Effects of Ambient Temperature on Egg and Larval Development of the Invasive Emerald Ash Borer (Coleoptera: Buprestidae): Implications for Laboratory Rearing Author(s): Jian J. Duan, Tim Watt, Phil Taylor, Kristi Larson, and Jonathan P. Lelito Source: Journal of Economic Entomology, 106(5):2101-2108. Published By: Entomological Society of America URL: http://www.bioone.org/doi/full/10.1603/EC13131
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FOREST ENTOMOLOGY
Effects of Ambient Temperature on Egg and Larval Development of the Invasive Emerald Ash Borer (Coleoptera: Buprestidae): Implications for Laboratory Rearing JIAN J. DUAN,1,2 TIM WATT,3 PHIL TAYLOR,1 KRISTI LARSON,3
AND
JONATHAN P. LELITO4
J. Econ. Entomol. 106(5): 2101Ð2108 (2013); DOI: http://dx.doi.org/10.1603/EC13131
ABSTRACT The emerald ash borer, Agrilus planipennis Fairmaire, an invasive beetle from Asia causing large scale ash (Fraxinus) mortality in North America, has been extremely difÞcult to rear in the laboratory because of its long life cycle and cryptic nature of immature stages. This lack of effective laboratory-rearing methods has not only hindered research into its biology and ecology, but also mass production of natural enemies for biological control of this invasive pest. Using sticks from the alternate host plant, Fraxinus uhdei (Wenzig) Lingelsh, we characterized the stage-speciÞc development time and growth rate of both emerald ash borer eggs and larvae at different constant temperatures (12Ð35⬚C) for the purpose of developing effective laboratory-rearing methods. Results from our study showed that the median time for egg hatching decreased from 20 d at 20⬚C to 7 d at 35⬚C, while no emerald ash borer eggs hatched at 12⬚C. The developmental time for 50% of emerald ash borer larvae advancing to third, fourth, and J-larval stages at 20⬚C were 8.3, 9.1, and 12.3 wk, respectively, approximately two times longer than at 30⬚C for the corresponding instars or stages. In contrast to 30⬚C, however, the development times of emerald ash borer larvae advancing to later instars (from oviposition) were signiÞcantly increased at 35⬚C, indicating adverse effects of this high temperature. The optimal range of ambient temperature to rear emerald ash borer larvae should be between 25Ð30⬚C; however, faster rate of egg and larval development should be expected as temperature increases within this range. KEY WORDS Agrilus planipennis, development, Fraxinus uhdei, invasive, laboratory rearing
The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is a relatively new invasive forest pest in North America, where it has caused large scale ash (Fraxinus) mortality and spread to 19 U.S. states (Kovacs et al. 2010, Emerald Ash Borer Information 2013) and two Canadian provinces (Canadian Food Inspection Agency 2013) since it was Þrst discovered in Michigan (Haack et al. 2002). In most of the infested areas, emerald ash borer adults emerge in late spring and early summer (MayÐJune), and feed on ash foliage for at least 2 wk before mating and laying eggs in crevices and under bark ßakes on limbs and trunks of ash trees in early summer (JuneÐJuly). After eclosion, Þrst instar larvae chew through the bark to reach the phloem, where they feed and develop throughout the growing season (Cappaert et al. 2005). After larvae go through three molts, the mature fourth instar larvae chew pupation chambers in the outer sapwood or bark. The mature larvae then develop a folded ap1 USDAÐARS, BeneÞcial Insects Introduction Research Unit, 501 South Chapel St., Newark, DE 19713. 2 Corresponding author, e-mail: [email protected]. 3 University of Delaware, Department of Entomology and Wildlife Ecology, Newark, DE 19713. 4 USDAÐAPHIS PPQ Emerald Ash Borer Biocontrol Laboratory, Brighton, MI 48116.
pearance for overwintering via obligatory diapause (J.J.D. and J.P.L., unpublished data). Larvae at this J-shaped stage were termed “J-larvae” by Duan et al. (2010), but called “prepupae” by Chamorro et al. (2012). Usually after a winter chill period, J-larvae develop into prepupae, which are visibly shorter and more cylindrical. Pupation generally occurs in early spring, with adults emerging from late spring through early summer. Adults live for several weeks feeding on mature ash leaves, and rarely cause any signiÞcant damage to the host tree (Bauer et al. 2004). Larvae, in contrast, feed and develop for several months on ash tree phloem, creating extensive galleries under the bark. When emerald ash borer populations are high, larval consumption of tree phloem is substantial, resulting in tree girdling and death in 3Ð 4 yr (Poland and McCullough 2006, McCullough et al. 2009). Emerald ash borer populations in the newly invaded North America (e.g., Cappaert et al. 2005, Siegert et al. 2007, Duan et al. 2010) as well as in its native North East Asia (e.g., Liu et al. 2007, Wei et al. 2007, Duan et al. 2012) may require 1Ð2 yr to complete development. Although climatic conditions in different geographic regions as well as host tree conditions may have contributed to the dichotomy of emerald ash borer life cycle, few studies have examined how cli-
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matic conditions (particularly ambient temperature) inßuence its development and diapause of immature stages. This lack of study on climatic conditions (particularly temperature) is largely because of our inability to rear emerald ash borer in laboratory conditions. Recently, classical biological control efforts against emerald ash borers in the United States have led to the introduction and release of three species of parasitoids from the pestÕs native range (Northeast Asia): the idiobiont egg parasitoid Oobius agrili Zhang and Huang (Encyrtidae) and the larval parasitoids Tetrastichus planipennisi Yang (Eulophidae) and Spathius agrili Yang (Braconidae) (Zhang et al. 2005, Liu et al. 2007, U.S. Department of AgricultureÐAnimal and Plant Health Inspection Service [USDAÐAPHIS] 2007). To produce suitable emerald ash borer hosts for mass production of those introduced biocontrol agents, researchers have obtained various stages of emerald ash borer larvae or adults by collecting infested ash (Fraxinus) logs. The Þeld-collected ash logs are then stored in a 3Ð5⬚C cooler (to slow the larval development, break the larval diapause, or both), and incubated in room temperature to obtain emergence of adult emerald ash borers (e.g., Yang et al. 2012, J.P.L. unpublished data). Some researchers have experimented with “artiÞcial infestation,” where a series of slits are cut into an ash log, emerald ash borer larvae are secured within the slits, and the larvae are then allowed to develop within their natural host wood, or presented to parasitoids as need be (e.g., Ulyshen et al. 2010a,b; Duan and Oppel 2012). Although these methods have allowed researchers to produce emerald ash borer larvae and adults, they are resource intensive and limited by seasonality. Historically, wood-boring buprestids such as the emerald ash borer have been extremely difÞcult to rear in the laboratory largely because of its long life cycle and cryptic nature of immature stages. Recently, Duan et al. (2011) reported on an alternative host plant-based emerald ash borer rearing method, which is considerably streamlined in comparison with present rearing strategies. The alternative host plant used in rearing emerald ash borer is the evergreen tropical ash, Fraxinus uhdei (Wenzig) Lingelsh, which can be cultivated year around in greenhouses without dormancy in growth. In the current study, we examine the effect of ambient temperature on the development of emerald ash borer eggs and larvae when using this alternate host plant-based method for rearing. Specifically, we characterize the stage-speciÞc development time and growth rate of emerald ash borer larvae on infested tropical ash sticks at different temperature regimes. This information is not only important to our understanding of emerald ash borer ecology (e.g., the effect of climatic condition on its population growth), but also critical to production of suitable stages of larvae for various research programs such as massrearing egg and larval parasitoids for biological control of this invasive pest (USDAÐAPHIS 2007).
