The Pennsylvania State University
The Graduate School
Department of Entomology
EFFECTS OF TEMPERATURE ON DEVELOPMENT AND FITNESS OF ASIAN
GYPSY MOTH AND THE BIOCONTROL AGENT OF HEMLOCK WOOLLY
ADELGID, SCYMNUS CAMPTODROMUS
A Dissertation in
Entomology
by
Samita Limbu
2017 Samita Limbu
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
May 2017
The dissertation of Samita Limbu was reviewed and approved* by the following:
Kelli Hoover Professor of Entomology Dissertation Advisor Chair of Committee
Melody Keena Research Entomologist Adjunct Faculty
Mary Barbercheck Professor of Entomology
Edwin G. Rajotte Professor of Entomology
James Sellmer Professor of Horticulture
Gary W. Felton Professor of Entomology Department Head of Entomology
*Signatures are on file in the Graduate School
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ABSTRACT
The recent rise in mean annual temperature along with growth and advancement in international trade have accelerated the rate of invasion by exotic species throughout the world. Around 50,000 non-native species have been estimated to be introduced into the US (Pimental et al. 2004). Exotic species have often become pests and have caused substantial disturbances to forest and agricultural ecosystem, threatened the biodiversity, and has the potential for severe economic impacts. Hemlock woolly adelgid, Adelges tsugae (Annand) (Hemiptera: Adelgidae) and gypsy moth (Lymantria dispar L.) are examples of two non-native insects that have seriously threatened urban and forest ecosystem in the US. Hemlock woolly adelgid (HWA) since its initial introduction has steadily expanded its range in the eastern US and caused extensive damage to hemlock stands. This insect is considered as the single most important threat to native hemlock stands in the eastern US. Similarly, gypsy moth is a major defoliator of numerous tree species in most of the northern hemisphere. In North America, the gypsy moth of European origin has been present for over 145 years and has decimated hardwood forests. Recently, Asian subspecies of L. dispar have been detected in North America possibly through international trade and is considered a greater threat to the commercial and urban forest than the European counterparts.
Not all non-native species have a negative impact because most of them do not survive in a new environment. Environmental barriers can prevent introduced species from establishing in a new location. Thus, invasion success is more likely when climatic conditions in the introduced area are similar to the native habitat of the exotic species (Walther et al. 2009). Because insects are ectotherms, temperature has a significant impact on their basic physiological functions. Understanding the potential of the non- native species to colonize and establish in a new environment requires a thorough knowledge on effects of temperature on the life history of the insect. We examined the effects of temperature on growth, development and fitness of two insect species: Scymnus camptodromus and Asian gypsy moth.
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In an attempt to control HWA population in the eastern US, several predators were introduced from outside the pest’s native range and Scymnus (Neopullus) camptodromus Yu and Liu (Coleoptera: Coccinellidae) was one of them. It is a predacious lady beetle brought to the US from China as one of the potential biological control agents of HWA. The lady beetle’s phenology is closely synchronized with that of HWA and has several characteristics of a promising biological control agent. As a prerequisite to field release, we first evaluated the potential non-target impacts of S. camptodromus. In host range studies, the predator was given the choice of adelgid and non-adelgid prey items. Non-target testing showed that S. camptodromus will feed to some degree on other adelgid species, but highly prefers HWA. We also evaluated the larval development of the predator on other adelgid species and only a small proportion of predator larvae were able to develop to adulthood. S. camptodromus showed minimum interest in feeding on the non-adelgid species tested in choice and no-choice experiments. S. camptodromus females did not oviposit on any host material other than HWA infested hemlock. Under the circumstances of the study, S. camptodromus appears to be a specific predator of HWA, with minimal risk to non-target species.
We also evaluated the effect of temperature on S. camptodromus larval development time and predation by instar and strain (geographical population). We observed that temperature had significant effects on the predator's life history. The larvae tended to develop faster and consume more eggs of HWA per day as rearing temperature increased. Mean egg consumption per day of HWA was significantly less at 15 than at 20 C. However, since larvae took longer to develop at the lower temperature, the total number of eggs consumed per instar during larval development did not differ significantly between the two temperatures. The lower temperature threshold for predator larval development was estimated to be 5 C and the accumulated degree-days for 50% of the predator neonates to reach adulthood were estimated to be 424. Although temperature had a significant effect on larval development and predation, it did not impact survival, size or sex ratio of the predator at 15 and 20 C. Furthermore, no remarkable distinctions were observed among different geographical populations of the
v predator. These findings on developmental rates, degree-day requirements, and predator consumption provide baseline data for developing mass rearing procedures and planning field releases of S. camptodromus.
For Asian gypsy moth, because it is not yet an established pest, our goal was to develop phenology models of AGM strains in response to temperature to facilitate detection and management/eradication efforts. Predicting phenology of the Asian gypsy moth is critical for monitoring and management. For instance, Bacillus thuringiensis kurstaki (Btk), a preferred treatment for Asian gypsy moth control, is most effective if applied to the early second instar (Reardon et al. 1994). Currently, phenology of the gypsy moth is predicted based on the European subspecies and by climate matching, which may not be accurate for the Asian subspecies. In this study we evaluated the development of eight strains of AGM from a broad range of geographic latitudes reared on artificial diet at five constant temperatures (10-30 °C). Our results suggest that AGM larvae developed faster as rearing temperature increased until it reached an optimum at 29 °C. Larvae displayed significant molting problems at the highest and lowest temperatures tested (10 and 30 °C), and at 30 °C female fitness was markedly compromised, as evidenced by reduced fecundity and fertility. These findings suggest that development and survival of Asian gypsy moth may be limited by summer temperature extremes in the southern US. We also determined the degree-day requirements for two critical life stages (from egg hatch to second instar and egg hatch to adult) to predict the timing for both bio-pesticide application and adult trapping. Our data will benefit pest managers in developing management strategies, pest risk assessments, and timing for implementation of management tactics.
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TABLE OF CONTENTS
List of Figures ...... x
List of Tables ...... xiii
Acknowledgements ...... xvi
Chapter 1 Introduction ...... 1
1.1 Impacts of temperature on exotic insects ...... 1 1.2 Hemlock woolly adelgid (HWA; Adelges tsugae) ...... 3 1.2.1 Importance of hemlock trees in North America ...... 4 1.2.2 Life history of HWA ...... 5 1.2.3 Current management practices against HWA ...... 6 1.2.3.1 Chemical control ...... 6 1.2.3.2 Cultural control ...... 7 1.2.3.3 Biological control of HWA ...... 7 1.2.3.3.1 Classical biological control ...... 8 1.2.3.3.1.1 Scymnus camptodromus (Coleoptera: Coccinellidae) as a potential biological control of HWA ...... 9 1.3 Lymantria dispar (Lepidoptera: Erebidae) in North America...... 11 1.3.1 Asian vs. European/North American gypsy moth ...... 13 1.3.2 Life history of Asian gypsy moth ...... 14 1.3.3 Management practices for AGM ...... 14 References ...... 16
Chapter 2 Host range specificity of Scymnus camptodromus (Coleoptera: Coccinellidae), a predator of hemlock woolly adelgid ...... 24
2.1 Abstract ...... 24 2.2 Introduction ...... 25 2.3 Materials and Methods ...... 28 2.3.1 Predator and Prey Source ...... 28 2.3.2 Prey Preference (Choice Tests) ...... 29 2.3.3 Four-way choice tests ...... 30 2.3.4 Three-way choice tests ...... 31 2.3.5 Paired choice tests ...... 33 2.3.6 Prey Acceptance (No-Choice Tests) ...... 33 2.3.7 Prey suitability...... 35 2.4 Results ...... 36 2.4.1 Choice test with four adelgids ...... 36 2.4.2 Choice test with three adelgids ...... 36 2.4.3 Paired choice tests ...... 37 2.4.4 No choice test with adelgid prey items ...... 37 2.4.5 No choice test with non-adelgid prey items ...... 38 2.4.6 Prey suitability...... 38
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2.5 Discussion ...... 39 References ...... 42
Chapter 3 Scymnus camptodromus (Coleoptera: Coccinellidae) Larval Development and Predation of Hemlock Woolly Adelgid ...... 45
3.1 Abstract ...... 45 3.2 Introduction ...... 46 3.3 Materials and Methods ...... 48 3.3.1 Source of predators and rearing...... 48 3.3.2 Prey provisioning ...... 49 3.3.3 Development time of S. camptodromus larvae by temperature and strain ...... 50 3.3.4 Statistical Analysis ...... 50 3.3.4.1 Estimating degree-day requirements (DD) for larvae ...... 52 3.4 Results ...... 53 3.4.1 Effect of temperature on predator survival and development time ...... 53 3.4.2 Predation by S. camptodromus larvae ...... 54 3.4.3 Estimation of degree-day requirement ...... 56 3.4.4 Effects of temperature, sex, and strain on adult beetle size ...... 56 3.5 Discussion ...... 57 References ...... 67
Chapter 4 Effects of Temperature on Development of Lymantria dispar asiatica and Lymantria dispar japonica (Lepidoptera: Erebidae) ...... 69
4.1 Abstract ...... 69 4.2 Introduction ...... 70 4.3 Materials and methods ...... 72 4.3.1 Gypsy moth populations ...... 72 4.3.2 Larval rearing at different temperatures ...... 73 4.3.3 Statistical Analyses ...... 74 4.4 Results ...... 78 4.4.1 Survival ...... 78 4.4.2 Development Time from Hatch to Second Instar and to Adult Emergence .... 79 4.4.3 Rate of Larval Development and Estimated Degree-Day Requirements ...... 80 4.4.4 Pupal and Adult Body Weight of L. d. asiatica ...... 81 4.4.5 Effect of Temperature and Population on Pupal and Adult Fitness ...... 82 4.5 Discussion ...... 83 References ...... 125
Appendix A Efficacy test of Scymnus camptodromus in the field using confined releases of predator adults and larvae ...... 128
A.1 Introduction ...... 128 A.2 Materials and Methods ...... 129 A.2.1 Study sites and predator population ...... 129 A.2.2 Spring 2012 confined release of S. camptodromus ...... 129 A.2.3 Estimation of average HWA eggs consumed by S. camptodromus adults and larvae ...... 130
