published by Elsevier Inc.

1. Introduction It is sometimes beneficial to introduce multiple populations of a biological control agent with different phenotypes or biological characteristics to achieve control of a target weed that has a wide distribution across a variety of climatic conditions (see examples in Nechols et al., 1995: Julien and Griffiths, 1998). Prior to release, each population of the agent should be tested for host specificity due to the possibility of cryptic species with potential variability in efficacy and feeding behavior (Harley and Forno, 1992). After thorough evaluation of host range (DeLoach et al., 2003; Lewis et al., 2003a), the leaf beetle Diorhabda elongata (Brullé) (Chrysomelidae) from Fukang, China and Chilik, Kazakhstan was released at multiple locations in the western United States in 2001 for the control of saltcedar (DeLoach et al., 2004; Carruthers et al., 2008).

* Corresponding author. Address: USDA-ARS Western Regional Research Center, Exotic and Invasive Weeds Research Unit, 800 Buchanan Street, Albany, CA 94710, USA. Fax: +1 510 559 6193. E-mail address: [email protected] (J.C. Herr). 1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2009.07.015

Saltcedar (Tamarix spp., Tamaricaceae) is an exotic tree from Eurasia that is highly invasive in riparian ecosystems across the western United States, where it has depleted water reserves, increased soil salinity, altered flooding and fire frequency, and displaced native species (Fraiser and Johnsen, 1991; DiTomaso, 1998; Lovich and DeGouvenain, 1998; Bossard et al., 2000). D. elongata established in most of the release sites north of the 37th parallel (in Nevada, Utah, Wyoming and Colorado), but failed to establish in more southern sites in Texas and California, apparently due to diapause characteristics that were not compatible with the climatic conditions in these areas (Bean et al., 2007). It was postulated that introductions of additional D. elongata populations from regions more climatically similar to the southern target areas would result in successful establishment due to diapause induction cues which match the shorter summer day lengths in these locations (Bean et al., 2007). Therefore, six additional Diorhabda populations were imported by the USDA-ARS in 2001 and 2002 for the evaluation of host range and potential efficacy in the USDA quarantine facilities at Albany, California and Temple, Texas. At the time of importation, it was believed that these beetle

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collections represented populations of one widely distributed species, D. elongata. However, a recent taxonomic revision (Tracy and Robbins, 2009) determined that they are actually four closely related species of Diorhabda (see Section 2 for the list of populations and corresponding species names). Previous host range testing on Diorhabda from China showed that although the beetles feed and reproduce almost exclusively on Tamarix species, they will feed, oviposit and complete development on native North American non-target plants in the genus Frankenia (Frankeniaceae), which are clearly within their physiological (laboratory) host range (DeLoach et al., 2003; Lewis et al., 2003a). The genus Frankenia contains ca. 80 species world-wide; all are halophytic shrubs with distributions limited to temperate and subtropical sea coasts, salt lakes, and salt deserts (Willis, 1973). Frankeniaceae and Tamaricaceae are the only two families in the order Tamaricales (Spichiger and Savolainen, 1997), and Frankenia is the only genus in this order with native North American species (Whalen, 1987). Six Frankenia species occur in North America: Frankenia salina (Molina) I.M. Johnston in California and Mexico; F. palmeri S. Watson in California and Mexico; F. johnstonii Correll in Texas and Mexico; F. jamesii Torrey ex A. Gray in Texas, New Mexico and Colorado; F. gypsophila I.M. Johnston in Mexico; and F. margaritae F. Gonzales Medrano in Mexico (Whalen, 1987). Although none of the U.S. Frankenia are currently federally listed species, F. johnstonii has a limited distribution and was previously listed as endangered (Janssen and Williamson, 1996), F. palmeri is rare in California but common in parts of Mexico (Skinner and Pavlik, 1994), and F. gypsophila and F. margaritae are rare in Mexico, with the later species known from only a single locality (Whalen, 1987). In contrast, F. salina is widely distributed in California coastal salt marshes and inland alkali flats (Hickman, 1993) and has been reported from 35 counties in the state (CalFlora, 2006). Inland populations of F. salina were formerly classified as F. grandiflora Cham. & Schlecht. var. campestris Gray (Munz and Keck, 1968), and have a more upright bush-like growth habit than the sprawling form found in coastal populations. The distributions of Frankenia and Tamarix overlap in many locations in the western United States and in limited sites in northern Mexico, and thus the true ecological host range of D. elongata with respect to Frankenia must be determined in order to accurately assess the risk to these nontarget species. The potential for weed biological control introductions to cause environmental damage through impact to non-target native plants has been addressed in a series of publications (McEvoy and Coombs, 2000; Strong and Pemberton, 2000; Louda et al., 2003; Pearson and Callaway, 2005). Although standardized host range testing protocols are followed by weed biological control researchers (USDA APHIS PPQ, 1998), current regulations do not contain clear guidelines on how to determine if agents are ‘‘safe enough” for release if they are not strictly host-specific. Several weed biological control agents that were known to utilize native species prior to release have been established in the U.S., and the majority have not caused significant non-target impacts (Pemberton, 2000). However, the example of Rhinocyllus conicus Frölich, introduced to control invasive thistles and now widespread in native California thistle populations (Herr, 2004) and causing apparent decline in native Nebraska thistle abundance (Louda et al., 2005), illustrates that population-level impacts can occur. Similarly, the pyralid moth Cactoblastis cactorum Bergroth, which has been used to successfully control invasive Opuntia spp. in many regions world-wide, is currently threatening endangered native cacti in Florida, although it was not intentionally released as a biological control agent in the U.S. (Pemberton, 1995). More recently, Bruchidius villosus Fabricius has been reared from a non-target plant in New Zealand, and subsequent studies showed that host preference varied between different populations of this weevil (Sheppard et al., 2006). Therefore, it is prudent to