Vol. 106, no. 5 Materials and Methods
Emerald Ash Borer. All adults used for egg and larval production in this study originated from emerald ash borer-infested green ash (Fraxinus pennsylvanica Marshall) trees collected in Maryland. Green ash trees with apparent emerald ash borer infestation symptoms (e.g., woodpecker scaling and feeding damage to trunks, epicormic growth, or reduced crowns) were felled from late fall (November) to later winter (February) of each year, cut to pieces of meter-long logs, and then stored in a climatically controlled walk-in cooler (Polar King International, Fort Wayne, IN) at 3Ð5⬚C for season-long production of emerald ash borer adults. For adult production, stored-ash logs were placed into emerging cages made of cardboard Sonotube (1.2 m long by 50 cm in diameter), and then incubated at 26 Ð29⬚C with a photoperiod of 16:8 (L:D) h in an environmentally controlled room at Maryland Department of Agriculture (Annapolis, MD). Emerging emerald ash borer adults from incubated-ash logs were collected every 1 or 2 d at the Maryland Department of Agriculture, and immediately sent to USDAÐAgricultural Research Service (ARS) BeneÞcial Insects Research Unit (Newark, DE) via same day or overnight delivery. On receipt at the USDAÐARS BeneÞcial Insects Research Unit, adults were immediately transferred to rearing containers (clear 3.5liter Jars with screen-ventilated lids) at the density of ⬇20 females ⫻ 20 males per container. Before oviposition, all adults were fed with bouquets of fresh green (F. pennsylvanica) or tropical (F. uhdei) ash leaves, which were replaced every 2 or 3 d. After approximately 2 wk of maturation feeding, gravid females were then transferred to 1-liter ventilated plastic (oviposition) cups at a density of ⬇5 females ⫻ 2Ð3 males per cup, and provided with fresh host plant leaves for egg production. To obtain emerald ash borer eggs, the mouth of the oviposition cup was covered with nylon screen (mesh size ⬇1 mm2) and then with a sheet of coffee Þlter paper (8.25 cm base, HomeLife, Eden Prairie, MN) on the top to serve as a suitable oviposition site (Duan et al. 2011). Gravid emerald ash borer females were attracted to lay eggs on the screen-covered substrates such as coffee Þlter paper (Yang et al. 2012). Emerald ash borer eggs freshly laid (within 24 h) on the coffee Þlter paper were then used in different egg and larval development experiments with ambient temperature treatments. Effect of Ambient Temperature on Egg Development. Coffee Þlter papers with freshly laid emerald ash borer eggs (within 24 h) were cut into small individual pieces (⬇3 mm long by 3 mm wide), each with one egg (for easy observation under stereomicroscope), and placed inside 8-cm (diameter) petri dishes lined with wet Þlter paper (for humidity). Petri dishes containing emerald ash borer eggs (10 Ð20 eggs per dish) were then placed inside ventilated Crisper Boxes, which were housed in growth chambers (ARB366, Percival ScientiÞc Inc., Perry, IA) set for different constant-temperature treatments with constant rela-
October 2013
DUAN ET AL.: METHODS FOR LABORATORY REARING OF EMERALD ASH BORER
tive humidity (RH; 60 ⫾ 5%) and a photoperiod of 16:8 (L:D) h. In total, 30 Ð70 emerald ash borer eggs (in three to Þve petri dishes) were tested at each of the six treatment temperatures: 12, 20, 25, 27, 30, and 35⬚C. On hatching, neonate emerald ash borer larvae chewed through the side of the egg shell that was attached to the Þlter paper and then penetrated the Þlter paper leaving an easily recognizable exit hole on the egg shell and Þlter paper. Observation of neonate emerald ash borer larvae hatching from eggs laid on coffee Þlter papers was conducted daily under the stereomicroscope for a maximum 30 d. At each observation day, the number of hatched eggs was recorded and then removed from the holding petri dishes. Effect of Ambient Temperature on Larval Development. Using tropical ash sticks, we measured the development (stage or instars) and growth (biomass and gallery width) of emerald ash borer larvae reared at Þve different ambient (constant) temperatures: 20, 25, 27, 30, and 35⬚C. The Þlter paper with emerald ash borer eggs was cut into small pieces (each with one to three emerald ash borer eggs), which were then placed closely against the surface of tropical ash sticks (20 Ð25 cm long by 1.5Ð3.5 cm in diameter) with the bottom side of eggs (attached to Þlter paper) against the bark of the sticks and wrapped with ParaÞlm strips (BEMIS, BEMIS Flexible Packaging, Neenah, WI). Across all temperature treatments, the number of eggs placed on each stick varied with the size of the ash sticks; generally smaller sticks (with a diameter ⬍2.0 cm) were infested with fewer eggs (5Ð10 eggs) and larger sticks (diameter ⬎2.0 up to 3.5 cm) with more eggs (12Ð20 eggs). Approximately 50 sticks were used for each of the Þve treatment temperatures throughout the study. Although the size of sticks varied across different temperature treatments, the density of infested eggs or larvae was adjusted per unit surface area of stick phloem, and was approximately the same (⬇320 eggs/m2 of surface area) among different treatments. After placement of eggs, ash sticks were placed upright in ßoral foam bricks (OASIS, SmithersÑOasis Company, Kent, OH) in storage boxes (58.4 by 41.3 by 31.4 cm, Sterilite, Sterilite Corporation, Townsend, MA) saturated with distilled water and a 0.1% solution of methylparaben to prevent molds. Storage boxes containing the emerald ash borer-infested ash sticks were then incubated in different environmental chambers set with the target ambient temperature but at the same RH (65 ⫾ 10%) and a photoperiod of 16:8 (L:D) h. Beginning 2Ð 4 wk after egg deposition, 20 Ð25 emerald ash borer larvae were dissected out of 5 to 10 ash sticks from each temperature on a weekly or biweekly basis. The dissection of ash sticks was done by peeling off the entire bark layer (including the cambium tissue) with a utility knife. On removal of the bark layer, exposed emerald ash larvae were removed from their galleries, scored for their stadium (instars) based on the width of their head capsules (Cappaert et al. 2005, Wang et al. 2010) and then weighed with an electronic balance (AB135-S/FACT, Mettler Toledo AG Labo-
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ratory Weighing and Technology, Greifensee, Switzerland) for their biomass. In addition, we also measured the width of each larvaÕs gallery at the position where its head is located, with a digital caliper (No. 147-Digital Fractional caliper Stainless Steel Ultra Tech, General Tools and Instruments, New York, NY). When reaching mature stage, fourth instar emerald ash larvae normally make chambers inside the stick (sapwood) and then fold into a J-shape (termed as J-larvae) for overwintering (via obligatory diapause). We removed the J-larvae from their chambers for measurement by splitting the sapwood tissue with a hand pruner and utility knife. We then measured the width of J-larval gallery before the entrance of their chambers, and weighed their biomass with the balance. Data Analysis. The time-related hatching event of emerald ash borer egg cohorts at different ambient temperatures was analyzed using survival analyses (PROC LIFETEST, SAS Institute 2012). The procedure Þrst estimated the time at 25th, 50th, and 75th percentile of hatching events for each temperature treatment based on the survival function computed by the product-limit procedure (also called the KaplanÐ Meier method), and then compared the hatching event probability curve by the Log-Rank test. As no eggs hatched at 12⬚C during the entire observation period (30 d), data for this temperature treatment were not included in the analysis. Unhatched eggs for all other temperature treatments were excluded in the survival analysis. We used the normal logistic regression model (P ⫽ 1/(1 ⫹ e⫺(a⫹bt)) to analyze the relationship between the probability (P) of an emerald ash borer larva developing into late (third, fourth, or mature J-shaped) instar and incubation time (t) for each ambient temperature, where a is the constant and b the coefÞcient of the logistic curve. The developmental time (DT50) and 95% CI for 50% of emerald ash borer larvae advancing to each late instar stage were then inversely predicted using the Þtted logistic regression model for each ambient temperature. A multiple logistic regression model was used to analyze the effect of both temperature and observation time on the probability of an emerald ash borer larva reaching a target instar or stage. Throughout the experiment, 4 Ð21% larva mortality was observed across different ambient temperature treatments; those dead larvae were excluded from the logistical regression analysis, as we could not determine exactly when those larvae had died. We used a multiple linear regression model (y ⫽ c ⫹ ax ⫹ bt, c ⫽ model intercept, a ⫽ coefÞcient for ambient temperature, b ⫽ coefÞcient for observation time) to evaluate the effect of ambient temperature (x) and observation time (t) on biomass or gallery width (y). We then used the simple linear regression model (y ⫽ bt) to estimate the rate of changes (b) in biomass or gallery width (y) per week (t). For convenience, we called this rate (b) of changes in biomass or gallery per week width as weekly growth rate.
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Vol. 106, no. 5
Fig. 1. Probability of emerald ash borer egg hatching (event) at different ambient temperatures.