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A.2.4 Field release of S. camptodromus 2013 ...... 131 A.2.5 Statistical analyses ...... 132 A.3 Results ...... 133 A.3.1 Confined release 2012 ...... 133 A.3.2 Confined release 2013 ...... 133 A.3.2 Survival and oviposition ...... 134 A.4 Discussion ...... 135 References ...... 140
Appendix B Petition for Unconfined Field Release of the Exotic Predator Scymnus camptodromus for Biological Control of Hemlock Woolly Adelgid (Adelges tsugae), in the Eastern United States ...... 141
B.1. Introduction ...... 141 B.1.1 Proposed Action ...... 141 B.1.2 Proposed of release ...... 141 B.1.3 Need for release ...... 142 B.1.4 Reasons for choice of the agent ...... 143 B.1.5 Specific location of rearing/quarantine facility and name of qualified personnel operating the facility ...... 144 B.1.6 Timing of the release ...... 144 B.1.7 Methods to be used ...... 145 B.1.8 Disposal of any host material and pathogens, parasites, hyperparasitoids of agent accompanying import shipment ...... 146 B.1.9 Agencies and/or individuals that will be involved in the release and monitoring ...... 146 B.2. Target Pest Information ...... 147 B.2.1 Taxonomy ...... 147 B.2.2 Economic impact of pest and benefits of the target organism ...... 148 B.2.3 Economic impact of pest and benefits of the target organism ...... 149 B.2.4 Distribution of pest ...... 149 B.2.5 Regulatory and/or pest status of the target in state, provincial or federal law ...... 149 B.2.6 Knowledge of status of other biological control organisms (indigenous and introduced) that attack the pest...... 150 B.3. Biological Control Agent Information ...... 152 B.3.1 Taxonomy ...... 152 B.3.2 Methods used to identify the agent ...... 153 B.3.3 Location of voucher specimens ...... 153 B.3.4 Natural geographic range, other areas of introduction, and expected attainable range in North America (also habitat preference and climatic requirements of the agent) ...... 153 B.3.5 Source of the culture/agent in nature (name of collector, name of identifier) ...... 154 B.3.6 Host/biocontrol agent interaction (e.g., parasitoid, pathogen, parasite, competitor, antagonist, etc.) ...... 154 B.3.7 Life history (including dispersal capability and damage inflicted on host insect or mite) ...... 155
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B.3.8 Known host organism based on valid literature records, host data from museum specimens, and unpublished records...... 156 B.3.9 History of past use of the biological control agent...... 156 B.3.10 Pathogens, parasites, hyperparasitoids of agent and how to eliminate them from a culture of the agent ...... 156 B.3.11 Standard Operating Procedure stating how agent will be handled in quarantine ...... 157 B.3.12 Other closely related genera, sibling species or closely-similar species in North America ...... 157 B.4. Environmental & Economic Impacts of the Proposed Release ...... 158 B.4.1 Known impact on vertebrates including humans ...... 158 B.4.2 Direct impact of the organism (e.g. intended effects on targets, direct effects on non-targets) ...... 158 B.4.3 Alternate Hosts Tested ...... 159 B.4.4 Choice Tests ...... 160 B.4.5 No-choice Tests (Feeding) ...... 160 B.4.6 No-choice Tests (Larval development) ...... 161 B.4.7 Possible direct or indirect effects on threatened and endangered species in North America ...... 161 B.5. Post Release Monitoring (In designing monitoring plans please note that pre- release baseline measurements of targets and non-targets provide for better monitoring data and documentation of effects. Also, some effects may take years or decades to manifest while others may not be long lasting) ...... 162 References ...... 174
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LIST OF FIGURES
Figure 1-1. Hemlock woolly adelgid distribution in eastern North America in 2015 (USDA 2015a) ...... 21
Figure 1-2. a) Hemlock woolly adelgid infested twig, b) First instar hemlock woolly adelgid (Havill 2015) ...... 21
Figure 1-3. Life cycle of HWA in the native range (D’Amico and Havill 2016) ...... 22
Figure 1-4. Scymnus camptodromus life stages. a) Egg b) larva c) pupa d) adult ...... 22
Figure 1-5. Gypsy moth quarantine areas in North America (USDA APHIS 2015b) ...... 23
Figure 1-6. a) Gypsy moth life-cycle (Cloyd & Nixon 2015), AGM life stages; b) first- instars emergence from an egg mass, c) larvae, d) Female (left) male (right), e) Pupa ... 23
Figure 2-1. Reduction in mean number of adelgid eggs (± SE) after 48 h when four adelgid species were provided simultaneously to S. camptodromus. Different letters represent significant differences in mean egg reduction (G2 = 44.5; P < 0.01)...... 41
Figure 2-2. Influence of prey combination on the mean number (± SE) of A. tsugae eggs consumed by S. camptodromus. Prey combination presented as Set A (A. tsugae, A. laricis and A. cooleyi), Set B (A. tsugae, P. strobi and A. cooleyi) and Set C (A. tsugae, P. strobi and A. laricis). Different letters represent a significant difference in mean number of A. tsugae eggs consumed by S. camptodromus within each set (G2 = 14.1, df = 3, P = 0.003)...... 42
Figure 3-1. Average eggs consumed per day among different S. camptodromus strains and instars. The solid line represents egg consumption per day at 20 ºC and the dashed line represents egg consumption per day at 15 ºC...... 64
Figure 3-2. Relationship between temperature and development rate (1/d) of S. camptodromus from neonate to adult. The line represents the simple linear regression combining all predator strains; some circles represent multiple data points at a given temperature due to overlap...... 65
Figure 3-3. Relationships between temperature and S. camptodromus larval (DTP) and pupal (DP) rates (1/days) of development. Lines represent the estimated linear relationships combining all the predator strains (DGS, LJS and MNP). Each diamond represents mean days to pupation and each triangle represents days as pupa at each temperature...... 65
Figure 3-4. Cumulative proportion of S. camptodromus neonates to reach adulthood over accumulated degree-days. Open circles represent individuals reared at 15, 20, and 25 °C; there is considerable overlap of circles...... 66
Figure 4-1. Percentage (mean ± SE) survivorship of L. dispar asiatica neonates to the pupal stage (a) and to adult (b) reared at four constant temperatures. The mean
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percentage survivorship of larvae to pupae (F = 3.27; df = 21, 288; P < 0.0001) and adult stages (F = 4.74; df = 21, 288; P < 0.0001) were significantly affected by the temperature and population interaction. Error bars represent the 95% confidence interval...... 117
Figure 4-2. Developmental rates (1/d) of L. dispar asiatica larvae from the R1 and C2 populations from neonate to the second instar in response to constant temperatures. The solid and dashed lines represent linear regression of R1 and C2, respectively. Data points at each temperature represent mean developmental rate of each sex, open circles for the R1 population, and solid triangles for the C2 population (See methods for detail)...... 118
Figure 4-3. Cumulative proportion of L. dispar asiatica R1 and C2 individuals to reach the second instar over accumulated degree-days. Open circles represent R1 and open triangles represent C2 individuals reared at 10, 15, 20, 25 and 30 °C. Solid lines were fitted to cumulative proportion of individuals using Gompertz function, P = exp [- exp(- bDD +a] ...... 119
Figure 4-4. Developmental rates (1/d) of L. dispar asiatica larvae from the R1 and C2 populations from neonate to adulthood in response to constant temperatures. The dashed and solid lines were fitted to Shi/Performance model of R1 and C2, respectively. Data points at each temperature represent mean developmental rate by ultimate instar; open circles for the R1 and solid triangles for the C2 population. (See methods for detail)...... 119
Figure 4-5. Cumulative proportion of L. dispar asiatica individuals from the R1 and C2 populations to complete development to adult over accumulated degree-days. Different data points represent individuals reared at 15, 20, 25, 30 °C of two populations of L. dispar asiatica, circles for the R1 and triangles for the C2 (Open marker for females and solid for males). Solid lines were fitted to cumulative proportion of female and dotted lines to male using Gompertz function: P = exp [- exp(- bDD +a]...... 120
Figure 4-6. Body weight (Mean ± SEM) of male and female L. dispar asiatica pupae reared at different constant temperatures. Different data points represent the ultimate instars within each sex (open markers female and solid markers male) for two populations of L. dispar asiatica, circles for the C2 and triangles for the R1. Solid lines were fitted to female weight and dotted lines to male weight using function: y = a (T – Tmin) (Tmax – T) (Lysyk 2001)...... 121
Figure 4-7. Body weight (Mean ± SEM) of male and female L. dispar asiatica adults reared at different constant temperatures. Different letters above bars represent significant differences at P < 0.05 using Tukey-Kramer post hoc test...... 122
Figure 4-8. Percentage of (Mean ± SE) L. dispar asiatica pupal mortality (a) and deformed adults (b) reared at five constant temperatures. Percentage of pupal mortality (F = 4.16; df = 21, 280; P < 0.0001) and deformed adults (F = 1.81; df = 21, 271; P < 0.0181) was significantly affected by the interaction of temperature and population. Error bars represent the 95% confidence interval...... 123
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Figure 4-9. Estimated overall performance of Asian gypsy moth by population at different temperatures. The estimated overall performance was significantly affected by an interaction of temperature and population (F = 4.50; df = 21, 280; P < 0.0001). Error bars represent the 95% confidence interval...... 124
Figure. A-1. Mean reduction in number of HWA eggs by treatment at the end of two months of predation by S. camptodromus in the field. Total HWA eggs lost was significantly greater in bags with S. camptodromus adults (F = 16.7; df = 2, 27; P < 0.0001) compared to the two controls. * indicates significant difference at P < 0.05. .... 139
Figure A-2. HWA eggs that remained per ovisac on each experimental branch after eight weeks of exposure to S. camptdromus adult confined to HWA-infested branches in the field. HWA eggs remaining in bags significantly differed by time (F = 201; df = 3, 131; P < 0.0001) and by treatment (F = 3.42; df = 2, 131; P = 0.04) with a significant reduction in HWA eggs in bags with beetles. There was no significant interaction between time and treatment (F = 1.9; df = 6, 131, P = 0.0847). *indicates significant difference of beetle treatment compared to controls at P < 0.05...... 140
Figure A-3. Percentage of adult S. camptodromus that survived confined release in the field at each 2 week check in 2013...... 140
Figure B-1. Map showing the primary collection sites in China of three species of Scymnus brought to the eastern U.S. (Montgomery and Keena 2011) ...... 173