thoroughly scrutinize the host specificity of widely distributed populations of any new weed biological control agent, particularly those that have the demonstrated ability to utilize native non-target species in the laboratory. The objectives of this study were to determine the host specificity of seven populations of D. elongata through the use of the following test procedures: (1) Larval development tests to determine if representative agricultural crops, native riparian plants and Frankenia spp. are within the physiological host range of the beetle and (2) choice and no-choice tests measuring adult ovipositional preference between Tamarix and Frankenia spp. in the laboratory. These tests were also used to determine if non-target host utilization varied significantly between Diorhabda populations. This paper presents the results of a series of host range tests conducted between 2002 and 2008, including four larval development tests, three adult no-choice tests, two adult paired-choice tests, and one adult multiple-choice test. These studies primarily focused on potential impact to F. salina, whereas a parallel set of experiments (Milbrath and DeLoach, 2006) evaluated risk to F. jamesii and F. johnstonii. Subsequent work more directly addressed the ecological host range of D. elongata under field conditions (Moran et al., 2009; Herr and Carruthers, in press). 2. Materials and methods 2.1. Study organism Diorhabda elongata eggs are laid on saltcedar leaves, where they typically hatch within 5 days at 24 °C (Lewis et al., 2003b). Larvae mature through three instars feeding primarily on leaves, shoots and occasionally flowers, and then pupate in the leaf litter and soil adjacent to defoliated trees. The developmental time from egg to adult is 34 days at 24 °C (Lewis et al., 2003b), however, temperature-dependent growth and development (Herrera et al., 2005) affects the phenology of these beetles under field conditions. In the regions where the beetles have been released in the U.S., two generations are typically seen each growing season in the more northern areas, however, in more southern areas up to five generations can be produced during the spring through early fall (Milbrath et al., 2007). Emerging adults often form large aggregations and can rapidly defoliate localized stands of Tamarix spp. (Carruthers et al., 2008). Where defoliation is heavy, adults typically disperse over wide areas searching for healthy saltcedar as fresh ovipositional sites. Following the last larval generation, adults emerge, feed heavily and overwinter in the upper soil profile and/or leaf litter, from which they emerge the following spring in close temporal synchrony with saltcedar bud break (see Carruthers et al., 2008, and Tracy and Robbins, 2009, for more life cycle details). 2.2. Beetle colonies Diorhabda elongata were collected from Tamarix spp. at seven locations, along a latitudinal gradient ranging from western China, through the Middle East and Eastern Europe to Tunisia in Northern Africa. At the time that these beetle populations were collected, it was determined that they represented different biotypes of a single species, D. elongata. Series of voucher specimens from each population were sent to the USDA-ARS Systematic Entomology Laboratory, Beltsville, Maryland, where they were identified as D. elongata by A.S. Konstantinov. However, a recent taxonomic revision by Tracy and Robbins (2009) demonstrated that the seven populations we tested are composed of four distinct Diorhabda species. For the purposes of this paper, we will simply refer to the populations by the name of the collection location (in quotation marks below). Species names from Tracy and Robbins (2009) are also provided for future ref-

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erence. Collecting locations and date of collection are as follows: 7 km west of Fukang, China, 8/12/02 [‘‘Fukang” = D. carinulata (Desbrochers)]; 10 km southeast of Turpan, China, 6/19/02 [‘‘Turpan” = D. carinulata (Desbrochers)]; 7 km west of Karshi, Uzbekistan, 9/26/02 [‘‘Karshi” = D. carinata (Faldermann)]; 5 km northwest of Buchara, Uzbekistan, 9/27/02 [‘‘Buchara” = D. carinata (Faldermann)]; Possidi beach, Halkidiki, Greece, 10/2/02 [‘‘Greece” = D. elongata (Brullé)]; 3 km west of Sfakaki, Crete, Greece, 4/5/02 [‘‘Crete” = D. elongata (Brullé)]; 15 km south of Sfax, Tunisia, 9/21/02 [‘‘Tunisia” = D. sublineata (Lucas)]. All populations except Fukang were imported from foreign collecting sites to the USDA-ARS Biological Control of Weeds Quarantine Facility, Albany, California in 2002. The Fukang colony was initiated with beetles collected at a field release site near Lovelock, Nevada, where they have been established since 2001 (Carruthers et al., 2008). In the Albany quarantine, foreign shipments were cleared of parasitoids (Erynniopsis antennata (Rondani), Diptera: Tachinidae) by manual removal, and a Nosema spp. pathogen by rearing under a high-temperature thermoperiod (40 °C for 8 h per day, 28 °C for 16 h) for one generation, followed by screening for presence of spores in feces. Beetles were reared on T. parviflora de Candolle and T. ramosissima Ledebour cuttings collected from California field sites at Cache Creek (Yolo County) and Tinemaha Reservoir (Inyo County), respectively.