Results Effect of Temperature on Egg Development. The relationship between emerald ash borer egg hatching event (probability) and observation time differed with ambient temperature treatments (Fig. 1). LogRank tests (survival analyses) showed a highly significant effect of ambient temperature at the test range (20 Ð35⬚C) on emerald ash borer egg development as measured by time-related egg hatching events (2 ⫽ 66.22; df ⫽ 4; P ⬍ 0.0001). Although no eggs hatched at 12⬚C throughout the entire observation time (30 d), ⬎84% of eggs successfully hatched at the higher ambient temperature (20 Ð35⬚C) treatments (Fig. 1). The median time (at 50th percentile) for egg hatching decreased from 20 d at 20⬚C to 7 d at 35⬚C (Table 1), indicating the faster egg development as ambient temperature increases from 20 to 35⬚C. Effect of Temperature on Larval Development. The progress in different stages (or instars) of emerald ash borer larvae over time was drastically different across different ambient temperature treatments (Fig. 2). The multiple logistic regression analysis showed that both ambient temperature and time signiÞcantly affect the probability of emerald ash borer (from oviposition) developing to later instars (Likelihood Ratio 2 tests: 2 ⫽ 174.5; df ⫽ 1; P ⬍ 0.0001 for temperature; 2 ⫽ 517.35; df ⫽ 1; P ⬍ 0.0001 for time). The DT50 for
50% of emerald ash borer larvae (from oviposition) advancing to third or later instars decreased sharply as ambient temperature increased (Table 2). While the 20⬚C treatment resulted in the longest DT50 for emerald ash borer advancing to third or later instars (8.3 wk for third instars, 9.1 wk for fourth instars, and 12.3 wk for J-larvae), the 30⬚C treatment resulted in shortest DT50 for the corresponding instars (3.2 wk for third instars, 3.7 wk for fourth instars, and 6.3 wk for J-larvae). In contrast to 30⬚C, DT50 for third, fourth, and J-larvae at 35⬚C increased signiÞcantly to 4.7, 5.5, and 8.0 wk, respectively (no overlapping of 95% CI with those at 30⬚C), indicating that the development of emerald ash borer larvae was adversely affected by this high temperature treatment. Mean biomass and gallery width of observed emerald ash borer larvae over time at each of the temperature treatments also varied signiÞcantly with temperature treatments (Fig. 3A for biomass: F ⫽ 29.469; df ⫽ 1, 862; P ⬍ 0.0001 and Fig. 3B for gallery width: F ⫽ 152.27; df ⫽ 1, 948; P ⬍ 0.0001). Results from simple linear regression analyses showed that weekly growth or change rates (linear coefÞcients) in both biomass and gallery width increased almost twofold, when ambient temperature increased from 20 to 25⬚C (Table 3). The weekly growth rates for biomass and gallery width at 25⬚C were 0.0115 and 0.4425, respec-
Table 1. Point estimates of hatching time (HT) and 95% CI (day) of emerald ash borer eggs at 25th, 50th, and 75th percentile estimated with survival analysis model for different ambient temperatures Temperature (⬚C)
N
No. eggs censored (unhatched)
HT25 with 95% CI (days)a
HT50 with 95% CI (days)a
HT75 with 95% CI (days)a
20 25 27 30 35
46 63 57 62 30
5 5 6 6 5
19 (18Ð19) 14 (14Ð15) 12 (10Ð11) 9 (9Ð10) 7 (5Ð7)
20 (19Ð25) 16 (15Ð16) 13 (12Ð13) 10 (10Ð11) 7 (5Ð9)
26 (26Ð28) 17 (16Ð17) 14 (13Ð16) 12 (11Ð14) 9 (8Ð9)
a The LIFETEST analysis is a nonparametric procedure, and treats the time as a discrete ordinary data; thus, the whole number is used for all the estimates. Log-Rank test for equality of hatching (survival) curves over different temperature treatments: 2 ⫽ 66.22; df ⫽ 4; P ⬍ 0.0001.
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DUAN ET AL.: METHODS FOR LABORATORY REARING OF EMERALD ASH BORER
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Fig. 2. Distribution of different stadium (instars) of emerald ash borer larvae at different ambient temperatures.
tively, approximately twice as those at 20⬚C (0.0050 for biomass and 0.2269 for gallery width). When ambient temperature increased from 25 to 30⬚C, however, the
growth rate in both biomass and gallery width only changed slightly, indicating slower rates of growth in both biomass and gallery width in those ranges of
Table 2. Developing time (DT) and 95% CI limit (weeks) for 50% emerald ash borer population reaching mature J-larval stage (from adult oviposition) Temperature (⬚C)
N
DT50 (95% CI) for third instars (weeks)a
DT50 (95% CI) for fourth instars (weeks)a
DT50 (95% CI) for J-larvae (weeks)a
20 25 27 30 35
177 209 220 265 134
8.3 (7.8Ð8.7) 4.7 (4.5Ð4.9) 4.1 (3.8Ð4.4) 3.2 (3.0Ð3.5) 4.7 (4.0Ð5.1)
9.1 (8.7Ð9.5) 5.6 (5.3Ð5.9) 5.1 (4.9Ð5.3) 3.7 (3.4Ð4.0) 5.5 (5.1Ð6.0)
12.3 (11.9Ð12.7) 7.1 (6.9Ð7.3) 7.0 (6.7Ð7.3) 6.3 (5.9Ð6.7) 8.0 (7.4Ð8.9)
a Estimated by inverse prediction of developmental times (weeks) with normal logistic regression model at 50% observed larvae reaching the target stage or instar for each temperature. All normal logistic regression models for different ambient temperatures showed signiÞcant relationship between developmental time and the probability of the larvae reaching each target instar or stage (likelihood 2 tests: P ⬍ 0.0001 for all temperature treatments).
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Vol. 106, no. 5
Fig. 3. Effect of ambient temperatures on emerald ash borer larval growth: (A) larval biomass and (B) width of larval gallery.
temperature. While the growth rate in gallery width was slightly lower (0.4688) at 35⬚C than that at 30⬚C (0.4864), the growth rate in biomass at 35⬚C (0.0077) was ⬇34% lower than that at 30⬚C (0.0116), indicating that 35⬚C might have adverse effects on the biomass accumulation of emerald ash borer larvae.
Table 3. Linear regression coefficients (or growth rates) for biomass (g) and gallery width (millimeters) of emerald ash borer larvae over time (week) at different ambient temperatures Temperature (⬚C) Biomass 20 25 27 30 35 Gallery width 20 25 27 30 35
CoefÞcient (growth rate)
SE
t-ratio
P ⬎ ta
0.0050 0.0115 0.0113 0.0116 0.0075
0.0002 0.0011 0.0004 0.0004 0.0004
20.39 10.40 24.59 28.67 16.74
⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001
0.2269 0.4425 0.5558 0.4864 0.4681
0.0045 0.0075 0.0089 0.0060 0.0126
50.93 59.20 62.34 60.30 37.02
⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001
a All t-ratio statistics indicate signiÞcant linear relationships between biomass or gallery width and time (week).
Discussion Findings from our study indicate that development, growth, or both, of eggs and larvae of emerald ash borers are strongly inßuenced by ambient temperature. The median time for egg hatching as well as the development time for larvae (from oviposition) advancing to later (third and fourth) instars (including mature J-larvae) varied greatly with ambient temperature. While no eggs hatched at 12⬚C, the median time for egg hatching decreased from 20 d at 20⬚C to 7 d at 35⬚C. The similar pattern holds true for the development of emerald ash borer larvae. Developmental times for 50% of emerald ash borer larvae advancing to third, fourth, and J-larval stages at 20⬚C were 8.3, 9.1, and 12.3 wk, respectively, approximately two times longer than at 30⬚C for the corresponding instars or stages. In contrast to 30⬚C, however, the development time of emerald ash borer larvae advancing to later instars was apparently longer at 35⬚C, indicating that high (⬎30⬚C) temperature had adverse effects on the larval development. The appropriate range of ambient temperature to rear emerald ash borer larvae may be between 25Ð30⬚C; however, faster rate of egg and larval development should be expected as temperature increases in this range.