Figure B-2 Average monthly temperatures (º C) in selected locations in China and in potential release sites in the U.S. (Mongomery and Keena 2011) ...... 174
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LIST OF TABLES
Table 3-1a. GLIMMIX model for mean development time of S. camptodromus after eclosion...... 60
Table 3-1b. Mean development time [mean days ± SE (n)] by life stage of S. camptodromus reared at 15 or 20 °C after eclosion ...... 60
Table 3-2a. GLIMMIX model for mean number of hemlock woolly adelgid eggs consumed by S. camptodromus after eclosion...... 61
Table 3-2b. Mean number [mean ± SE (n)] of hemlock woolly adelgid eggs consumed by S. camptodromus by instar reared at 15 °C or 20 °C after larval eclosion...... 62
Table 3-3. GLIMMIX model for mean number of hemlock woolly adelgid egg consumed per day by S. camptodromus after eclosion...... 63
Table 3-4. Mean size [mean ± SE (n)] of S. camptodromus following adult eclosion reared at 15 °C or 20 °C...... 63
Table 4-1. Approximate location (latitude and longitude), designation of the source population, collection date, and laboratory generation of gypsy moth evaluated in this study, arranged by latitude from north to south...... 89
Table 4-2. Mathematical models used to describe relationships between temperature and developmental rate of Asian gypsy moths...... 91
Table 4-3. Proportion of each population of Asian gypsy moth categorized (using HSPLIT in SAS) into each of the nodes based on the developmental criteria listed for each temperature. Criteria for 10 °C are based on days in the first instar (D1), while the other temperatures are based on days to adult (DA)...... 92
Table 4-4. Mean [± SE (n)] time spent (d) by L. dispar asiatica in the first instar at different temperatures by sex and population...... 94
Table 4-5. Mean [± SE (n)] time spent (d) by L. dispar asiatica from egg hatch to adult at different temperatures, by population and ultimate instar attained...... 95
Table 4-6. Parameter values for linear and non-linear models used to describe the relationship between temperature (°C) and developmental rate of Asian gypsy moth life stages by population and sex...... 96
Table 4-7. Estimated accumulated degree-days (± SE) required to reach second instar and adult emergence by L. d. asiatica larvae from egg hatch for 10, 50, 90 and 99% of the population...... 97
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Table 4-8. Mean [± SE (n)] pupal weight (g) of L. dispar asiatica at different temperatures, by population, ultimate instar attained and sex...... 98
Table 4-9. Parameter values for Lysyk models used to describe the relationship between temperature (°C) and pupal weight (g) of Asian gypsy moth by population and sex...... 100
Table 4-10. Percentage (Mean + SE [n]) of embryonated eggs of L. dispar asiatica populations by temperatures...... 101
SI 1. Development time [(Mean ± SEM) n] spent as first instar by L. dispar asiatica at different temperatures by sex, and population...... 102
SI 2. Development time [(Mean ± SEM) n] spent as second instar by L. dispar asiatica at different temperatures by sex, and population...... 104
SI 4. Development time [(Mean ± SEM) n] spent as fourth instar by L. dispar asiatica at different temperatures by sex, and population...... 108
SI 5. Development time [(Mean ± SEM) n] spent as fifth instar by L. dispar asiatica at different temperatures by sex, and population...... 110
SI 6. Development time [(Mean ± SEM) n] spent as sixth instar by L. dispar asiatica at different temperatures and population ...... 112
SI 7. Development time [(Mean ± SEM) n] spent as pupa by L. dispar asiatica at different temperatures by sex, and population...... 113
SI 8. Total development time [(Mean ± SEM) n] of L. dispar asiatica from egg hatch to adult at different temperatures by population, and ultimate instar...... 115
Table A-1. Four branches from each tree were selected and randomly assigned for the different treatments...... 137
Table A-2. Mean [± SEM (n)] HWA eggs consumed by three different strains of S. camptodromus adults per day and mean eggs oviposited in 72 hours at 20 °C in the lab...... 137
Table A-3. Mean (± SE) HWA life stages remaining on experimental branches containing S. camptodromus adult as measured every 2 weeks over an 8-week period in 2013...... 137
Table A-4. Mean (± SE) HWA stages on bagged and unbagged experimental branches after 6 weeks of predation by S. camptodromus larvae in the field in 2013...... 138
Table B-1. Location of the source populations of Scymnus camptodromus from China (Keena et al. 2012) ...... 163
Table B-2. Important species of conifer-feeding adelgids (Havill & Foottit, 2007) ...... 164
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Table B-3. Mean development time [mean days ± SE (n)] by life stage of S. camptodromus reared at 15 or 20 °C after eclosion (Limbu et al. 2015)...... 165
Table B-4. Mean number [mean ± SE (n)] of hemlock woolly adelgid eggs consumed by S. camptodromus by instar reared at 15 °C or 20 °C after larval eclosion (Limbu et al. 2015)...... 165
Table B-5. Listing of all the Scymnus species in IT IS database...... 166
Table B-6. Mean [± SEM (n)] HWA eggs consumed by three different strains of S. camptodromus adults per day in lab...... 171
Table B-7. Test prey on associated host plant, native range of test prey, and rationale for selection of prey item used in host specificity tests (Limbu et al. 2016)...... 172
Table B-8. Mean (±S.E) number of non-adelgid prey items eaten in 72 h in paired choice tests...... 173
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ACKNOWLEDGEMENTS
First, I would like to sincerely thank my advisor, Kelli Hoover for giving me the opportunity to work with her. She has helped me learn the fundamentals of research and has always motivated me to achieve my goals. I am very grateful for the guidance she has provided me in every step of the way. Her ideas and teachings will remain valuable throughout my life.
My sincere thanks to my collaborator and my mentor, Melody Keena, for letting me work at her research facility in USDA forest Service, Ansonia. Her support and training have been very valuable. I wouldn’t have achieved my research goals without her constant support, advice, and training.
I would like to thank current and past members of Hoover’s lab: David Long, Francine
McCullough, and Erin Scully for helping me in multiple projects and being there for me when I needed them. Thanks to Charlie Mason, and Ikkei Shikano for their insightful discussions about my research data and their advice. I would like to acknowledge members of USDA Forest Service lab: Paul Moore, Jessica Richards, Vicente Sánchez, and Greg Bradford, for providing assistance in the laboratory. My sincere thanks to Edwin Rajotte and Robert (Talbot) Trotter III for their support and contributions to my research projects.
My graduate research would not have been possible without generous funding from various sources. The experiments and write-up portions in chapters 2 and 3 were funded in part by two USDA Forest Service Northeastern Area (NA) State and Private Forestry (S&PF)
Technology Development competitive grants to KH: 13-CA-11422224-060 part of the Joint
Hemlock Woolly Adelgid Initiative funding and 09-DG-1142004-330 with allocation through PA
DCNR to Improve Management of Hemlock Woolly Adelgid. The experiments and write-up in chapter 4 were funded in part by USDA APHIS Plant Protection and Quarantine Science and
Technology Cooperative Agreement 14-8130-0451-CA to KH.
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Finally, I would like to thank my family back home and my friends here in Penn State for their continuous love and support.
1
Chapter 1
Introduction
1.1 Impacts of temperature on exotic insects
Temperature has significant impact on regulating the survival, growth, development, fecundity, spread and migration ectotherms such as insects (Walther et al. 2009, Beck 1983).
There is increasing interest in investigating insect responses to changing temperature because it influences insects’ physiological processes, such as metabolic and respiratory rates, as well as functions of the nervous and endocrine systems (Neven 2000). Each insect has an optimum range of temperatures in which normal development, growth, and functioning of their physiological systems are achieved. Extreme thermal conditions can be fatal to insects if irreversible physiological changes occur. The majority of insects are well adapted to their native range and have evolved several mechanisms, such as diapause or quiescence, to survive under extreme thermal conditions, but they may struggle to survive in a new environment to which they are not adapted. In addition, the recent rise in mean annual temperatures associated with global climate change and frequent augmentation of exotic species due to trade and other human activities have increased the likelihood of establishment of exotic insects (Huang et al. 2011; Ponti et al. 2014).
Growth and advances in international trade have accelerated the rate of invasion by exotic species over the last 200 years (Liebhold et al. 1995a). Exotic species have often become pests and can cause substantial disturbances to an ecosystem with potential for severe economic impacts. Every year in the US, invasive arthropods are associated with $2.1 billion in losses, and threaten 42 % of native endangered species (Pimentel et al. 2000, Pimentel et al. 2005, Ponti et al.
2014). Environmental barriers can prevent introduced species from establishing in a new
2 location, thus, invasion success is more likely when climatic conditions in the introduced area are similar to the native habitat of the introduced species (Walther et al. 2009). Therefore, understanding the effects of temperature on insects is crucial, especially in cases of exotic insects, to estimate the risk of pest establishment (Ratte 1985, Nietschke et al. 2007, Naves and Sousa,
2009). Temperature based phenology models are especially useful to help predict the occurrence and seasonality of each developmental stage of an insect (Nietschke et al. 2007). Such models can be used in planning the timing for deploying biological control agents, insecticide sprays, and pheromone traps for efficient monitoring and management.
The goal of my dissertation research is to determine the effects of temperature on growth and development of two exotic insects Scymnus camptodromus (Coleoptera:
Coccinellidae) and Asian gypsy moth (Lepidoptera: Erebidae; Lymantria dispar spp.).
Scymnus camptodromus is an efficient predator of hemlock woolly adelgid (Hemiptera:
Adelgidae; Adelges tsugae) in China (Montgomery and Keena 2011) and this study explores the potentiality of this predator as a biocontrol agent if released against hemlock woolly adelgid in the eastern US. In contrast, Asian gypsy moth (AGM) presents a serious threat to the US and many other countries should it be introduced and become established (Matsuki 2001, Pitt 2007,
USDA 2016); therefore, I examined effects of temperature on development of AGM to predict its potential for establishment in different geographic regions of the US and to facilitate timing for treatments and trapping.