2.3.1. Crop plant and habitat associate tests The Crete Diorhabda population was tested in May and July of 2002 on the following economically important plant species: Prunus domestica L. (plum, ‘‘Muro”, Rosaceae), Fragaria X ananassa Duchesne (hybrid strawberry, ‘‘sequoia”, Rosaceae), Ulmus minor Miller (English elm, Ulmaceae), Solanum lycopersicum L. (tomato, ‘‘Ace vfn”, Solanaceae), Lactuca sativa L. (garden lettuce, Asteraceae), Brassica oleraceae L. var. capitata (cabbage, ‘‘early flat Dutch”, Brassicaceae), Cucumis sativus L. (cucumber, ‘‘lemon”, Cucurbitaceae), Vitis vinefera L. (grape, ‘‘red globe”, Vitaceae) and on T. ramosissima controls. This test used 20 larvae per sleeve, with three replicates of each plant species. Environmental conditions measured during the test were as follows. Mean (±SEM) temperature: 24.0 ± 0.1 °C, range 17.6–33.0; mean (±SEM) relative humidity: 56.9 ± 0.4%, range 10.9–87.3. The same plant species were used to test the Tunisia, Greece, Karshi, Buchara, and Turpan Diorhabda populations in June and July of 2003, with the addition of Helianthus annus L. (sunflower, ‘‘Taiyo”, Asteraceae), Populus fremontii S. Watson (Fremont cottonwood, Salicaceae), Salix exigua Nutt. (narrow-leaved willow, Salicaceae), Juglans californica S. Watson (California Black Walnut, Juglandaceae), and Quercus lobata Nee (Valley Oak, Fagaceae). Each test plant was inoculated with 10 neonate larvae and the entire set was replicated 3 times per Diorhabda population. Test conditions: 23.4 ± 0.1 °C, range 17.2–32.4; 52.0 ± 0.2% RH, range 27.0–73.4.

2.3. Larval survival tests

2.3.2. Frankenia tests A similar experimental design was used to test larvae from three Diorhabda populations (Fukang, Crete and Karshi) more extensively for their ability to develop to the adult stage on the following Tamarix and Frankenia species (plant collecting locations listed here pertain to material used in all tests presented in this paper): T. ramosissima (Tinemaha Reservoir, Inyo County, CA), T. parviflora (Cache Creek, Yolo County, CA), T. aphylla (Esparto, Yolo County, CA), F. jamesii (Pueblo, Pueblo County, CO) and F. salina (inland form: Independence, Inyo County, CA; coastal form: Pt. Isabel, Contra Costa County, CA). Both the inland and coastal forms of F. salina were used in these tests, although the Karshi population was tested only on the more susceptible inland form. Each test plant received 15 neonate larvae and was replicated 5 times per Diorhabda population. The Crete and Fukang populations were tested in September of 2002 (test conditions: 23.5 ± 0.1 °C, range 13.0–31.7; 54.3 ± 0.2% RH, range 25.2– 86.0) and Karshi was tested in June of 2003 (test conditions: 23.4 ± 0.1 °C, range 17.2–32.4; 53.8 ± 0.2% RH, range 29.8–73.4). In April of 2008, Crete Diorhabda larvae were tested for their ability to develop on F. palmeri. This native plant is rare in California, with only one known population near San Diego, but is common in portions of Mexico, including coastal Baja California and Sonora (Whalen, 1987; Skinner and Pavlik, 1994;). F. palmeri test plants were purchased from a nursery in San Diego and T. parviflora was used as a control, with five replicates of each plant species and 15 neonate larvae per plant.

Prior to release in 2001, DeLoach et al. (2003) extensively evaluated the larval host range of the Fukang Diorhabda population using 79 test plant species, and found that Frankenia spp. were the only North American non-target plants that could support development to the adult stage. Old World D. elongata host records also include the genus Myricaria (Tamaricaceae), whose distribution is limited to Eurasia. DeLoach et al. (2003) confirmed that Fukang D. elongata larvae can complete development on Myricaria germanica (L.), from China. The Asian genus Reaumuria (Tamaricaceae) contains the only other plant species closely related to saltcedar, although no members of this genus have been recorded as natural hosts for Diorhabda (Kovalev, 1995). We evaluated larvae from the six additional Diorhabda populations for their ability to feed and survive on a variety of agricultural plants commonly grown in California and on Tamarix spp. controls (including the invasive T. ramosissima and T. parviflora, as well as T. aphylla (L.) Karsten (athel), which is commonly used for windbreaks and as a shade tree in arid agricultural areas). Larvae were also tested on plants native to California that are habitat associates of saltcedar in riparian areas and on F. salina and F. palmeri from California and F. jamesii from Colorado. A total of 19 plant species from 12 families were used in these larval tests. To avoid variability associated with desiccation of cut test plant material, only whole potted plants were used in all tests. Plants were collected from the field or purchased from nurseries, and were grown in SupersoilÒ potting mix in 15  20 cm (height  diameter) pots, fertilized monthly with liquid fertilizer (Miracle-GroÒ 24-816), applied until the soil was saturated. Neonate larvae were contained on test plants in fine mesh sleeves, with a layer of sand at the bottom of each sleeve to provide a pupation substrate. Sleeved plants were held in a temperature-controlled greenhouse (typical temperature range: 17–33 °C) in the USDA-ARS quarantine facility in Albany under natural light augmented with high pressure sodium grow lights (photoperiod of 16:8 L:D), until larvae developed to adults or died. Temperature and humidity was monitored using data loggers (Hobo ProÒ, Onset Computer Corporation). Summaries of environmental conditions are provided in the description of each test, as follows.