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DUAN ET AL.: METHODS FOR LABORATORY REARING OF EMERALD ASH BORER
It is well documented that the life cycle of emerald ash borer populations varied from one to two generations in North America as well as North East Asia (Cappaert et al. 2005; Liu et al. 2007; Siergert et al. 2007; Wei et al. 2007; Duan et al. 2010, 2012). Emerald ash borers enter obligatory diapause as J-larvae and resume development after a period of cold (wintering) temperatures. Whether larvae are able to advance to the J-stage before winter likely determines whether the population completes development in 1 or 2 yr. A 1-yr (univoltine) life cycle might be expected in southern states (e.g., Tennessee, Missouri, Kentucky, and Maryland) whereas a 2-yr (semivoltine) generation may be expected in northern states where the insect may not reach the J-stage before winter. We recommend that future development of ecological models to predict the emerald ash borer phenology and population growth should incorporate the relationship between the developmental time and growth rate of both egg and larvae at different temperatures zones. Findings from our study also have important implications for laboratory rearing of emerald ash borer egg and larval parasitoids for biological control programs. For example, when rearing the egg parasitoid O. agrili, the window of host egg susceptibility to the parasitoid would become wider at the lower temperature (e.g., 20⬚C), as emerald ash borer eggs take much longer to hatch (and thus become not susceptible to parasitism) than at the higher temperature (e.g., 25⬚C or higher). However, the window of host egg susceptibility would become narrower at the higher temperature range (e.g., at 35⬚C), as the host eggs hatch much faster. Thus, the primary exposure time for egg parasitoid production should be adjusted according to the rearing temperature. The same implication also applies to laboratory rearing of larval emerald ash borer parasitoids such as T. planipennisi and S. agrili. For example, both T. planipennisi and S. agrili prefer attacking late (third to fourth) feeding instars of emerald ash borer (Ulyshen et al. 2010b, Gould et al. 2011), but are not able to attack J-larvae in their sap-wood chambers (because they do not feed). Thus, production of suitable stages of emerald ash borer larvae for primary exposure to parasitoids should consider the potential effect of ambient temperature on the host larval development.
Acknowledgments We thank Jacqueline Hoban and Susan Barth (all University of Delaware employees) for their great assistance in all phases of the study. We appreciate the great assistance of Dick Bean, Kim Rice, and Charles Pickett (all at Maryland Department of Agriculture) in producing and shipping adult emerald ash borer to us for this study. We are also grateful to Roger Fuester and Doug Luster (USDAÐARS, BeneÞcial Insects Introduction Research Units) for helpful comments to the earlier version of the manuscript.
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References Cited Bauer, L. S., H.-P. Liu, R. A. Haack, D. L. Miller, and T. R. Patrice. 2004. Natural enemies of emerald ash borer in southeastern Michigan, p. 33. In V. Mastro and R. Reardon (eds.), Proceedings, Emerald Ash Borer Research and Technology Development Meeting, USDA FS FTET2004 Ð 02, Port Huron, MI. Canadian Food Inspection Agency. 2013. Emerald Ash BorerÐAgrilus planipennis. (http://www.inspection.gc.ca/ english/plaveg/pestrava/agrpla/agrplae.shtml). Cappaert, D., D. G. McCullough, T. M. Poland, and N. W. Siegert. 2005. Emerald ash borer in North America: a research and regulatory challenge. Am. Entomol. 51: 152Ð 165. Chamorro, M. L., M. G. Volkovitsh, T. M. Poland, R. A. Haack, and S. W. Lingafelter. 2012. Preimaginal stages of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae: Agrilinae): an invasive species of ash trees (Fraxinus). PLoS ONE 7: e33185. (doi: 10.1371/journal.pone.0033185). Duan, J. J., and C. B. Oppel. 2012. Critical rearing parameters of Tetrastichus planipennisi (Hymenoptera: Eulophidae) as affected by host-plant substrate and host-parasitoid group structure. J. Econ. Entomol. 105: 792Ð 801. Duan, J. J., M. D. Ulyshen, L. S. Bauer, J. Gould, and R. Van Driesche. 2010. Measuring the impact of biotic factors on populations of immature emerald ash borer (Coleoptera: Buprestidae). Environ. Entomol. 39: 1513Ð1522. Duan, J. J., T. Watt, and C. B. Oppel. 2011. An alternative host plant-based method for laboratory rearing of emerald ash borer to produce larval parasitoids for biological control, p. 3. In G. Parra, D. Lance, V. Mastro, R. Reardon, and C. Benedict (eds.), Proceedings, Emerald Ash Borer Research and Technology Meeting, 10Ð23 October 2011, USDA Forest Service/APHIS/FHTET-2011-06, Wooster, OH. Duan, J. J., G. Yurchenko, and R. W. Fuester. 2012. Occurrence of emerald ash borer (Coleoptera: Buprestidae) and biotic factors affecting its immature stages in the Russian Far East. Environ. Entomol. 41: 245Ð254. Emerald Ash Borer Information 2013. Emerald ash borer information. (http://www.emeraldashborer.info/ homeownerinfo.cfm). Gould, J. R., T. Ayer, and I. Fraser. 2011. Effect of rearing conditions on reproduction by Spathius agrili (Hymenoptera: Braconidae), a parasitoid of the emerald ash borer (Coleoptera: Buprestidae). J. Econ. Entomol. 104: 379 Ð387. Haack, R. A., J. E. Jendek, H. Liu, K. R. Marchant, T. R. Petrice, R. M. Poland, and H. Ye. 2002. The emerald ash borer: a new exotic pest in North America. Newsl. Mich. Entomol. Soc. 47: 1Ð5. Kovacs, F. K., R. G. Haight, D. G. McCullough, R. J. Mercader, N. W. Siegert, and A. M. Leibhold. 2010. Cost of potential emerald ash borer damage in U.S. communities, 2009 Ð2019. Ecol. Econ. 69: 569 Ð578. Liu, H.-P., L. S. Bauer, D. L. Miller, T.-H. Zhao, R.-T. Gao, L. Song, Q. Luan, R. Jin, and C. Gao. 2007. Seasonal abundance of Agrilus planipennis (Coleoptera: Buprestidae) and its natural enemies Oobius agrili (Hymenoptera: Encyrtidae) and Tetrastichus planipennisi (Hymenoptera: Eulophidae) in China. Biol. Control 42: 61Ð71. McCullough, D. G., T. M. Poland, and D. Cappaert. 2009. Attraction of the emerald ash borer to ash trees stressed by girdling, herbicide treatment, or wounding. Can. J. For. Res. 39: 1331Ð1345.
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Poland, T. M., and D. G. McCullough. 2006. Emerald ash borer: invasion of the urban forest and the threat to North AmericaÕs ash resource. J. For. 104: 118 Ð124. SAS Institute. 2012. JMP PRO modeling and multivariate methods, version 10. SAS Institute, Cary, NC. Siergert, N. W., D. G. McCullough, and A. R. Tluczek. 2007. Two years under the bark: towards understanding multiple-year development of emerald ash borer larvae, pp. 39 Ð36. In V. Mastro, D. Lance, R. Reardon, and G. Parra (eds.), Proceedings, Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, 29 OctoberÐ2 November 2006, Cincinnati, OH. FHTET-2007-04, USDA 361 Forest Service Forest Health Technology Enterprise Team, Morgantown, WV. (USDA–APHIS) U.S. Department of Agriculture–Animal and Plant Health Inspection Service. 2007. The proposed release of three parasitoids for the biological control of the emerald ash borer (Agrilus planipennis) in the continental United States: environmental assessment. Fed. Regist. 72: 28947Ð28948, Docket No. APHIS-20070060. Ulyshen, M. D., J. J. Duan, and L. S. Bauer. 2010a. Interactions between Spathius agrili (Hymenoptera: Braconidae) and Tetrastichus planipennisi (Hymenoptera: Eulophidae), larval parasitoids of Agrilus planipennis (Coleoptera: Buprestidae). Biol. Control 52: 188 Ð193.
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Ulyshen, M. D., J. J. Duan, L. S. Bauer, and F. Ivich. 2010b. Suitability and accessibility of immature Agrilus planipennis (Coleoptera: Buprestidae) stages to Tetrastichus planipennisi (Hymenoptera: Eulophidae). J. Econ. Entomol. 103: 1080 Ð1085. Wang X. Y., Z. Q. Yang, J. R. Gould, Y. N. Zhang, G. J. Liu, and E. S. Liu. 2010. The biology and ecology of the emerald ash borer, Agrilus planipennis, in China. J. Insect Sci. 10: 128. Wei, X., W. Yun, R. Reardon, T.-H. Sun, M. Lu, and J.-H. Sun. 2007. Biology and damage traits of emerald ash borer (Agrilus planipennis Fairmaire) in China. Insect Sci. 14: 367Ð373. Yang, S., J. J. Duan, T. Watt, K. J. Abell, and R. van Driesche. 2012. Responses of an idiobiont ectoparasitoid Spathius galinae to host larvae parasitized by the koinobiont endoparasitoid Tetrastichus planipennisi: implications for biological control of the emerald ash borer (Coleoptera: Buprestidae). Environ. Entomol. 41: 925Ð932. Zhang, Y.-Z., D.-W. Huang, T.-H. Zhao, H.-P. Liu, and L. S. Bauer. 2005. Two new species of egg parasitoids (Hymenoptera: Encyrtidae) of wood-boring beetle pests from China. Phytoparasitica 53: 253Ð260. Received 17 March 2013; accepted 27 June 2013.