For purposes of continuity, I will focus first on hemlock woolly adelgid and a predator from its native range, S. camptodromus, followed by a discussion of Asian gypsy moth.
3 1.2 Hemlock woolly adelgid (HWA; Adelges tsugae)
Hemlock woolly adelgid (HWA) is a hemipteran pest belonging to the family Adelgidae.
It is native to the western US and Asia but was introduced into the eastern US. A phylogenetic study revealed that HWA in the eastern US originated from southern Japan, probably in nursery stock (Havill et al. 2006). It was first reported in Richmond, Virginia on ornamental hemlock species in 1951 (Gouger 1971) and now occurs in 19 states in eastern North America (Figure 1-
1). Recently, HWA was also detected in western Michigan where efforts to eradicate the isolated infestations were initiated (Michigan Invasive Species 2016). Eastern hemlock, Tsuga canadensis
(L.) Carriere, and Carolina hemlock, Tsuga caroliniana Engelmann, are the two susceptible hemlock species affected by HWA infestations in the eastern US.
As a hemipteran, hemlock woolly adelgid has piercing and sucking mouthparts, similar to those of aphids (Figure 1-2). However, HWA stylet length is much longer than that of aphids, which enables HWA to gain access to stored nutrients in xylem ray parenchyma cells. The stylet bundle is comprised of two outer mandibular stylets and two inner maxillary stylets protected by the salivary sheath (Young et al. 1995). A study on feeding biology of HWA suggests that these insects may be able to perform extra-oral digestion and induce systemic defense responses in host plants (Oten et al. 2014). HWA feeding on hemlock results in yellowing and desiccation of the hemlock needles and can kill the tree in 1-3 years in its southern range and 5-15 years in its northern range (Ellison et al. 2010).
Currently, available control measures are not sufficiently effective to reduce the spread of
HWA in the eastern US. Systemic insecticides are effective against HWA, but successes are generally limited to ornamental plantings (Cheah et al. 2004). For example, imidacloprid is a commonly used systemic insecticide against HWA in high values trees. However, the insecticide residues can persist in hemlock needles for extended periods of time, which can affect the
4 arthropod community in these trees (Kung et al. 2015). This raises concern for non-target arthropods and the broader ecological impacts of insecticides in forests.
HWA is not a pest in its native range, which suggests that pest populations are being kept in check by natural enemies and/or host plant resistance in the native range (McClure 1987).
While development of host plant resistance is still limited to breeding studies, for several years research has focused on developing a complex of natural enemies of HWA for reducing pest populations. Currently, eight species of predatory insects have been released in the northeastern
US, with three showing evidence of establishment. Two of these predator species were imported from Japan, four from the northwestern US, and two from China (Keena et al. 2012, Houtman
2015, NPS 2013). My thesis explores the feasibility of using another predatory beetle from
China, Scymnus camptodromus, as a component of the complex of biocontrol agents for HWA in the northeastern US.
1.2.1 Importance of hemlock trees in North America
Hemlocks are evergreen conifers belonging to the family Pinaceae. They are an important foundation tree species and dominate about 1 million hectares of eastern US forests
(Domec et al. 2013). Hemlocks can live 800 years or more and can reach 175 feet in height and 5 feet in diameter (Ward et al. 2004). There are nine hemlock species; among these four are native to North America. Eastern hemlock (T. canadensis) and Carolina hemlock (T. caroliniana) are found in the eastern US, whereas Mountain hemlock, Tsuga mertensiana, and Western hemlock,
Tsuga heterophylla, are endemic to the western US (Clepper 1994).
Hemlocks are the fifth longest-lived and most abundant tree species in the eastern US
(Pederson et al. 2012), having strong relationships with their associated community of other plants and animals, indicating their importance to forest ecology. Moreover, hemlocks are shade
5 tolerant trees, co-evolved in mixed deciduous forests, and are known for their ability to mediate soil moisture, stabilize stream base-flow, and regulate stream temperatures. They also create cool, moist microclimates with slow rates of nitrogen cycling because of deep shade, resulting in slow decomposition of acidic organic litter that is unique to hemlock dominated forests (Ellison et al.
2010). Thus, hemlocks provide and support a unique community of terrestrial and aquatic organisms; there are no other known tree species that can replace its ecological functions (Ward et al. 2004).
1.2.2 Life history of HWA
Hemlock woolly adelgids have a complex holocyclic life cycle in their native range and an abbreviated life cycle in their introduced range (Figure 1-3). HWA requires two hosts and two years to complete its holocyclic life cycle, with Picea being its primary and Tsugae being the secondary host. In the introduced range, survival or reproduction of the sexual form is unknown due to lack of a suitable host.
In the introduced range of HWA, two apterous, parthenogenetic generations occur: sistens and progrediens. The sistens resumes development after summer aestivation slowly in the late fall and start laying eggs in January in the South and in March in the North. The sistens generation produces more eggs compared to the progrediens (about 300 and 100 eggs per female, respectively) (McClure et al. 2001). Thus, in the introduced range, the sistens are considered the key stage in the adelgids’ life cycle to target for management. The eggs of the sistens’ give rise to progrediens and the flying generation. Due to lack of suitable hosts, the flying generation is not known to reproduce, so there is no sexual generation in the introduced range. The progrediens lay sistens eggs that oversummer as settled crawlers that do not become active again until fall.
6 1.2.3 Current management practices against HWA
1.2.3.1 Chemical control
Horticultural oils, insecticidal soaps, malathion, diazinon, fluvalinate, imidacloprid and other pesticides can be used to significantly reduce HWA populations on hemlocks (Wallace and
Hain 2000). Contact insecticides such as insecticidal oils, soaps, and other petrochemicals were used in earlier attempts to control HWA and were successful if the full cover of foliage was obtained, which is very difficult to accomplish in forest settings. Currently, imidacloprid is the most commonly used systemic insecticide against HWA in high-value trees and is quite effective
(Webb et al. 2003). Also, imidacloprid and its metabolite, olefin, persist in tree foliage for a long time, suppressing HWA populations, but these compounds can be a threat to aquatic and other non-target organisms due to leaching. Chemical controls can effectively reduce HWA populations, but at the same time, HWA is more problematic in forested areas where the application of insecticide is not practical due to economic and ecological issues (Montgomery and
Lyon 1995, Wallace and Hain 2000, Butin et al. 2004). Recently, studies have indicated that using low rates of imidacloprid on hemlock trees provided sufficient control over HWA populations, promoted hemlock health, and supported post-treatment HWA predators after chemical protection had diminished in the trees (Eisenback et al. 2010, Eisenback et al. 2014,
Mayfield et al. 2015). This indicates that chemical and biological control may be compatible for the management of HWA; however, further studies are required to determine the selective timing for application of these treatments.
7 1.2.3.2 Cultural control
Silvicultural practices and development of HWA-resistant or tolerant hemlock hybrids are two of the approaches that have been explored for HWA control, yet both are limited to research studies because it takes a very long time to develop tree lines and treatment protocols.
Silvicultural treatments have been applied and are being evaluated (Fajvan 2008). For example, reducing stand density for better hemlock growth has been evaluated, but it is not known if the effort will make hemlock less or more attractive to HWA. Further studies are needed to evaluate the effects of thinning on HWA populations, which can alter forest climate, soil, and foliar nutrient cycles (Fajvan 2008). As for hybrid development, Tsuga chinensis (Franch.) E. Pritz is a hemlock species found in China, and hybrids of T. chinensis with T. caroliniana are comparatively more resistant to A. tsugae than native species in the eastern US (Montgomery et al. 2009). These hybrids may be suitable for landscape or horticultural use but their effectiveness in HWA-invaded forest settings and success in replacing native hemlocks is still being evaluated.
Impacts of replacement on biodiversity and sustainability of hybrids in eastern forests have yet to be explored.
1.2.3.3 Biological control of HWA
As a biological control agent, entomopathogenic fungi are being studied to control HWA.
Several entomopathogenic fungi associated with HWA have been identified from the eastern US and southern China and some were found to be more effective than others in a laboratory study
(Reid et al. 2002). Entomopathogenic fungi have specific temperature and moisture requirements to be efficacious, and field conditions may not be as ideal as laboratory conditions. A study is still
8 underway on the production and application of appropriate myco-insecticides that can survive variable field conditions and be effective for control of HWA.
Despite a concerted effort, no parasitoids associated with HWA have been found (Zilahi-
Balogh et al. 2002, Cheah et al. 2004, Keena et al. 2012). However, there are several predators that are present in the native range in Asia and on the west coast of the US. Although natural enemies found in the US, such as Harmonia axyridis (Coleoptera: Coccinellidae), lacewings
(Neuroptera: Chrysopidae and Hemerobiidae) and gall gnats (Diptera: Cecidomyiidae) prey on
HWA, they alone are not sufficient to control HWA. Moreover, these predators tend to be generalists and are more active during the time period that the sistens generation of HWA has laid a large number of eggs (Wallace and Hain 2000). Because native predators cannot sufficiently reduce HWA in the eastern US, classical biological control strategies focused on non-native natural enemies are under development as a potentially sustainable approach to control HWA populations (Montgomery and Lyon 1995, Wallace and Hain 2000, Keena et al. 2012).
1.2.3.3.1 Classical biological control
Classical biological control takes advantage of natural enemies that keep an exotic pest under control in the pest’s native range. Exotic species can become pests if the environment is conducive for their growth and development and if they lack natural controls in their introduced range. Natural enemies can be imported to control the invasive species, when a native species suddenly becomes a pest, or when there are no natural enemies associated with the native pest.
The concept of classical biological control was first introduced in the 1800s to manage cottony cushion scale (Icerya purchasi Maskell) in citrus in California and gained popularity after its incredible success using the predatory vedalia beetle (Vedalia cardinalis) (Caltagirone and Doutt
1989). The introduction of exotic natural enemies, however, requires a significant amount of data
9 to convince regulatory government agencies that the natural enemy has a narrow host range and that non-target effects will be minimal or non-existent.