2.4. Adult ovipositional tests The ovipositional preference of Diorhabda adults was assessed in the USDA-ARS Albany quarantine greenhouse, under conditions as described previously for the larval survival tests. Greenhouse temperature was set to reach 33 °C for 5 h per day to insure that these desert-adapted beetles were warm enough to fly within their cages. Aluminum screen cages (16  18 mesh, BioquipÒ) were used to contain beetles and test plants in both choice and no-choice experimental designs. The majority of ovipositional tests used a cage size of 61  61  61 cm, but larger cages measuring 92 deep  92 wide  122 cm tall were used for choice tests when multiple species of plants were offered simultaneously. Higher numbers of beetles per

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cage were used in the large cage tests, in order to keep the relative density of beetles per cage volume similar between tests. An equal number of male and female beetles were placed in each cage (male D. elongata are easily distinguished by an apical notch in the last visible abdominal sternite (Tracy and Robbins, 2009)). Within each test, an effort was made to choose plants of similar size and structure, and the Frankenia spp. with low, sprawling growth forms were elevated on inverted pots to help standardize test plant height. All tests were replicated with 3–5 randomly placed cages per treatment, with all treatments running concurrently. After an exposure period that typically lasted 1–2 weeks, depending upon observed fecundity levels, all Diorhabda egg clusters were removed from test plants and cage walls, and individual eggs were counted under a dissection microscope. The adult experiments were focused on determining the relative ovipositional preference for Tamarix spp. and Frankenia spp., and did not include crop plants or native habitat associates, except as negative controls in some of our tests. These tests primarily used Frankenia spp. that are native to California, whereas Frankenia native to Texas, Colorado and New Mexico were tested by Milbrath and DeLoach (2006). 2.4.1. No-choice tests The ovipositional response of the Crete Diorhabda population was evaluated in August of 2002 using a no-choice design with 1 plant per cage (61  61  61 cm), 20 adult beetles per cage, replicated 3 times per plant species. Although the value of no-choice tests has been questioned by some, we felt it was important to evaluate the beetle’s response to Frankenia spp. with this test design, since rapid defoliation of saltcedar by D. elongata under natural conditions could potentially create no-choice situations in the field. Plant species used for the test were as follows: T. ramosissima, T. parviflora, T. aphylla, F. jamesii, F. salina (both coastal and inland forms), and U. minor (English elm, a known negative control). Test duration was 10 days. Environmental conditions measured during the experiment were: 24.7 ± 0.2 °C, range 17.1–31.1; 52.6 ± 0.5% RH, range 23.8–78.2. For comparative purposes, the same test plant species were used in a no-choice design to evaluate the ovipositional response of the Fukang and Karshi Diorhabda populations. In September of 2003, the Fukang beetles were tested in 61  61  61 cm cages containing 14 beetles, with each plant species replicated 3 times over test period of 7 days. Environmental conditions measured during the experiment were: 25.5 ± 0.3 °C, range 18.3–33.6; 48.5 ± 1.0% RH, range 19.0–82.9. In August of 2003, a similar test was conducted with the Karshi population using three replicates of T. ramosissima, T. parviflora, T. aphylla, F. jamesii, and F. salina (inland form only). In addition to the primary test plant, each cage (61  61  61 cm) also contained a known negative control plant, S. exigua (narrow-leaved willow), to help us better evaluate the significance of eggs laid on inert substrates, such as the cage walls. Each cage contained 20 beetles and the test was run for 6 days under the following conditions: 24.8 ± 0.2 °C, range 18.4–33.5; 52.8 ± 0.9% RH, range 20.7–75.8. Within each no-choice test, the ovipositional response was assessed by comparing the number of eggs laid on each plant species. In order to make comparisons between beetle populations with respect to their relative acceptance of F. salina, the number of eggs laid on this plant was converted to a percentage of total eggs laid in each replicate. This conversion standardizes differences in test duration and density of adults per cage and also minimizes the effect of possible confounding factors such as variable fecundity rates between beetle populations. 2.4.2. Choice tests A multiple-choice test using seven plant species per cage (92  92  122 cm) was used to test the host preference of the