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FOREST ENTOMOLOGY
Effects of Ambient Temperature on Egg and Larval Development of the Invasive Emerald Ash Borer (Coleoptera: Buprestidae): Implications for Laboratory Rearing JIAN J. DUAN,1,2 TIM WATT,3 PHIL TAYLOR,1 KRISTI LARSON,3
AND
JONATHAN P. LELITO4
J. Econ. Entomol. 106(5): 2101Ð2108 (2013); DOI: http://dx.doi.org/10.1603/EC13131
ABSTRACT The emerald ash borer, Agrilus planipennis Fairmaire, an invasive beetle from Asia causing large scale ash (Fraxinus) mortality in North America, has been extremely difÞcult to rear in the laboratory because of its long life cycle and cryptic nature of immature stages. This lack of effective laboratory-rearing methods has not only hindered research into its biology and ecology, but also mass production of natural enemies for biological control of this invasive pest. Using sticks from the alternate host plant, Fraxinus uhdei (Wenzig) Lingelsh, we characterized the stage-speciÞc development time and growth rate of both emerald ash borer eggs and larvae at different constant temperatures (12Ð35⬚C) for the purpose of developing effective laboratory-rearing methods. Results from our study showed that the median time for egg hatching decreased from 20 d at 20⬚C to 7 d at 35⬚C, while no emerald ash borer eggs hatched at 12⬚C. The developmental time for 50% of emerald ash borer larvae advancing to third, fourth, and J-larval stages at 20⬚C were 8.3, 9.1, and 12.3 wk, respectively, approximately two times longer than at 30⬚C for the corresponding instars or stages. In contrast to 30⬚C, however, the development times of emerald ash borer larvae advancing to later instars (from oviposition) were signiÞcantly increased at 35⬚C, indicating adverse effects of this high temperature. The optimal range of ambient temperature to rear emerald ash borer larvae should be between 25Ð30⬚C; however, faster rate of egg and larval development should be expected as temperature increases within this range. KEY WORDS Agrilus planipennis, development, Fraxinus uhdei, invasive, laboratory rearing
The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is a relatively new invasive forest pest in North America, where it has caused large scale ash (Fraxinus) mortality and spread to 19 U.S. states (Kovacs et al. 2010, Emerald Ash Borer Information 2013) and two Canadian provinces (Canadian Food Inspection Agency 2013) since it was Þrst discovered in Michigan (Haack et al. 2002). In most of the infested areas, emerald ash borer adults emerge in late spring and early summer (MayÐJune), and feed on ash foliage for at least 2 wk before mating and laying eggs in crevices and under bark ßakes on limbs and trunks of ash trees in early summer (JuneÐJuly). After eclosion, Þrst instar larvae chew through the bark to reach the phloem, where they feed and develop throughout the growing season (Cappaert et al. 2005). After larvae go through three molts, the mature fourth instar larvae chew pupation chambers in the outer sapwood or bark. The mature larvae then develop a folded ap1 USDAÐARS, BeneÞcial Insects Introduction Research Unit, 501 South Chapel St., Newark, DE 19713. 2 Corresponding author, e-mail: [email protected]. 3 University of Delaware, Department of Entomology and Wildlife Ecology, Newark, DE 19713. 4 USDAÐAPHIS PPQ Emerald Ash Borer Biocontrol Laboratory, Brighton, MI 48116.
pearance for overwintering via obligatory diapause (J.J.D. and J.P.L., unpublished data). Larvae at this J-shaped stage were termed “J-larvae” by Duan et al. (2010), but called “prepupae” by Chamorro et al. (2012). Usually after a winter chill period, J-larvae develop into prepupae, which are visibly shorter and more cylindrical. Pupation generally occurs in early spring, with adults emerging from late spring through early summer. Adults live for several weeks feeding on mature ash leaves, and rarely cause any signiÞcant damage to the host tree (Bauer et al. 2004). Larvae, in contrast, feed and develop for several months on ash tree phloem, creating extensive galleries under the bark. When emerald ash borer populations are high, larval consumption of tree phloem is substantial, resulting in tree girdling and death in 3Ð 4 yr (Poland and McCullough 2006, McCullough et al. 2009). Emerald ash borer populations in the newly invaded North America (e.g., Cappaert et al. 2005, Siegert et al. 2007, Duan et al. 2010) as well as in its native North East Asia (e.g., Liu et al. 2007, Wei et al. 2007, Duan et al. 2012) may require 1Ð2 yr to complete development. Although climatic conditions in different geographic regions as well as host tree conditions may have contributed to the dichotomy of emerald ash borer life cycle, few studies have examined how cli-
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matic conditions (particularly ambient temperature) inßuence its development and diapause of immature stages. This lack of study on climatic conditions (particularly temperature) is largely because of our inability to rear emerald ash borer in laboratory conditions. Recently, classical biological control efforts against emerald ash borers in the United States have led to the introduction and release of three species of parasitoids from the pestÕs native range (Northeast Asia): the idiobiont egg parasitoid Oobius agrili Zhang and Huang (Encyrtidae) and the larval parasitoids Tetrastichus planipennisi Yang (Eulophidae) and Spathius agrili Yang (Braconidae) (Zhang et al. 2005, Liu et al. 2007, U.S. Department of AgricultureÐAnimal and Plant Health Inspection Service [USDAÐAPHIS] 2007). To produce suitable emerald ash borer hosts for mass production of those introduced biocontrol agents, researchers have obtained various stages of emerald ash borer larvae or adults by collecting infested ash (Fraxinus) logs. The Þeld-collected ash logs are then stored in a 3Ð5⬚C cooler (to slow the larval development, break the larval diapause, or both), and incubated in room temperature to obtain emergence of adult emerald ash borers (e.g., Yang et al. 2012, J.P.L. unpublished data). Some researchers have experimented with “artiÞcial infestation,” where a series of slits are cut into an ash log, emerald ash borer larvae are secured within the slits, and the larvae are then allowed to develop within their natural host wood, or presented to parasitoids as need be (e.g., Ulyshen et al. 2010a,b; Duan and Oppel 2012). Although these methods have allowed researchers to produce emerald ash borer larvae and adults, they are resource intensive and limited by seasonality. Historically, wood-boring buprestids such as the emerald ash borer have been extremely difÞcult to rear in the laboratory largely because of its long life cycle and cryptic nature of immature stages. Recently, Duan et al. (2011) reported on an alternative host plant-based emerald ash borer rearing method, which is considerably streamlined in comparison with present rearing strategies. The alternative host plant used in rearing emerald ash borer is the evergreen tropical ash, Fraxinus uhdei (Wenzig) Lingelsh, which can be cultivated year around in greenhouses without dormancy in growth. In the current study, we examine the effect of ambient temperature on the development of emerald ash borer eggs and larvae when using this alternate host plant-based method for rearing. Specifically, we characterize the stage-speciÞc development time and growth rate of emerald ash borer larvae on infested tropical ash sticks at different temperature regimes. This information is not only important to our understanding of emerald ash borer ecology (e.g., the effect of climatic condition on its population growth), but also critical to production of suitable stages of larvae for various research programs such as massrearing egg and larval parasitoids for biological control of this invasive pest (USDAÐAPHIS 2007).