1.2.3.3.1.1 Scymnus camptodromus (Coleoptera: Coccinellidae) as a potential biological control of HWA
In an attempt to control HWA population in the eastern US, several predators were introduced from outside the pest’s native range. To date, seven non-native predator species have been released with three successfully establishing in the eastern US. Among the seven Scymnus
(Neopullus) species collected from hemlock in China, three of them: -- Scymnus (Neopullus) camptodromus Yu and Liu, S. (N.) sinuanodulus Yu & Yao and S. (N.) ningshanesis Yu & Yao - were considered the most promising for classical biological control of HWA (Montgomery and
Keena 2011). S. sinuanodulus and S. ningshanesis were released into eastern North America in
2004 and 2007, respectively, and are not known to have established (Montgomery and Keena
2011, Keena et al. 2012). The third species, S. camptodromus, exhibits a different life history trait compared to the other two Neopullus species that synchronize their development with that of
HWA (Figure 1-4). S. camptodromus has an egg diapause, which is not common among the
Coccinellidae, and its diapause is synchronous with HWA summer aestivation. Eggs deposited in the spring or summer hatch the following spring and attack the key life stage of HWA, which is the eggs. These predators are abundant over a broad geographic range and habitats, and at variable HWA densities in its native range (Montgomery and Keena 2011, Keena et al. 2012).
These unique characteristics make this predator a promising candidate for biological control of
HWA.
S. camptodromus were transported for the first time in the US in 1995 and placed in quarantine at the US Forest Service facility in Ansonia, CT. However, they were difficult to rear
10 in the laboratory because of their egg diapause. Research on rearing revealed that the eggs require a chill period (5 ºC) to break diapause, followed by warmer temperature (10 ºC) to enhance hatching (Montgomery and Keena 2011, Keena et al. 2012). These small lady beetles, measure about 2.7 mm in length, and 1.4 mm at the widest part of their body (Figure 1-4). They are shiny brown/ dark orange in color with an elliptical dark brown/black spot on the dorsum of their elytra.
Sexual dimorphism is almost nonexistent in these predators. The head color and abdominal apex are distinguishing characteristics that can be used to separate males from females (Yu et al.
1997). Although the head color is not enough to identify the sex of these beetles, shape of the abdominal apex is helpful to separate the sexes (sometimes).
Adult females oviposit about three weeks after eclosion if mated, but their eggs enter diapause and hatch coinciding with the sistens oviposition stage of HWA (Keena et al. 2012). The larvae of S. camptodromus have four instars and the time in each instar varies by instar and is temperature dependent (Limbu et al. 2015). The larvae start feeding as soon they hatch. Scymnus adults and larvae will feed on all life stages of HWA but the larvae prefer HWA eggs (Limbu et al. 2016). Adult Scymnus will chew on eggs, but the larvae suck the egg contents out after partially digesting them first leaving empty chorions behind (referred to as extra-oral feeding).
This predator has been released from quarantine but is not yet approved for field release.
S. camptodromus has a high likelihood of establishing in the US as a major natural enemy of
HWA for several reasons: its phenology is closely synchronized with HWA, it occurs over a broad geographic area and in diverse habitats in its native range, beetle larvae are present at a key point in the life cycle of HWA, and the adults feed on HWA throughout most of the year
(Montgomery and Keena, 2011). To assess the efficacy of the predatory larvae in the laboratory, my objectives were to (1) Evaluate potential non-target impacts of S. camptodromus; (2)
Determine the effects of temperature on larval development and predation of HWA by S.
11 camptodromus in a laboratory environment; and (3) prepare a NAPPO Petition for permission to conduct field releases of S. camptodromus as a biological control agent of HWA.
1.3 Lymantria dispar (Lepidoptera: Erebidae) in North America
Gypsy moth (Lymantria dispar/ Lymantria spp.) is a well-known lepidopteran pest in the family Erebidae, capable of major defoliation of numerous tree species in forest and urban ecosystems (Campbell and Sloan 1977; Peterson and Smitley 1991). Gypsy moths are native to
Europe and Asia; the population of gypsy moths present in North America originated from
Western Europe. The European gypsy moth (EGM: L. dispar dispar) was accidently introduced to Medford, MA in 1869 by Leopold Trouvelot. Since then, it has gradually expanded its range at the rate of 6-18 km/year (Tobin et al. 2007) and currently occupies 19 eastern and mid-western states in the US (Figure 1-5) (Maloney et al. 2010). It is currently found as far south as North
Carolina, as far west as Wisconsin (USDA 2015), and has established populations in the southern parts of Canada east to Lake Superior (Régnière et al. 2009).
European gypsy moth in the US is also referred to as North American gypsy moth. The moth has been in the US for over 145 years, defoliating a million acres of forest trees every year
(Gypsy Moth Digest, 2015). Establishment and spread of gypsy moth has been facilitated by its polyphagous nature. It can feed on > 300 different host species; including oaks, willows, poplars, birches, and apples as major hosts, but it prefers oaks (Quercus spp.) (Liebhold et al. 1995b).
Since 1970, gypsy moths have defoliated 81 million acres of forest and suppression projects have cost about $271 million to state and federal governments since 1980 (Gypsy Moth Digest 2015).
Gypsy moth outbreaks have impacted forest ecosystems (Campbell 1977) and timber production, and in urban areas, ornamental trees, creating a nuisance for homeowners, especially those with allergies to the larvae or adults (Bigsby et al. 2014, Twery 1991).
12 More recently, introductions of gypsy moths of Asian origin into non-native habitats have gained global attention (Matsuki 2001, Pitt 2007, USDA 2015). Asian gypsy moths (AGM) currently include two Lymantria dispar subspecies (L. dispar asiatica, L. dispar japonica) and three other Lymantria species (L. albescens, L. postalba, and L. umbrosa) (Pogue and Schaefer
2007, USDA 2015, Stewart et al. 2016). The two L. dispar subspecies have distinct distributions;
L. d. asiatica occurs to the east of the Ural Mountains and in China and Korea, whereas L. d. japonica is endemic to all main islands in Japan, except Hokkaido (Pogue and Schaefer 2007,
Keena et al. 2008). The remaining three Lymantria species are distributed over two main islands
(Hokkaido and South Kyushu) and Ryukyu Islands of Japan (Pogue and Schaefer, 2007). AGM is not known to be established in North America; however, it has been detected in several locations on multiple occasions. AGM was identified in North America for the first time in 1991 in British
Columbia, Canada (Bogdanowicz 1993, Savotikov 1995), but control measures were quickly undertaken and the pest was successfully eradicated.
Despite efforts to exclude AGM, there have been multiple introductions of the pest in
North America through international trade (Gibbons 1992, USDA 2016, Matsuki et al. 2001). A recent study on potential global distribution using ecological niche modeling suggests that AGM can occupy almost all temperate regions of the world (Peterson et al. 2007). However, survival and establishment of the Asian subspecies of gypsy moth in a new area depend on several factors.
For example, larval development is influenced by temperature, foliage suitability, geographical origin, larval density, and sex (Casagrande 1987, Hough and Pimentel 1978, Honěk 1996,
Campbell 1978, Jarošík and Honek, 2007). Because insects are ectothermic, the temperature has been reported to be the primary factor influencing gypsy moth larval development (Casagrande
1987, Johnson et al. 1983).
13 1.3.1 Asian vs. European/North American gypsy moth
There are no distinct morphological characters to accurately distinguish life-stages of
AGM from European or North American gypsy moth. However, minor distinctions exist between two L. dispar species, which could be used for preliminary identification. For example; slight differences were observed in female wing area in different gypsy moth species. Asian gypsy moth females have a larger wing area than females of European and North American gypsy moths (Shi et al. 2015). Also, there are some larval color morphs of gypsy moth in the fourth or fifth instars that are not found in North America (Keena 2004). However, visible identification characteristics are often not available because male gypsy moths captured in pheromone traps are usually damaged, the larvae of Asian gypsy moth can take all described color-forms, and hybrids of Asian and European gypsy moth can be intermediate in characters between two parents (Keena et al. 2001). Therefore, DNA analysis is performed to accurately identify gypsy moths of various origins (Garner and Slavicek 1996, Stewart et al. 2016).
Despite the morphological similarities, AGM is considered a greater threat to commercial and urban forests than European gypsy moth, in part because unlike EGM, AGM females are capable of sustained flight (Wallner et al. 1995, Keena 2008). AGM strains also have a broader host range than their European counterparts, including more coniferous hosts (Baranchikov
1989), and the eggs of some strains of AGM can hatch after a shorter exposure to low temperatures in comparison with European populations (Keena 2015). Additionally, the Asian and European subspecies can successfully hybridize and the initial hybrids are able to glide varying distances, indicating better dispersal capabilities in hybrid offspring than the non-flying parent (Keena et al. 2001). Therefore, the introduction of the Asian subspecies in areas where the
European subspecies is already present could make limiting the spread of gypsy moth much more difficult, and require more aggressive management strategies. Taken together, these
14 characteristics of AGM subspecies increase the chances of establishment in new areas, where they have the potential for faster dispersion and represent a major economic and ecological threat to hardwood and coniferous forests.
1.3.2 Life history of Asian gypsy moth
Being holometabolous, AGM has four life stages: egg, larva, pupa and adult. Adult moths do not feed. They find a mate as soon as they emerge, and females lay hundreds of eggs in a single egg mass (USDA 2015). Eggs, which are laid during the late summer months, do not hatch until the following spring in close synchrony with bud break of their host plants. There are generally 5-6 larval instars (females have an extra instar) and pupation occurs in late June to early
July, or later depending on temperatures (Figure 1-6).
1.3.3 Management practices for AGM
Strategies to manage gypsy moth in the US include; i) suppression of gypsy moth populations in areas where they are established, ii) eradication of isolated populations of gypsy moth from uninfested areas, and iii) slow the rate of spread from infested areas by monitoring and treating gypsy moths in transition areas (areas next to infested areas). Several treatments are used under all three strategies, which include the use of pheromone traps baited with DisparlureTM for detection, mating disruption and mass trapping of male adults, as well as biopesticides and insect growth regulators used against the early larval stages of gypsy moth (USDA 2012).