Crete Diorhabda population in July and August of 2002. The plant species used in this test were the same as those used in the Crete no-choice test. Each set of plants was replicated 5 times, and each cage held 100 beetles. The experiment was run for a total of 18 days, but egg collections were made on three occasions during this period (at day 7, 11, and 18) to prevent feeding damage by hatched larvae. Egg totals from all three collections were combined for analysis. Test conditions were: 24.0 ± 0.1 °C, range 17.6–30.6; 56.7 ± 0.4% RH, range 23.8–87.3. A paired-choice design with two plant species per cage was used to compare Crete Diorhabda ovipositional preference between T. ramosissima and F. palmeri in September of 2006. The experiment was replicated 5 times, each cage (92  92  122 cm) contained 40 beetles, and the test period was 13 days. Test conditions were: 22.9 ± 0.2 °C, range 16.0–29.9; 53.5 ± 0.5% RH, range 22.6–81.3. A similar paired-choice design (two plant species per cage, a cage size of 61  61  61 cm, 20 beetles per cage, 5 replicates per beetle population, 6–7 day exposure period) was used to compare the preference between T. ramosissima and F. salina (inland variety) for all seven Diorhabda populations between March and May of 2003. Due to space limitations in the quarantine greenhouse, we were able to test only three of the populations concurrently. Therefore, the Fukang population was included in each experimental group as an internal control to check for possible differences in beetle behavior in response to subtle changes in environmental conditions between test periods. We chose T. ramosissima and F. salina (inland form) as a representative target/non-target pair of host plants because these two species are known to co-occur in parts of California. The environmental conditions measured during each of the three beetle population tests were as follows: Turpan/Fukang/Tunisia, 3/6/03 to 3/12/ 03: 23.0 ± 0.2 °C, range 17.5–30.3; 43.7 ± 0.5% RH, range 25.2–66.9. Crete/Fukang/Karshi, 4/18/03 to 4/25/03: 23.3 ± 0.3 °C, range 14.8– 36.0; 40.2 ± 0.6% RH, range 19.0–66.9. Greece/Fukang/Buchara, 5/ 14/03 to 5/20/03: 24.2 ± 0.2 °C, range 17.7–31.8; 39.7 ± 0.5% RH, range 18.6–61.3. 2.5. Statistical analysis Larval survival and adult ovipositional data were analyzed with a t-test or a one-way ANOVA, blocked by replicate. A separate analysis was performed for each beetle population. Mean separation was determined by the Tukey–Kramer HSD test. Before analysis, percentage data were converted to proportions and transformed by arcsine square root and egg count data were transformed by square root (x + 0.5) to improve the independence of variance and means (Little and Hills, 1978). Analysis was performed with JMP version 4.0.2 statistical package (SAS Institute, 2000). 3. Results 3.1. Larval survival tests 3.1.1. Crop plant and habitat associate tests No larvae survived on any of the agricultural or native habitat associate test plants. In all cases, larvae appeared to be unable to feed, and died within 3 days without causing any damage to the plants. These results illustrate that all the California crop plants we tested are safe from attack by D. elongata, and confirm the findings of previous studies that showed that none of the Diorhabda populations are able to survive on any plant species outside of the order Tamaricales (DeLoach et al., 2003; Lewis et al., 2003a; Milbrath and DeLoach, 2006). Percent survival to the adult stage on T. ramosissima controls was (mean ± SEM): Turpan 96.67 ± 3.33, Karshi 73.33 ± 8.82, Buchara 83.33 ± 12.02, Crete 61.67 ± 24.89, Greece 83.33 ± 12.02, and Tunisia 80.00 ± 0.00.

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3.1.2. Frankenia tests In the Frankenia spp. tests with Tamarix spp. controls, significant differences in larval survival to the adult stage were found between some plant species for all three Diorhabda populations tested (Fig. 1) (ANOVA, Crete: P = 0.0061, F = 4.315, df = 5, 24; Fukang: P = 0.0004, F = 6.974, df = 5, 24; Karshi: P = 0.0075, F = 4.738, df = 4, 20). For all populations, the most successful survival was recorded on T. ramosissima, where mean (±SEM) levels ranged from 86.7 ± 4.2% in the Fukang population, to 96.0 ± 4.0% in Karshi. Similarly, the host plant species that produced the lowest survival was also consistent across Diorhabda populations: larvae reared on F. jamesii had 14.7 ± 5.7% survival in the Fukang population and 66.7 ± 5.6% in Karshi. However, many of the target and non-target species produced larval survival rates that were not significantly different from each other, illustrating that the Frankenia species tested are clearly within the physiological host range of all the beetle populations evaluated in this experiment. For example, the high survival rates recorded on the inland variety of F. salina (Crete: 80.00 ± 7.60%; Fukang: 84.00 ± 6.86%; Karshi: 92.00 ± 1.33%) were not significantly different from any of the three Tamarix species, in all three Diorhabda populations tested. When comparing survival rates between Diorhabda populations on each test plant species, no significant differences were found except on F. jamesii, where survival of Karshi larvae was significantly higher than Fukang larvae (ANOVA, P = 0.0050, F = 8.499, df = 2, 12). In the F. palmeri test with Crete Diorhabda, larval survival was only 38.7 ± 8.3% on F. palmeri, compared with 92.0 ± 6.5% on T. parviflora controls (t-test, P = 0.0010, t = 5.080, df = 8). Survival rates on Frankenia spp. measured in our studies were consistently more than twice as high as those recorded in a previous study (Milbrath and DeLoach, 2006), using the same Diorhabda populations and Frankenia spp. Differences in host plant quality and test methods (whole plant vs. cut foliage in vials) are thought to have contributed to this discrepancy.