Vol. 106, no. 5 Materials and Methods
Emerald Ash Borer. All adults used for egg and larval production in this study originated from emerald ash borer-infested green ash (Fraxinus pennsylvanica Marshall) trees collected in Maryland. Green ash trees with apparent emerald ash borer infestation symptoms (e.g., woodpecker scaling and feeding damage to trunks, epicormic growth, or reduced crowns) were felled from late fall (November) to later winter (February) of each year, cut to pieces of meter-long logs, and then stored in a climatically controlled walk-in cooler (Polar King International, Fort Wayne, IN) at 3Ð5⬚C for season-long production of emerald ash borer adults. For adult production, stored-ash logs were placed into emerging cages made of cardboard Sonotube (1.2 m long by 50 cm in diameter), and then incubated at 26 Ð29⬚C with a photoperiod of 16:8 (L:D) h in an environmentally controlled room at Maryland Department of Agriculture (Annapolis, MD). Emerging emerald ash borer adults from incubated-ash logs were collected every 1 or 2 d at the Maryland Department of Agriculture, and immediately sent to USDAÐAgricultural Research Service (ARS) BeneÞcial Insects Research Unit (Newark, DE) via same day or overnight delivery. On receipt at the USDAÐARS BeneÞcial Insects Research Unit, adults were immediately transferred to rearing containers (clear 3.5liter Jars with screen-ventilated lids) at the density of ⬇20 females ⫻ 20 males per container. Before oviposition, all adults were fed with bouquets of fresh green (F. pennsylvanica) or tropical (F. uhdei) ash leaves, which were replaced every 2 or 3 d. After approximately 2 wk of maturation feeding, gravid females were then transferred to 1-liter ventilated plastic (oviposition) cups at a density of ⬇5 females ⫻ 2Ð3 males per cup, and provided with fresh host plant leaves for egg production. To obtain emerald ash borer eggs, the mouth of the oviposition cup was covered with nylon screen (mesh size ⬇1 mm2) and then with a sheet of coffee Þlter paper (8.25 cm base, HomeLife, Eden Prairie, MN) on the top to serve as a suitable oviposition site (Duan et al. 2011). Gravid emerald ash borer females were attracted to lay eggs on the screen-covered substrates such as coffee Þlter paper (Yang et al. 2012). Emerald ash borer eggs freshly laid (within 24 h) on the coffee Þlter paper were then used in different egg and larval development experiments with ambient temperature treatments. Effect of Ambient Temperature on Egg Development. Coffee Þlter papers with freshly laid emerald ash borer eggs (within 24 h) were cut into small individual pieces (⬇3 mm long by 3 mm wide), each with one egg (for easy observation under stereomicroscope), and placed inside 8-cm (diameter) petri dishes lined with wet Þlter paper (for humidity). Petri dishes containing emerald ash borer eggs (10 Ð20 eggs per dish) were then placed inside ventilated Crisper Boxes, which were housed in growth chambers (ARB366, Percival ScientiÞc Inc., Perry, IA) set for different constant-temperature treatments with constant rela-
October 2013
DUAN ET AL.: METHODS FOR LABORATORY REARING OF EMERALD ASH BORER
tive humidity (RH; 60 ⫾ 5%) and a photoperiod of 16:8 (L:D) h. In total, 30 Ð70 emerald ash borer eggs (in three to Þve petri dishes) were tested at each of the six treatment temperatures: 12, 20, 25, 27, 30, and 35⬚C. On hatching, neonate emerald ash borer larvae chewed through the side of the egg shell that was attached to the Þlter paper and then penetrated the Þlter paper leaving an easily recognizable exit hole on the egg shell and Þlter paper. Observation of neonate emerald ash borer larvae hatching from eggs laid on coffee Þlter papers was conducted daily under the stereomicroscope for a maximum 30 d. At each observation day, the number of hatched eggs was recorded and then removed from the holding petri dishes. Effect of Ambient Temperature on Larval Development. Using tropical ash sticks, we measured the development (stage or instars) and growth (biomass and gallery width) of emerald ash borer larvae reared at Þve different ambient (constant) temperatures: 20, 25, 27, 30, and 35⬚C. The Þlter paper with emerald ash borer eggs was cut into small pieces (each with one to three emerald ash borer eggs), which were then placed closely against the surface of tropical ash sticks (20 Ð25 cm long by 1.5Ð3.5 cm in diameter) with the bottom side of eggs (attached to Þlter paper) against the bark of the sticks and wrapped with ParaÞlm strips (BEMIS, BEMIS Flexible Packaging, Neenah, WI). Across all temperature treatments, the number of eggs placed on each stick varied with the size of the ash sticks; generally smaller sticks (with a diameter ⬍2.0 cm) were infested with fewer eggs (5Ð10 eggs) and larger sticks (diameter ⬎2.0 up to 3.5 cm) with more eggs (12Ð20 eggs). Approximately 50 sticks were used for each of the Þve treatment temperatures throughout the study. Although the size of sticks varied across different temperature treatments, the density of infested eggs or larvae was adjusted per unit surface area of stick phloem, and was approximately the same (⬇320 eggs/m2 of surface area) among different treatments. After placement of eggs, ash sticks were placed upright in ßoral foam bricks (OASIS, SmithersÑOasis Company, Kent, OH) in storage boxes (58.4 by 41.3 by 31.4 cm, Sterilite, Sterilite Corporation, Townsend, MA) saturated with distilled water and a 0.1% solution of methylparaben to prevent molds. Storage boxes containing the emerald ash borer-infested ash sticks were then incubated in different environmental chambers set with the target ambient temperature but at the same RH (65 ⫾ 10%) and a photoperiod of 16:8 (L:D) h. Beginning 2Ð 4 wk after egg deposition, 20 Ð25 emerald ash borer larvae were dissected out of 5 to 10 ash sticks from each temperature on a weekly or biweekly basis. The dissection of ash sticks was done by peeling off the entire bark layer (including the cambium tissue) with a utility knife. On removal of the bark layer, exposed emerald ash larvae were removed from their galleries, scored for their stadium (instars) based on the width of their head capsules (Cappaert et al. 2005, Wang et al. 2010) and then weighed with an electronic balance (AB135-S/FACT, Mettler Toledo AG Labo-
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ratory Weighing and Technology, Greifensee, Switzerland) for their biomass. In addition, we also measured the width of each larvaÕs gallery at the position where its head is located, with a digital caliper (No. 147-Digital Fractional caliper Stainless Steel Ultra Tech, General Tools and Instruments, New York, NY). When reaching mature stage, fourth instar emerald ash larvae normally make chambers inside the stick (sapwood) and then fold into a J-shape (termed as J-larvae) for overwintering (via obligatory diapause). We removed the J-larvae from their chambers for measurement by splitting the sapwood tissue with a hand pruner and utility knife. We then measured the width of J-larval gallery before the entrance of their chambers, and weighed their biomass with the balance. Data Analysis. The time-related hatching event of emerald ash borer egg cohorts at different ambient temperatures was analyzed using survival analyses (PROC LIFETEST, SAS Institute 2012). The procedure Þrst estimated the time at 25th, 50th, and 75th percentile of hatching events for each temperature treatment based on the survival function computed by the product-limit procedure (also called the KaplanÐ Meier method), and then compared the hatching event probability curve by the Log-Rank test. As no eggs hatched at 12⬚C during the entire observation period (30 d), data for this temperature treatment were not included in the analysis. Unhatched eggs for all other temperature treatments were excluded in the survival analysis. We used the normal logistic regression model (P ⫽ 1/(1 ⫹ e⫺(a⫹bt)) to analyze the relationship between the probability (P) of an emerald ash borer larva developing into late (third, fourth, or mature J-shaped) instar and incubation time (t) for each ambient temperature, where a is the constant and b the coefÞcient of the logistic curve. The developmental time (DT50) and 95% CI for 50% of emerald ash borer larvae advancing to each late instar stage were then inversely predicted using the Þtted logistic regression model for each ambient temperature. A multiple logistic regression model was used to analyze the effect of both temperature and observation time on the probability of an emerald ash borer larva reaching a target instar or stage. Throughout the experiment, 4 Ð21% larva mortality was observed across different ambient temperature treatments; those dead larvae were excluded from the logistical regression analysis, as we could not determine exactly when those larvae had died. We used a multiple linear regression model (y ⫽ c ⫹ ax ⫹ bt, c ⫽ model intercept, a ⫽ coefÞcient for ambient temperature, b ⫽ coefÞcient for observation time) to evaluate the effect of ambient temperature (x) and observation time (t) on biomass or gallery width (y). We then used the simple linear regression model (y ⫽ bt) to estimate the rate of changes (b) in biomass or gallery width (y) per week (t). For convenience, we called this rate (b) of changes in biomass or gallery per week width as weekly growth rate.
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Vol. 106, no. 5
Fig. 1. Probability of emerald ash borer egg hatching (event) at different ambient temperatures.