AGM is not yet established in the US; however, adults of AGM in its native range often deposit egg masses on ships and containers in port areas (Wallner et al. 1995). Thus, international trade, especially via shipping, is a major pathway for new AGM introductions in the US.
15 Currently, government agencies are primarily focused on limiting the introduction of Asian subspecies into new locations. Phytosanitary procedures are followed during international trade to regulate the movement of AGM from its native range. Also, several ports in the AGM native range (Asia) are monitored through an international collaborative program to limit the entry of the moths from the area during the high-risk female flight period (Hajek and Tobin 2009). In North
America, the Asian subspecies or its hybrids are considered high-risk pests and when detected through trapping males in pheromone traps, the area is more heavily monitored to delineate the introduction. Then in subsequent years, the area is sprayed with biopesticides, primarily Bacillus thuringiensis kurstaki (Btk) and/or with insect growth regulators, to eradicate the infestation. The surveillance and eradication of AGM cost millions of dollars to governments in North America every year (Gray 2004).
Temperature requirements can prevent introduced species from establishing in a new location, thus, invasion success is more likely when climatic conditions in the introduced area are similar to the native habitat of an introduced species (Walther et al. 2009). There have been studies on the effect of temperature on European subspecies, but none on Asian subspecies.
Predicting phenology of AGM is critical for monitoring and management. For instance, Bacillus thuringiensis kurstaki (Btk), a preferred treatment for AGM populations, is most effective if applied to early second instars (Reardon et al. 1994; Peacock et al. 1998). Currently, phenology of the gypsy moth is predicted based on the European subspecies and by climate matching, which may not be accurate for the Asian subspecies. Therefore, our goal is to investigate the effects of temperature on larval development of AGM as one component that can be used to predict the potential for AGM establishment in different geographic regions of the US and to facilitate timing for treatments and trapping.
16
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17 Garner, K. J., and J. M. Slavicek. 1996. Identification and characterization of a RAPD‐PCR marker for distinguishing Asian and North American gypsy moths. Insect molecular biology, 5: 81-91. Gibbons, A. 1992. Asian gypsy moth jumps ship to US. Science, 255: 526. Gray, D. R. 2004. The gypsy moth life stage model: landscape-wide estimates of gypsy moth establishment using a multi-generational phenology model. Ecol. Model. 176: 155-171. Gouger, R.J. 1971. Control of Adelges tsugae on hemlock in Pennsylvania Scientific Tree Topics 3: 9. Gypsy moth digest. 2015. http://na.fs.fed.us/fhp/gm/index.shtml Hajek, A. E., and P. C. Tobin. 2009. North American eradications of Asian and European gypsy moth. In Use of microbes for control and eradication of invasive arthropods (pp. 71-89). Springer Netherlands. Havill, N. P., and M. E. Montgomery. 2006. Mitochondrial DNA from hemlock woolly adelgid (Hemiptera: Adelgidae) suggests cryptic speciation and pinpoints the source of the introduction to eastern North America. Annals of the Entomological Society of America 99: 195-203. Honěk, A. 1996. Geographical variation in thermal requirements for insect development. Eur. J. Entomol. 93: 303–312. Hough, J. A., and D. Pimentel. 1978. Influence of host foliage on development, survival, and fecundity of the gypsy moth. Environ. Entomol. 7: 97-102. Houtman, N., 2015. Scientists release predatory flies to protect eastern hemlock from insect attack. Available at http://oregonstate.edu/ua/ncs/archives/2015/jun/scientists-release- predatory-flies-protect-eastern-hemlocks-insect-attack. Huang, C., Barnett, A. G., Wang, X., Vaneckova, P., FitzGerald, G., & Tong, S. 2011. Projecting future heat-related mortality under climate change scenarios: a systematic review. Environmental health perspectives, 119: 1681. Jarošík, V., and A. Honek, 2007. Sexual differences in insect development time in relation to sexual size dimorphism, pp 205-211. In Fairbairn, D. J., W. U. Blackenhorn, and T. Székely (eds.), Sex, Size and Gender Roles: Evolutionary Studies of Sexual Size Dimorphism, Oxford University Press, Oxford. Johnson, P. C., D. P. Mason, S. L. Radke, and K. T. Tracewski. 1983. Gypsy moth, Lymantria dispar (L.)(Lepidoptera: Lymantriidae), egg eclosion: degree-day accumulation. Environ. Ent. 12: 929-932. Keena, M. A., W. E. Wallner, P. S. Grinberg, and R. T. Cardé. 2001. Female flight propensity and capability in Lymantria dispar (Lepidoptera: Lymantriidae) from Russia, North America, and their reciprocal F1 hybrids. Environ. Entomol. 30: 380-387. Keena, M., 2004. Identification of gypsy moth larval color forms. Available online at http://www.forestpests.org/gypsymth/ Keena, M.A., R. T. Trotter III, C. Cheah, and M. E. Montgomery. 2012. Effects of temperature and photoperiod on the aestivo-hibernal egg diapause of Scymnus camptodromus (Coleoptera: Coccinellidae). Environ. Entomol. 41: 1662-1671. Keena, M. A., M. J. Côté, P. S. Grinberg, and W. E. Wallner. 2008. World distribution of female flight and genetic variation in Lymantria dispar (Lepidoptera: Lymantriidae). Environ. Entomol. 37: 636-649. Keena, M. A. 2015. Inheritance and World Variation in Thermal Requirements for Egg Hatch in Lymantria dispar (Lepidoptera: Erebidae). Environ. Entomol. 45: 1-10. Kung, W. Y., K. Hoover, R. Cowles, and R. T. Trotter III. 2015. Long-Term Effects of Imidacloprid on Eastern Hemlock Canopy Arthropod Biodiversity in New England. Northeastern Naturalist. 22: NENHC-40-NENHC-55.
18 Liebhold, A. M., MacDonald, W. L., Bergdahl, D., and Mastro, V. C. 1995a. Invasion by exotic forest pests: a threat to forest ecosystems. Forest Science, 41: a0001-z0001. Liebhold A. M., K. W. Gottschalk, R. M. Muzika, M. E. Montgomery, R. Young, K. O’Day, and B. Kelley. 1995b. Suitability of North American tree species to the gypsy moth: a summary of field and laboratory tests. USDA For. Serv. Gen. Tech. Rep. GTR-NE-211: 1-34. Limbu, S., M. Keena, D. Long, N. Ostiguy, and K. Hoover. 2015. Scymnus camptodromus (Coleoptera: Coccinellidae) larval development and predation of hemlock woolly adelgid. Environ. Entomol. 44: 81-89. Limbu, S., Cassidy, K., Keena, M., Tobin, P., & Hoover, K. 2016. Host Range Specificity of Scymnus camptodromus (Coleoptera: Coccinellidae), A Predator of Hemlock Woolly Adelgid (Hemiptera: Adelgidae). Environ entomol. 45: 94-100. Maloney, K., Baressi, J., Schneeberger, N.F., 2010. Reducing Damage and Slowing the Spread. National Gypsy Moth Management Program. USDA Forest Service Special Initiative Brief. Available online at http://www.na.fs.fed.us/ra/specialinitiatives/gm/ gysp_moth_featured_brief11.pdf. Matsuki, M., M. Kay, J. Serin, R. Floyd, and J. K. Scott. 2001. Potential risk of accidental introduction of Asian gypsy moth (Lymantria dispar) to Australasia: effects of climatic conditions and suitability of native plants. Agric. For. Ent. 3: 305-320. Mayfield, A. E., Reynolds, B. C., Coots, C. I., Havill, N. P., Brownie, C., Tait, A. R., and Galloway, A. B. (2015). Establishment, hybridization, and impact of Laricobius predators on insecticide-treated hemlocks: Exploring integrated management of the hemlock woolly adelgid. Forest Ecol. Manag. 335: 1-10. Montgomery, M. E., and M. A. Keena. 2011. Chapter 5: Scymnus (Neopullus) lady beetles from China, pp. 53-76. In R. Reardon and B. onken (eds.), Implementation and status of biological control of the hemlock woolly adelgid. FHTET 2011-04. USDA Forest Service, Morgantown, WV. Montgomery, M. E., and S. M. Lyon. 1995. Natural enemies of adelgids in North America: their prospect for biological control of Adelges tsugae (Homoptera: Adelgidae), pp. 89-102. In S. M. Salom, T. C. Tigner, and R. C. Reardon (eds.), Proceedings of the First Hemlock Woolly Adelgid Review. Charlottesville, VA, U.S. Department of Agriculture Forest Service, Forest Health Technology Enterprise Team, Morgantown, WV. Montgomery, M. E., S. E. Bentz, and R. T. Olsen. 2009. Evaluation of hemlock (Tsuga) species and hybrids for resistance to Adelges tsugae (Hemiptera: Adelgidae) using artificial infestation. J. Econ. Entomol. 102: 1247–1254. McClure, M. S. 1987. Biology and control of hemlock woolly adelgid (Vol. 851). New Haven, CT: Connecticut Agricultural Experiment Station. McClure, M. S. 2001. Biological control of hemlock woolly adelgid in the eastern US. FHTET- 2000-08. USDA Forest Service, Forest Health Technology Enterprise Team. Morgantown, WV. Naves, P. and E. Sousa. 2009. Threshold temperatures and degree-day estimates for development of post-dormancy larvae of Monochamus galloprovincialis (Coleoptera: Cerambycidae). Journal of pest science, 82: 1-6. Neven, L. G. 2000. Physiological responses of insects to heat. Postharvest Biology and Technology, 21: 103-111. Nietschke, B. S., Magarey, R. D., Borchert, D. M., Calvin, D. D., and Jones, E. 2007. A developmental database to support insect phenology models. Crop Protection, 26: 1444- 1448.