3.2. Adult ovipositional tests 3.2.1. No-choice tests The Crete Diorhabda population showed very few differential responses between Tamarix and Frankenia species in the no-choice test (Fig. 2). Although the ANOVA was highly significant (P = 0.0001, F = 13.32, df = 6, 14), the mean (±SEM) number of eggs laid on the inland variety of F. salina (187.3 ± 38.2) and on F. jamesii

Fig. 1. Larval survival rates in three Diorhabda populations when reared on nontarget native Frankenia spp. and Tamarix spp. controls. Means of five replicates per test plant species per beetle population, with 15 larvae per plant (the Karshi population was not tested on the coastal form of F. salina). Bars marked with the same letter within a Diorhabda population are not significantly different, by ANOVA (see text for test statistics). Error bars indicate ± SEM.

Fig. 2. Ovipositional preference of the Crete, Fukang, and Karshi Diorhabda populations measured in no-choice tests comparing target and non-target plants. Means marked with the same letter within a beetle population are not significantly different by ANOVA (see text for test statistics). Error bars indicate ± SEM.

(125.7 ± 15.2) was not significantly different from the ovipositional level recorded on any of the Tamarix species (means ranged from 83.0 ± 20.0 to 298.0 ± 48.8 eggs per plant). Only the non-host U. minor (English elm), which received no eggs, was significantly different from all the other test plants. These results indicate that in the absence of preferred host plants in the field, Crete Diorhabda may oviposit on some Frankenia species. The mean number of eggs laid on cage walls was 23.2 ± 4.87 (n = 21 cages), and was not significantly different between any of the test plant treatments (ANOVA, P = 0.5590, F = 0.84, df = 6, 14), indicating that confined D. elongata commonly lay a small number of eggs on inert substrates, regardless of the relative suitability of the host plant that they are caged with. Similar results were obtained in the Fukang no-choice test (Fig. 2), where oviposition on the inland form of F. salina was not significantly different from that on any of the Tamarix spp. (ANOVA, P < 0.0001, F = 11.73, df = 6, 14). However, the coastal form of F. salina and F. jamesii received significantly fewer eggs than either T. ramosissima or T. parviflora. While these results indicate that there is minimal risk to F. jamesii, it is worth noting that this non-target received a mean of 28.7 (±13.9) eggs per plant, which raises the possibility of potential impact in portions of its native range where the Fukang beetles have been established, such as near Pueblo, Colorado. To date, however, no damage to F. jamesii

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has been found in the vicinity of this site (A. Sher and M. DePrenger-Levin, Denver Botanic Garden, personal communication, 2008), despite the build up of large beetle populations sufficient to cause localized defoliation of saltcedar (DeLoach et al., 2008). In the Karshi population no-choice test (Fig. 2), oviposition was significantly higher on T. parviflora (193.3 ± 17.4 eggs per plant) and T. aphylla (138.7 ± 33.5) when compared to the inland variety of F. salina (19.3 ± 2.3) (ANOVA, P = 0.0008, F = 11.94, df = 4, 10). However, there was no significant difference between the number of eggs laid on F. salina and T. ramosissima (80.7 ± 36.1) suggesting that in the absence of saltcedar, Karshi Diorhabda might also utilize this non-target plant in the field. No eggs were laid on F. jamesii in this test, confirming the lack of response to this native plant found by Milbrath and Deloach (2006). No eggs were laid on the negative control S. exigua (narrow-leaved willow), providing further evidence that Diorhabda will not utilize plants outside the order Tamaricales. As in the Crete no-choice test, the number of eggs laid by the Karshi beetles on the cage walls was not significantly different between plant treatments (ANOVA, P = 0.8859, F = 0.27, df = 4, 10). In order to test for differences between the Crete, Fukang, and Karshi populations with respect to their ovipositional response to F. salina (inland form), the number of eggs laid on this plant was converted to a percentage of total eggs laid on all plants and cage walls in each replicate. The resulting percentages were as follows (mean ± SEM): Crete 18.5 ± 5.5; Fukang 12.8 ± 3.7; Karshi 2.5 ± 0.6. The response to F. salina by the Crete population was significantly higher than that of Karshi, but the Fukang population response was not significantly different from that of the other two populations (ANOVA, P = 0.0310, F = 6.55, df = 2, 6). Because T. aphylla (athel) is occasionally considered beneficial for windbreaks and shade, we compared the preference for it between beetle populations using the same procedure. The percent of total eggs laid on T. aphylla was as follows: Crete 7.3 ± 0.8; Fukang 7.8 ± 2.2; Karshi 16.9 ± 2.1. The Karshi beetles laid a significantly higher proportion of their eggs on T. aphylla than the Crete and Fukang Diorhabda (ANOVA, P = 0.0156, F = 9.01, df = 2, 6), indicating that they may have a higher chance of attacking athel in the field than the other two beetle populations.