Results Effect of Temperature on Egg Development. The relationship between emerald ash borer egg hatching event (probability) and observation time differed with ambient temperature treatments (Fig. 1). LogRank tests (survival analyses) showed a highly significant effect of ambient temperature at the test range (20 Ð35⬚C) on emerald ash borer egg development as measured by time-related egg hatching events (2 ⫽ 66.22; df ⫽ 4; P ⬍ 0.0001). Although no eggs hatched at 12⬚C throughout the entire observation time (30 d), ⬎84% of eggs successfully hatched at the higher ambient temperature (20 Ð35⬚C) treatments (Fig. 1). The median time (at 50th percentile) for egg hatching decreased from 20 d at 20⬚C to 7 d at 35⬚C (Table 1), indicating the faster egg development as ambient temperature increases from 20 to 35⬚C. Effect of Temperature on Larval Development. The progress in different stages (or instars) of emerald ash borer larvae over time was drastically different across different ambient temperature treatments (Fig. 2). The multiple logistic regression analysis showed that both ambient temperature and time signiÞcantly affect the probability of emerald ash borer (from oviposition) developing to later instars (Likelihood Ratio 2 tests: 2 ⫽ 174.5; df ⫽ 1; P ⬍ 0.0001 for temperature; 2 ⫽ 517.35; df ⫽ 1; P ⬍ 0.0001 for time). The DT50 for
50% of emerald ash borer larvae (from oviposition) advancing to third or later instars decreased sharply as ambient temperature increased (Table 2). While the 20⬚C treatment resulted in the longest DT50 for emerald ash borer advancing to third or later instars (8.3 wk for third instars, 9.1 wk for fourth instars, and 12.3 wk for J-larvae), the 30⬚C treatment resulted in shortest DT50 for the corresponding instars (3.2 wk for third instars, 3.7 wk for fourth instars, and 6.3 wk for J-larvae). In contrast to 30⬚C, DT50 for third, fourth, and J-larvae at 35⬚C increased signiÞcantly to 4.7, 5.5, and 8.0 wk, respectively (no overlapping of 95% CI with those at 30⬚C), indicating that the development of emerald ash borer larvae was adversely affected by this high temperature treatment. Mean biomass and gallery width of observed emerald ash borer larvae over time at each of the temperature treatments also varied signiÞcantly with temperature treatments (Fig. 3A for biomass: F ⫽ 29.469; df ⫽ 1, 862; P ⬍ 0.0001 and Fig. 3B for gallery width: F ⫽ 152.27; df ⫽ 1, 948; P ⬍ 0.0001). Results from simple linear regression analyses showed that weekly growth or change rates (linear coefÞcients) in both biomass and gallery width increased almost twofold, when ambient temperature increased from 20 to 25⬚C (Table 3). The weekly growth rates for biomass and gallery width at 25⬚C were 0.0115 and 0.4425, respec-
Table 1. Point estimates of hatching time (HT) and 95% CI (day) of emerald ash borer eggs at 25th, 50th, and 75th percentile estimated with survival analysis model for different ambient temperatures Temperature (⬚C)
N
No. eggs censored (unhatched)
HT25 with 95% CI (days)a
HT50 with 95% CI (days)a
HT75 with 95% CI (days)a
20 25 27 30 35
46 63 57 62 30
5 5 6 6 5
19 (18Ð19) 14 (14Ð15) 12 (10Ð11) 9 (9Ð10) 7 (5Ð7)
20 (19Ð25) 16 (15Ð16) 13 (12Ð13) 10 (10Ð11) 7 (5Ð9)
26 (26Ð28) 17 (16Ð17) 14 (13Ð16) 12 (11Ð14) 9 (8Ð9)
a The LIFETEST analysis is a nonparametric procedure, and treats the time as a discrete ordinary data; thus, the whole number is used for all the estimates. Log-Rank test for equality of hatching (survival) curves over different temperature treatments: 2 ⫽ 66.22; df ⫽ 4; P ⬍ 0.0001.
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DUAN ET AL.: METHODS FOR LABORATORY REARING OF EMERALD ASH BORER
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Fig. 2. Distribution of different stadium (instars) of emerald ash borer larvae at different ambient temperatures.
tively, approximately twice as those at 20⬚C (0.0050 for biomass and 0.2269 for gallery width). When ambient temperature increased from 25 to 30⬚C, however, the
growth rate in both biomass and gallery width only changed slightly, indicating slower rates of growth in both biomass and gallery width in those ranges of
Table 2. Developing time (DT) and 95% CI limit (weeks) for 50% emerald ash borer population reaching mature J-larval stage (from adult oviposition) Temperature (⬚C)
N
DT50 (95% CI) for third instars (weeks)a
DT50 (95% CI) for fourth instars (weeks)a
DT50 (95% CI) for J-larvae (weeks)a
20 25 27 30 35
177 209 220 265 134
8.3 (7.8Ð8.7) 4.7 (4.5Ð4.9) 4.1 (3.8Ð4.4) 3.2 (3.0Ð3.5) 4.7 (4.0Ð5.1)
9.1 (8.7Ð9.5) 5.6 (5.3Ð5.9) 5.1 (4.9Ð5.3) 3.7 (3.4Ð4.0) 5.5 (5.1Ð6.0)
12.3 (11.9Ð12.7) 7.1 (6.9Ð7.3) 7.0 (6.7Ð7.3) 6.3 (5.9Ð6.7) 8.0 (7.4Ð8.9)
a Estimated by inverse prediction of developmental times (weeks) with normal logistic regression model at 50% observed larvae reaching the target stage or instar for each temperature. All normal logistic regression models for different ambient temperatures showed signiÞcant relationship between developmental time and the probability of the larvae reaching each target instar or stage (likelihood 2 tests: P ⬍ 0.0001 for all temperature treatments).
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Fig. 3. Effect of ambient temperatures on emerald ash borer larval growth: (A) larval biomass and (B) width of larval gallery.
temperature. While the growth rate in gallery width was slightly lower (0.4688) at 35⬚C than that at 30⬚C (0.4864), the growth rate in biomass at 35⬚C (0.0077) was ⬇34% lower than that at 30⬚C (0.0116), indicating that 35⬚C might have adverse effects on the biomass accumulation of emerald ash borer larvae.
Table 3. Linear regression coefficients (or growth rates) for biomass (g) and gallery width (millimeters) of emerald ash borer larvae over time (week) at different ambient temperatures Temperature (⬚C) Biomass 20 25 27 30 35 Gallery width 20 25 27 30 35
CoefÞcient (growth rate)
SE
t-ratio
P ⬎ ta
0.0050 0.0115 0.0113 0.0116 0.0075
0.0002 0.0011 0.0004 0.0004 0.0004
20.39 10.40 24.59 28.67 16.74
⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001
0.2269 0.4425 0.5558 0.4864 0.4681
0.0045 0.0075 0.0089 0.0060 0.0126
50.93 59.20 62.34 60.30 37.02
⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001
a All t-ratio statistics indicate signiÞcant linear relationships between biomass or gallery width and time (week).
Discussion Findings from our study indicate that development, growth, or both, of eggs and larvae of emerald ash borers are strongly inßuenced by ambient temperature. The median time for egg hatching as well as the development time for larvae (from oviposition) advancing to later (third and fourth) instars (including mature J-larvae) varied greatly with ambient temperature. While no eggs hatched at 12⬚C, the median time for egg hatching decreased from 20 d at 20⬚C to 7 d at 35⬚C. The similar pattern holds true for the development of emerald ash borer larvae. Developmental times for 50% of emerald ash borer larvae advancing to third, fourth, and J-larval stages at 20⬚C were 8.3, 9.1, and 12.3 wk, respectively, approximately two times longer than at 30⬚C for the corresponding instars or stages. In contrast to 30⬚C, however, the development time of emerald ash borer larvae advancing to later instars was apparently longer at 35⬚C, indicating that high (⬎30⬚C) temperature had adverse effects on the larval development. The appropriate range of ambient temperature to rear emerald ash borer larvae may be between 25Ð30⬚C; however, faster rate of egg and larval development should be expected as temperature increases in this range.
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DUAN ET AL.: METHODS FOR LABORATORY REARING OF EMERALD ASH BORER
It is well documented that the life cycle of emerald ash borer populations varied from one to two generations in North America as well as North East Asia (Cappaert et al. 2005; Liu et al. 2007; Siergert et al. 2007; Wei et al. 2007; Duan et al. 2010, 2012). Emerald ash borers enter obligatory diapause as J-larvae and resume development after a period of cold (wintering) temperatures. Whether larvae are able to advance to the J-stage before winter likely determines whether the population completes development in 1 or 2 yr. A 1-yr (univoltine) life cycle might be expected in southern states (e.g., Tennessee, Missouri, Kentucky, and Maryland) whereas a 2-yr (semivoltine) generation may be expected in northern states where the insect may not reach the J-stage before winter. We recommend that future development of ecological models to predict the emerald ash borer phenology and population growth should incorporate the relationship between the developmental time and growth rate of both egg and larvae at different temperatures zones. Findings from our study also have important implications for laboratory rearing of emerald ash borer egg and larval parasitoids for biological control programs. For example, when rearing the egg parasitoid O. agrili, the window of host egg susceptibility to the parasitoid would become wider at the lower temperature (e.g., 20⬚C), as emerald ash borer eggs take much longer to hatch (and thus become not susceptible to parasitism) than at the higher temperature (e.g., 25⬚C or higher). However, the window of host egg susceptibility would become narrower at the higher temperature range (e.g., at 35⬚C), as the host eggs hatch much faster. Thus, the primary exposure time for egg parasitoid production should be adjusted according to the rearing temperature. The same implication also applies to laboratory rearing of larval emerald ash borer parasitoids such as T. planipennisi and S. agrili. For example, both T. planipennisi and S. agrili prefer attacking late (third to fourth) feeding instars of emerald ash borer (Ulyshen et al. 2010b, Gould et al. 2011), but are not able to attack J-larvae in their sap-wood chambers (because they do not feed). Thus, production of suitable stages of emerald ash borer larvae for primary exposure to parasitoids should consider the potential effect of ambient temperature on the host larval development.