19 (NPS) National Park Service. 2013. Park releases new species of predatory beetles to combat hemlock woolly adelgid. Available at. https://www.nps.gov/grsm/learn/news/new-hwa- beetle.htm Oten, K. L., Cohen, A. C., & Hain, F. P. 2014. Stylet bundle morphology and trophically related enzymes of the hemlock woolly adelgid (Hemiptera: Adelgidae). Annals of the Entomological Society of America, 107: 680-690. Peacock, J. W., D. F. Schweitzer, J. L. Carter, and N. R. Dubois. 1998. Laboratory assessment of the effects of Bacillus thuringiensis on native Lepidoptera. Environ. Entomol.27: 450- 457. Pederson, N., K. Tackett, R. W. McEwan, S. Clark, A. Cooper, G. Brosi, R. Eaton and R. D. Stockwell. 2012. Long-term drought sensitivity of trees in second-growth forests in a humid region. Canadian Journal of Forest Research, 42: 1837-1850. Peterson, N. C., & Smitley, D. R. (1991). Susceptibility of selected shade and flowering trees to gypsy moth (Lepidoptera: Lymantriidae). Journal of economic entomology, 84: 587-592. Peterson, A. T., R. Williams, and G. Chen. 2007. Modeled global invasive potential of Asian gypsy moths, Lymantria dispar. Entomol. Exp. Appl. 125: 39-44. Pimentel, D., Lach, L., Zuniga, R., & Morrison, D. 2000. Environmental and economic costs of nonindigenous species in the US. BioScience, 50: 53-65. Pimentel, D., Zuniga, R., & Morrison, D. 2005. Update on the environmental and economic costs associated with alien-invasive species in the US. Ecological economics, 52: 273-288. Pitt, J. P. W., J. Régnière, and S. Worner. 2007. Risk assessment of the gypsy moth, Lymantria dispar (L), in New Zealand based on phenology modelling. International journal of biometeorology, 51: 295-305. Ponti, L., Gutierrez, A. P., Ruti, P. M., & Dell’Aquila, A. (2014). Fine-scale ecological and economic assessment of climate change on olive in the Mediterranean Basin reveals winners and losers. Proceedings of the National Academy of Sciences, 111: 5598-5603. Pogue, M. G., and P. W. Schaefer. 2007. A review of selected species of Lymantria Hübner (1819) including three new species (Lepidoptera: Noctuidae: Lymantriinae) from subtropical and temperate regions of Asia, some potentially invasive to North America. FHTET-2006-07. USDA For. Serv., Forest Health Technology Enterprise Team, Morgantown, WV. Ratte, H. T. 1984. Temperature and insect development. In Environmental physiology and biochemistry of insects. Springer Berlin Heidelberg. pp. 33-66. Reardon, R., N. R. Dubois, and W. McLane. 1994. Bacillus thuringiensis for managing gypsy moth: a review. National Center of Forest Health Management FHM-NC-01-94. Reid, W., Parker, B. L., Skinner, M., Gouli, S., & Teillon, H. B. 2002. Insect-killing fungi for management of hemlock woolly adelgid: A review of progress. In Proceedings: Hemlock Woolly Adelgid in the Eastern United States Symposium (pp. 198-204). Rutgers University. Régnière, J., V. Nealis, and K. Porter. 2009. Climate suitability and management of the gypsy moth invasion into Canada. Biological Invasions, 11: 135-148. Stewart, D., R. Zahiri, A. Djoumad, L. Freschi, J. Lamarche, D. Holden, S. Cervantes, D. Ojeda, A. Potvin, A. Nisole, C. Beliveau, A. Capron, T. Kimoto, B. Day, H. Yueh, C. Duff, R. Levesque, R. C. Hamelin, M. Cusson. 2016. A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (Lymantria spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America. PloS one, 11: e0160878. Savotikov, I. F., A. I. Smetnik, and A. D. Orlinskii. 1995. Situation of the Asian form of gypsy moth (Lymantria dispar) in Russia and in the world. EPPO Bulletin, 25: 617-622.
20 Shi, J., F. Chen, and M. A. Keena. 2015. Differences in wing morphometrics of Lymantria dispar (Lepidoptera: Erebidae) between populations that vary in female flight capability. Annals of the Entomological Society of America, 108: 528-535. Tobin, P. C., S. L. Whitmire, D. M. Johnson, O. N. Bjørnstad, and A. M. Liebhold. 2007. Invasion speed is affected by geographical variation in the strength of Allee effects. Ecology Letters, 10: 36-43. Twery, M. J. 1991. Effects of defoliation by gypsy moth. In K.W. Gottschalk, M. J. Twery, S. I. Smith (eds.), Proceedings of the USDA interagency gypsy moth research review, January 22–25, 1990, East Windsor, CT, USDA Forest Service, Northeastern Forest Experiment Station, Radnor, PA (1991), pp. 27–39 general technical report NE-146 USDA APHIS. 2016. Pest Alert. https://www.aphis.usda.gov/publications/plant_health/content/printable_version/fs_phasi angm.pdf USDA. 2012. http://na.fs.fed.us/pubs/fhp/gm-management/record-of-decision-121211.pdf USDA. 2015a. http://na.fs.fed.us/fhp/hwa/maps/2015_HWA_Infestation_Map_20160502.pdf USDA. 2015b. https://www.aphis.usda.gov/plant_health/plant_pest_info/gypsy_moth/downloads/gypmo th.pdf Wallace, M. S., and F. P. Hain. 2000. Field surveys and evaluation of native and established predators of the hemlock woolly adelgid (Homoptera: Adelgidae) in the southeastern US. Environ. Entomol. 29: 638-644. Wallner, W. E., L. M. Humble, R. E. Levin, Y. N. Baranchikov, and R. T. Carde. 1995. Response of adult lymantriid moths to illumination devices in the Russian Far East. J. of Econ. Entomol. 88:337-342 Walther, G. R., A. Roques, P. E. Hulme, M. T. Sykes, P. Pyšek, I. Kühn, ... & J. Settele. 2009. Alien species in a warmer world: risks and opportunities. Trends Ecol. Evol. 24: 686-693. Ward, J. S., Montgomery, M. E., Cheah, C. A. J., Onken, B. P., & Cowles, R. S. 2004. Eastern hemlock forests: guidelines to minimize the impacts of hemlock woolly adelgid. Webb, R. E., J. R. Frank, and M. J. Raupp. 2003. Eastern hemlock recovery from hemlock wooly adelgid damage following imidaclopid therapy. Journal of Arboriculture, 29: 298-302. Young, R. F., K. S. Shields, and G. P. Berlyn. 1995. Hemlock woolly adelgid (Homoptera: Adelgidae): stylet bundle insertion and feeding sites. Ann. Ent. Soc. Am. 88: 827-835. Yu, G., Yao, D., & Liu, H. 1996. The coccinellidae collected from Tsuga with Adelges tsuga Annand (Homoptera: Adelgidae). Sci. Silvae Sin. 33: 432-440. Zilahi-Balogh, G. M. G., Kok, L. T., & Salom, S. M. (2002). Host specificity of Laricobius nigrinus Fender (Coleoptera: Derodontidae), a potential biological control agent of the hemlock woolly adelgid, Adelges tsugae Annand (Homoptera: Adelgidae). Biological control, 24: 192-198.
21
Figure 1-1. Hemlock woolly adelgid distribution in eastern North America in 2015 (USDA 2015a)
Figure 1-2. a) Hemlock woolly adelgid infested twig, b) First instar hemlock woolly adelgid (Havill 2015)
22
Figure 1-3. Life cycle of HWA in the native range (D’Amico and Havill 2016)
Figure 1-4. Scymnus camptodromus life stages. a) Egg b) larva c) pupa d) adult (Photos by Melody Keena)
23
Figure 1-5. Gypsy moth quarantine areas in North America (USDA APHIS 2015b)
Figure 1-6. a) Gypsy moth life-cycle (Cloyd & Nixon 2015), AGM life stages; b) first-instars emergence from an egg mass, c) larvae, d) Female (left) male (right), e) Pupa (Photos by Melody Keena)
24
Chapter 2
Host range specificity of Scymnus camptodromus (Coleoptera: Coccinellidae), a predator of hemlock woolly adelgid
Limbu, S., Cassidy, K., Keena, M., Tobin, P., & Hoover, K. 2016. Environ Entomol. 45: 94-100.
2.1 Abstract
Scymnus (Neopullus) camptodromus Yu and Liu (Coleoptera: Coccinellidae) was brought to the United States from China as a potential biological control agent for hemlock woolly adelgid (Adelges tsugae Annand) (Hemiptera: Adelgidae). Scymnus camptodromus phenology is closely synchronized with that of A. tsugae and has several characteristics of a promising biological control agent. As a prerequisite to field release, S. camptodromus was evaluated for potential non-target impacts. In host range studies, the predator was given the choice of sympatric adelgid and non-adelgid prey items. Non-target testing showed that S. camptodromus will feed to some degree on other adelgid species, but highly prefers A. tsugae. We also evaluated the larval development of S. camptodromus on Pineus strobi Hartig (Hemiptera:
Adelgidae) and Adelges laricis Vallot (Hemiptera: Adelgidae); a small proportion of predator larvae was able to develop to adulthood on P. strobi or A. laricis alone. Scymnus camptodromus showed no interest in feeding on Paraprociphilus tessellatus Fitch (Hemiptera: Aphididae) or
Eriosoma lanigerum Hausm. (Hemiptera: Aphididae), and minimal interest in Aphis gossypii
Glover (Hemiptera: Aphididae) in choice and no-choice experiments. Scymnus camptodromus
25 females did not oviposit on any host material other than A. tsugae infested hemlock. Under the circumstances of the study, S. camptodromus appears to be a specific predator of A. tsugae, with minimal risk to nontarget species. Although the predator can develop on P. strobi, the likelihood that S. camptodromus would oviposit on pine hosts of this adelgid is unlikely.
2.2 Introduction
Hemlock woolly adelgid (Adelges tsugae, Annand (Hemiptera: Adelgidae)) is a major pest of Eastern hemlock, Tsuga canadensis (L.) Carriére and Carolina hemlock, Tsuga caroliniana Engelmann in the eastern U. S., causing marked tree decline and mortality (McClure,
1991). It was originally introduced into the eastern U.S. from southern Japan (Havill et al.,
2006). Adelges tsugae is not considered a pest in its native range due to the presence of a complex of natural enemies and naturally resistant hemlocks (Cheah et al., 2004; Cheah &
McClure, 2000). Although A. tsugae is endemic to the western U.S., the biotype there is genetically distinct from the introduced A. tsugae in the eastern U. S. that causes severe injury and extensive tree decline (Orwig & Foster, 1998).