3.2.2. Choice tests When offered a set of host plants simultaneously in the multiple-choice test (Fig. 3), oviposition by Diorhabda from Crete was more selective than in the no-choice test, and they laid significantly more eggs on T. ramosissima than on any of the other test plant species (ANOVA, P < 0.0001, F = 37.28, df = 7, 32). However,

Fig. 3. Ovipositional preference of the Crete Diorhabda population measured in a multiple-choice test comparing Tamarix spp., Frankenia spp., and Ulmus minor (English elm, a negative control). Bars marked with the same letter are not significantly different by ANOVA (P < 0.0001, F = 37.28, df = 39). Error bars indicate ± SEM.

oviposition on T. parviflora and T. aphylla was not significantly different from the inland form of F. salina (which received 11.39 ± 1.57% of eggs), suggesting that we cannot rule out the possible utilization of this non-target plant in the field. In contrast, the response to both F. jamesii and the coastal form of F. salina was relatively low in this test (<1.4% of eggs), with both species receiving significantly fewer eggs than the other test plants (except the nonhost English elm, which received no eggs). The significantly higher preference for the inland form of F. salina when compared to the coastal form suggests that there may be biological differences between them that confer differential levels of host acceptance and risk of impact by the Crete Diorhabda. As noted above, these two forms were previously classified as different species within the genus Frankenia (Munz and Keck, 1968). The mean number of eggs laid on cage walls in this test was 148.4 ± 22.1, and was not significantly different from the number of eggs laid on the Frankenia spp. Although a previous study (Lewis et al., 2003a) used relative ovipositional rates on cage walls to indicate non-preference for particular host plants, this interpretation may or may not be valid. Since the surface area of cage walls is much greater than that of host plants, it is difficult to directly compare oviposition between them. In addition, the natural dispersal response of confined beetles results in relatively high numbers of adults resting on cage walls throughout the experiment, and a small percentage of these beetles will commonly lay eggs, even if they are not on a plant. To the extent that this ovipositional behavior might be attributable to relatively small cage size, more accurate measures of Diorhabda host plant preference should be obtained through field cage or open-field tests. In the paired-choice test, Crete Diorhabda also laid significantly more eggs on T. ramosissima (mean ± SEM of 1100.24 ± 61.17 eggs per plant) than F. palmeri (337.10 ± 33.91 eggs per plant) (t-test, P < 0.0001, t = 13.049, df = 8). However, the percentage of eggs laid on F. palmeri (mean of 23.6 ± 2.65%) was the highest recorded on a non-target plant in any of our choice tests, prompting concern for potential impact in portions of San Diego and Baja California, where saltcedar and F. palmeri are know to co-occur. Although the poor larval survival on F. palmeri reported above may indicate that utilization of this host is unlikely, additional tests were conducted using potted F. palmeri plants under open-field conditions to further assess the host preference of Crete Diorhabda in more realistic situations (Herr and Carruthers, in press). In the series of paired-choice tests used to evaluate all seven Diorhabda populations, significantly more eggs were laid on T. ramosissima than F. salina in every test (Fig. 4). ANOVA results for all populations were highly significant, with P < 0.001, df = 2, 12, and F ratios ranging from 12.13 to 115.89. Analysis of the three separate paired-choice tests with the Fukang population showed that there was no significant difference between the trials with respect to egg production (ANOVA, P = 0.7220, F = 0.33, df = 2, 12) or percent of eggs laid on F. salina (ANOVA, P = 0.7885, F = 0.24, df = 2, 12). These results indicate that test conditions did not vary significantly during the 3-month period in which the trials were conducted, allowing for valid comparisons between beetle populations tested in different months. For example, we analyzed fecundity rates and found that there were significant differences between some of the beetle populations in the number of eggs laid per female per day (Fig. 5) (ANOVA, P = 0.0003, F = 6.551, df = 6, 28). Egg production ranged from 4.7 ± 0.5 by the beetles from Tunisia to 13.9 ± 0.6 eggs/female/day by the Crete population. All beetle populations from Greece and Uzbekistan had significantly greater fecundity than the Tunisia population, and the Crete Diorhabda also had significantly greater egg production than beetles from Fukang. It was also observed that the Uzbekistan Diorhabda differed from the other populations in their habit of laying many of their eggs on the trunks and stems of plants, rather than on the foliage.

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In order to assess the relative level of acceptance that each Diorhabda population has for F. salina, the number of eggs per plant was converted to the percentage of total eggs laid per cage (since some of the beetle populations have different fecundity rates, it would not be valid to directly compare egg counts). The percentage of eggs (mean ± SEM) laid on F. salina ranged from 0.79 ± 0.79% by the Karshi population to 15.74 ± 4.09% by the Tunisia population. The Crete beetles laid 10.74 ± 4.5% of their eggs on F. salina, a rate nearly identical to that recorded in the multiple-choice test. During analysis, the Diorhabda populations were pooled by species according to the recent revision by Tracy and Robbins (2009). Results of this analysis (Fig. 6) show that significant differences exist between some beetle populations with respect to their relative preference for the inland form of F. salina (ANOVA, P = 0.0050, F = 5.202, df = 3, 31). The Greek (including Greece and Crete populations) and Tunisian Diorhabda laid a significantly higher percentage of their eggs on F. salina than the Uzbekistan (Karshi and Buchara) beetles. However, the ovipositional response to F. salina by the Chinese Diorhabda (Fukang and Turpan populations) was not significantly different from any of the other beetle groups. These results are identical to those produced by the adult nochoice tests, where the response to F. salina was significantly higher in the Crete population than the Karshi population, but neither was significantly different from the Fukang population.