Acknowledgments We thank Jacqueline Hoban and Susan Barth (all University of Delaware employees) for their great assistance in all phases of the study. We appreciate the great assistance of Dick Bean, Kim Rice, and Charles Pickett (all at Maryland Department of Agriculture) in producing and shipping adult emerald ash borer to us for this study. We are also grateful to Roger Fuester and Doug Luster (USDAÐARS, BeneÞcial Insects Introduction Research Units) for helpful comments to the earlier version of the manuscript.
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References Cited Bauer, L. S., H.-P. Liu, R. A. Haack, D. L. Miller, and T. R. Patrice. 2004. Natural enemies of emerald ash borer in southeastern Michigan, p. 33. In V. Mastro and R. Reardon (eds.), Proceedings, Emerald Ash Borer Research and Technology Development Meeting, USDA FS FTET2004 Ð 02, Port Huron, MI. Canadian Food Inspection Agency. 2013. Emerald Ash BorerÐAgrilus planipennis. (http://www.inspection.gc.ca/ english/plaveg/pestrava/agrpla/agrplae.shtml). Cappaert, D., D. G. McCullough, T. M. Poland, and N. W. Siegert. 2005. Emerald ash borer in North America: a research and regulatory challenge. Am. Entomol. 51: 152Ð 165. Chamorro, M. L., M. G. Volkovitsh, T. M. Poland, R. A. Haack, and S. W. Lingafelter. 2012. Preimaginal stages of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae: Agrilinae): an invasive species of ash trees (Fraxinus). PLoS ONE 7: e33185. (doi: 10.1371/journal.pone.0033185). Duan, J. J., and C. B. Oppel. 2012. Critical rearing parameters of Tetrastichus planipennisi (Hymenoptera: Eulophidae) as affected by host-plant substrate and host-parasitoid group structure. J. Econ. Entomol. 105: 792Ð 801. Duan, J. J., M. D. Ulyshen, L. S. Bauer, J. Gould, and R. Van Driesche. 2010. Measuring the impact of biotic factors on populations of immature emerald ash borer (Coleoptera: Buprestidae). Environ. Entomol. 39: 1513Ð1522. Duan, J. J., T. Watt, and C. B. Oppel. 2011. An alternative host plant-based method for laboratory rearing of emerald ash borer to produce larval parasitoids for biological control, p. 3. In G. Parra, D. Lance, V. Mastro, R. Reardon, and C. Benedict (eds.), Proceedings, Emerald Ash Borer Research and Technology Meeting, 10Ð23 October 2011, USDA Forest Service/APHIS/FHTET-2011-06, Wooster, OH. Duan, J. J., G. Yurchenko, and R. W. Fuester. 2012. Occurrence of emerald ash borer (Coleoptera: Buprestidae) and biotic factors affecting its immature stages in the Russian Far East. Environ. Entomol. 41: 245Ð254. Emerald Ash Borer Information 2013. Emerald ash borer information. (http://www.emeraldashborer.info/ homeownerinfo.cfm). Gould, J. R., T. Ayer, and I. Fraser. 2011. Effect of rearing conditions on reproduction by Spathius agrili (Hymenoptera: Braconidae), a parasitoid of the emerald ash borer (Coleoptera: Buprestidae). J. Econ. Entomol. 104: 379 Ð387. Haack, R. A., J. E. Jendek, H. Liu, K. R. Marchant, T. R. Petrice, R. M. Poland, and H. Ye. 2002. The emerald ash borer: a new exotic pest in North America. Newsl. Mich. Entomol. Soc. 47: 1Ð5. Kovacs, F. K., R. G. Haight, D. G. McCullough, R. J. Mercader, N. W. Siegert, and A. M. Leibhold. 2010. Cost of potential emerald ash borer damage in U.S. communities, 2009 Ð2019. Ecol. Econ. 69: 569 Ð578. Liu, H.-P., L. S. Bauer, D. L. Miller, T.-H. Zhao, R.-T. Gao, L. Song, Q. Luan, R. Jin, and C. Gao. 2007. Seasonal abundance of Agrilus planipennis (Coleoptera: Buprestidae) and its natural enemies Oobius agrili (Hymenoptera: Encyrtidae) and Tetrastichus planipennisi (Hymenoptera: Eulophidae) in China. Biol. Control 42: 61Ð71. McCullough, D. G., T. M. Poland, and D. Cappaert. 2009. Attraction of the emerald ash borer to ash trees stressed by girdling, herbicide treatment, or wounding. Can. J. For. Res. 39: 1331Ð1345.
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Poland, T. M., and D. G. McCullough. 2006. Emerald ash borer: invasion of the urban forest and the threat to North AmericaÕs ash resource. J. For. 104: 118 Ð124. SAS Institute. 2012. JMP PRO modeling and multivariate methods, version 10. SAS Institute, Cary, NC. Siergert, N. W., D. G. McCullough, and A. R. Tluczek. 2007. Two years under the bark: towards understanding multiple-year development of emerald ash borer larvae, pp. 39 Ð36. In V. Mastro, D. Lance, R. Reardon, and G. Parra (eds.), Proceedings, Emerald Ash Borer and Asian Longhorned Beetle Research and Technology Development Meeting, 29 OctoberÐ2 November 2006, Cincinnati, OH. FHTET-2007-04, USDA 361 Forest Service Forest Health Technology Enterprise Team, Morgantown, WV. (USDA–APHIS) U.S. Department of Agriculture–Animal and Plant Health Inspection Service. 2007. The proposed release of three parasitoids for the biological control of the emerald ash borer (Agrilus planipennis) in the continental United States: environmental assessment. Fed. Regist. 72: 28947Ð28948, Docket No. APHIS-20070060. Ulyshen, M. D., J. J. Duan, and L. S. Bauer. 2010a. Interactions between Spathius agrili (Hymenoptera: Braconidae) and Tetrastichus planipennisi (Hymenoptera: Eulophidae), larval parasitoids of Agrilus planipennis (Coleoptera: Buprestidae). Biol. Control 52: 188 Ð193.
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Ulyshen, M. D., J. J. Duan, L. S. Bauer, and F. Ivich. 2010b. Suitability and accessibility of immature Agrilus planipennis (Coleoptera: Buprestidae) stages to Tetrastichus planipennisi (Hymenoptera: Eulophidae). J. Econ. Entomol. 103: 1080 Ð1085. Wang X. Y., Z. Q. Yang, J. R. Gould, Y. N. Zhang, G. J. Liu, and E. S. Liu. 2010. The biology and ecology of the emerald ash borer, Agrilus planipennis, in China. J. Insect Sci. 10: 128. Wei, X., W. Yun, R. Reardon, T.-H. Sun, M. Lu, and J.-H. Sun. 2007. Biology and damage traits of emerald ash borer (Agrilus planipennis Fairmaire) in China. Insect Sci. 14: 367Ð373. Yang, S., J. J. Duan, T. Watt, K. J. Abell, and R. van Driesche. 2012. Responses of an idiobiont ectoparasitoid Spathius galinae to host larvae parasitized by the koinobiont endoparasitoid Tetrastichus planipennisi: implications for biological control of the emerald ash borer (Coleoptera: Buprestidae). Environ. Entomol. 41: 925Ð932. Zhang, Y.-Z., D.-W. Huang, T.-H. Zhao, H.-P. Liu, and L. S. Bauer. 2005. Two new species of egg parasitoids (Hymenoptera: Encyrtidae) of wood-boring beetle pests from China. Phytoparasitica 53: 253Ð260. Received 17 March 2013; accepted 27 June 2013.