4. Discussion

Fig. 4. Ovipositional preference of six Diorhabda populations measured in pairedchoice tests comparing Tamarix ramosissima and Frankenia salina (inland form). The Fukang population was included in each group as an internal control for possible differences in environmental conditions between test dates. Bars marked with the same letter within a population are not significantly different by ANOVA (see text for test statistics). Error bars indicate ± SEM.

Fig. 5. Fecundity of different Diorhabda populations, as measured in a series of paired-choice tests (total eggs laid on both plant species and cage walls). Means marked with the same letter are not significantly different (ANOVA, P = 0.0003, F = 6.551, df = 34). Error bars indicate ± SEM.

The relatively high larval survival we recorded on F. salina indicates that D. elongata larvae can significantly damage this native plant, and reaffirms that adult selection of host plants for oviposition will determine the extent of potential non-target impact in the field. However, it is clearly possible that F. salina plants growing in close proximity to saltcedar in locations where these plants are known to co-occur (CalFlora, 2006) could sustain transient feeding damage by D. elongata larvae moving out of highly defoliated T. ramosissima. The lower survival rates on F. jamesii and F. palmeri may translate to a reduced risk of impact by D. elongata in the field. Both of these taxa are classified in a separate species group from F. salina within the American Frankenia (Whalen, 1987), so it is possible that they share biological characteristics that make them less suitable hosts. In all of the adult choice tests, significantly more eggs were laid on T. ramosissima than F. salina. However, it is important to note that statistical preference for saltcedar does not necessarily preclude biological impact to non-target species. For example, the Crete Diorhabda laid a mean of 106.2 ± 41.8 eggs per F. salina plant

Fig. 6. Percent of eggs laid on Frankenia salina (inland form) by four Diorhabda species, as measured in a paired-choice test comparing Tamarix ramosissima and F. salina. Means marked with the same letter are not significantly different by ANOVA (see text for test statistics). Error bars indicate ± SEM.

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in the paired-choice test, clearly a density sufficient to cause defoliation of caged plants. In order to determine if this ovipositional response was influenced by cage effects, large field cage and open-field tests were subsequently permitted and used to further evaluate the risk to Frankenia spp. by the Crete Diorhabda population that were selected for use in more southern U.S. locations (Moran et al., 2009; Herr and Carruthers, in press). Both these studies confirmed that T. ramosissima was the preferred host plant, although Herr and Carruthers (in press) found that F. salina did sustain feeding damage from both adults and larvae in an open-field test when beetle population levels were extremely high. Therefore, it is advised that no matter which Diorhabda populations are being considered for release, that follow-up tests and assessments should be conducted under realistic conditions in an isolated region, before widespread release and redistribution is warranted into natural areas containing sensitive native Frankenia species. Taken as a whole, the host range studies reported here show that all the Frankenia spp. we tested are within the physiological host range of the entire D. elongata species group, but that subtle differences between plant species and between beetle taxa may translate to varying degrees of potential non-target impact. The inland form of F. salina appears to be the non-target species most likely to be within the ecological host range of D. elongata, based on the relatively high levels of larval survival and adult oviposition, its co-occurrence with T. ramosissima, and its habitat, which is suitable for beetle development (in contrast to the coastal form, which often grows in tidal inundation zones that are unsuitable for beetle pupation). Differences between some of the beetle populations should be taken into account to help decrease environmental risk when planning releases for saltcedar biological control. For example, the Tunisia Diorhabda would appear to be a less desirable agent, based on its significantly lower fecundity and higher response to F. salina compared to the Karshi population. On the other hand, the Karshi beetles showed a significantly higher preference for T. aphylla than some of the other Diorhabda populations, indicating that they may not be the best choice for areas where athel is considered beneficial. These subtle differences in host specificity affirm the recommendation of Harley and Forno (1992) that host range testing should be conducted on all populations of a biological control agent that have been selected for release. Determining the boundaries of a population requiring separate testing may be difficult to accomplish, but could be guided by the degree of geographic isolation, relative position along a cline, differences in genetic variability, or observed differences in host plant utilization in the native range (as in the case of B. villosus (Sheppard et al., 2006)). At a minimum, we recommend that all populations of a prospective agent be tested against the non-target taxa that are most susceptible to attack, as was done in our tests with the inland form of F. salina. Acknowledgments We would like to thank Rouhollah Sobhian and Alan Kirk (USDA-ARS EBCL, Montferrier-sur-Lez, France), Massimo Cristofaro (ENEA C.R. Casaccia, Rome, Italy) and Li Bao Ping (Nanjing Agricultural University, Nanjing, China) for collecting populations of Eurasian Diorhabda; Alexander Konstantinov (USDA-ARS SEL, Washington, DC) for identification of insect specimens; John Gaskin (USDA-ARS Sidney, MT) for identification of saltcedar specimens; Debra Eberts (USDI Bureau of Reclamation, Denver, CO) for Frankenia jamesii plants; and the excellent technical staff at the USDA-ARS, Albany, CA, including: Tammy Chew and Julie Keller for insect colony maintenance, and Dave Leclergue, Hillary Thomas, Angelica Herrera, Anni Ala and Connie Ganong for assistance with the host range tests. Mike Pitcairn (California Dept. of Food and Agriculture, Sacramento, CA) and Andrew Lawson (California

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