Adelges tsugae has spread rapidly since its introduction; there are no known parasitoids, insufficient generalist native predators, and an abundance of susceptible hemlock stands in the eastern U.S. (Montgomery & Lyon, 1996; Wallace & Hain, 2000; Hakeem et al., 2013; Havill et al., 2014). This pest is currently distributed over 19 states in the eastern U. S., encompassing approximately 40% of the T. canadensis range and 100% of the T. caroliniana range (USFS,
2012; Preisser et al., 2014). The rate of spread has been estimated at about 15 km per year in the
South and 8 km per year in the North (Evans & Gregoire, 2007). The proliferation of A. tsugae is believed to be more rapid in the South due to the warmer climate, which allows prolonged
26 feeding and reduced winter mortality of adelgids resulting in faster hemlock decline (Parker et al.,
1999; Skinner et al., 2003; Ford et al., 2012).
Evaluation of classical biological control agents for managing A. tsugae populations has been progressing in earnest for several years (Butin et al., 2003; Butin et al., 2004; Vieira et al.,
2011; Story et al., 2012; Jones et al., 2014; Limbu et al., 2015) and is considered the most promising approach for forest settings (Onken & Reardon, 2011). The aim is to build a community of predators that collectively keeps the pest population below damaging levels.
Natural enemies introduced in the eastern U.S. for control of A. tsugae include several predatory insect species collected from the native range of A. tsugae worldwide (Cheah &
McClure,1998; Mausel et al., 2010; Montgomery & Keena, 2011; Montgomery et al., 2011;
Hakeem et al., 2013). Two of those predatory species, Laricobius nigrinus Fender (Coleoptera:
Derodontidae) and Sasajiscymnus tsugae Sasaji and McClure (Coleoptera: Coccinellidae), have been released, recovered and were found co-existing in the areas of release (Hakeem et al., 2011).
However, studies also suggest that predation is somewhat temperature dependent; for example, some predators are more active during cooler spring temperatures and some during warmer summer temperatures (Flowers et al., 2006). Therefore, a complex of natural enemies will likely be required to significantly reduce A. tsugae populations in different seasons and geographic locations.
Exploration for natural enemies of A. tsugae included a collection of Scymnus
(Neopullus) camptodromus Yu and Liu (Coleoptera: Coccinellidae) from the Sichuan and
Yunnan provinces in the southwestern China (Montgomery & Keena, 2011). Scymnus camptodromus is an efficient predator in its native ecosystem; the adults feed on all life stages of
A. tsugae throughout their development (Cheah et al., 2004). The larvae also feed on multiple life stages, although they feed most voraciously on A. tsugae eggs (Montgomery & Keena, 2011;
Limbu et al. 2015). This predator’s phenology is closely aligned with that of A. tsugae; S.
27 camptodromus eggs diapause while A. tsugae are in summer aestivation, hatching in spring as A. tsugae begin laying eggs (Keena et al., 2012). Also, the lower temperature threshold for development of S. camptodromus larvae is 5 C, which closely matches that of A tsugae progrediens, making it an attractive choice for biological control in the northeastern U.S. (Limbu et al. 2015).
Scymnus camptodromus is found over a broad geographic region and diverse habitats in its native range, yet unlike some Scymnus (Neopullus) species collected from China, it was not found in association with host trees other than hemlock, suggesting it may be a specialist predator of A. tsugae (Montgomery & Keena, 2011). However, as with introduction of any classical biological control agent, potential ecological impacts of the introduced species must be explored, particularly in respect to non-target species (Bellows, 2001; Van Lenteren et al., 2003). We studied the host range of S. camptodromus, including the possibility that it might feed on sympatric adelgid species, as well as four non-adelgid prey items including, Paraprociphilus tessellatus Fitch (Woolly alder aphid) in choice and no-choice tests. Paraprociphilus tessellatus is an important consideration for non-target feeding since the larvae of the only known carnivorous butterfly Fenisica tarquinius Fabricius (Lepidoptera: Lycaenidae) in the continental
U.S. feed almost exclusively on P. tessellatus (Scott, 1986; Hall et al., 2009; Butin et al., 2004).
These data were incorporated into an environmental assessment report for consideration for the release of S. camptodromus for control of A. tsugae in the field.
28 2.3 Materials and Methods
2.3.1 Predator and Prey Source
Scymnus camptodromus adults were collected in 2006-2007 from different geographic locations in China and transported to the USDA Forest Service quarantine facility in Ansonia, CT under permit. Voucher specimens of adults were deposited at the Entomology Division, Yale
Peabody Museum of Natural History, New Haven, CT. Five geographic populations of S. camptodromus were used in this study (Keena et al., 2012), including DGS (CHINA:Sichuan: 5-
X-06, 5-11-XI-06, 26-IV-07), MNP (CHINA:Yunnan: 20-IV-07, 13-VI-07, 23-XI-07), NBG
(CHINA:Sichuan: 5-X-06, 5-11-XI-06, 26-IV-07), LP (CHINA:Yunnan: 23-IX-05, 10-VI-07, 20-
IX-07),and LJS (CHINA:Yunnan: 21-22-IV-07, 25-V-07, 11-VI-07, 21-IX-07). The strains used in any one experiment depended on which strain had adults available to test at the time. Equal numbers of predators from each province were evaluated in each test whenever possible.
Information on test prey with the rationale for testing a given prey species are shown in
Table 1. The A. tsugae used in this study were collected from T. canadensis trees in 2007 and
2008 from the vicinity of Raleigh, NC and in 2009 and 2010 from Rothrock State Forest, PA.
Scymnus camptodromus was evaluated for non-target impacts on three sympatric adelgids collected in April (overwintering adelgid generation) or June (second adelgid generation) of
2007, 2010 and 2015. Pineus strobi Hartig (Hemiptera: Adelgidae) (pine bark adelgid) were collected from Pinus strobus L. in Hamden, CT, Ansonia, CT, Perry Township, PA, and Yale
Meyers Forest in CT. Adelges cooleyi Gillette (Cooley spruce gall adelgid) were collected from
Pseudotsuga menzesii (Mirb.) Franco in Hamden, CT, and Adelges laricis Vallot (Hemiptera:
Adelgidae) (larch adelgid) from the Lake Watrous area, CT from Larix kaempferi (Lamb.) Carr.
The predator was also tested using four native non-adelgid species: Fiorinia externa Ferris
29 (Hemiptera: Diaspididae) from A. tsugae in Mile Run exit, PA; Eriosoma lanigerum Hausm.
(Hemiptera: Aphididae) (woolly apple aphid), from Malus species, in the Soergel Orchards area,
PA; P. tessellatus (woolly alder aphid) from Alnus species in Scotia Barrens, PA collected in
February 2008; and Aphis gossypii Glover (Homoptera: Aphididae) (cotton aphid) obtained from honeydew melon (Cucumis melo L.) plants from a lab colony maintained at Penn State University
(University Park, PA) in March 2015.
2.3.2 Prey Preference (Choice Tests)
Choice tests were conducted in 150 × 15 mm petri dishes with filter paper placed in the bottom. Host material with prey items was placed inside the petri dish by trimming them to approximately equal size to minimize bias based on the amount of cover for prey or predator. All samples in each test were placed on top of the filter paper and arranged around the edges, making sure each sample touched the other samples as little as possible. The number of prey items presented to each predator was equalized as much as possible. Prior to placement in the choice test, each foliage sample was cleaned of any predators and other insects at the time it was collected.
The sex of each beetle, the foliage sample type, and the number of prey items present on each type of foliage were recorded prior to the start of each test. Each adult S. camptodromus was placed in the center of the test arena, equidistant from each sample, the lid of the petri dish was closed, and the dish edges were sealed with parafilm or tape to prevent insects from escaping.
To maintain humidity the petri dishes were placed in a plastic box on a screen over water and held in an incubator at 15 ± 1 C with a 12:12 (L:D) h for 48 h (four-way choice tests) or 72 h
(three-way choice tests).
30 After 48 or 72 hours, the predators were removed, and the final location of each predator
(on which sample or on the filter paper) was recorded. The predators were returned to holding cages, and the number of prey items remaining on each sample was recorded. Oviposition by S. camptodromus was noted and recorded.
2.3.3 Four-way choice tests
To evaluate the feeding preference of adult S. camptodromus among sympatric adelgid species and for different adelgid stages, choice tests were conducted by giving each S. camptodromus adult a choice of four prey items (A. laricis, P. strobi, A. tsugae, A. cooleyi) simultaneously without removing the mature adelgids that continued laying eggs. Since the choice test was performed with ovipositing adelgids, it was only held for 48 h to minimize oviposition and egg eclosion. Additionally, based on availability at the time of the test, the overwintering generation of P. strobi, A. tsugae and A. cooleyi and the second generation of A. laricis were used. A 1 cm long piece of infested hemlock, a 1 cm2 piece of P. strobi excised infested bark, one infested needle of P. menzesii, and 3-5 infested needles of L. kaempferi were spaced evenly around the margins of the petri dish. There were 10 replicates for this experiment, which included five MNP strain and five DGS strain females. After 48 h, the remaining adults, eggs and crawlers were counted, and the remaining crawlers were subtracted from the eggs left in the test. Because ovipositing prey adults were present during the course of this experiment, we first sought to determine if there were differences in oviposition by prey species and prey generation, which could alter subsequent analyses. To estimate the average number of eggs laid by each adelgid in 48 h, all eggs were removed from 10 adelgid females of each species and held in the same containers and conditions as used in the choice test. In this analysis, the number of
31 eggs laid by prey and prey generation was transformed using log10(y+1) and subjected to an analysis of variance.
Total eggs consumed of each adelgid species by each S. camptodromus adult was calculated by summing the number of eggs at the start plus the average number of eggs laid by a female of that species and then subtracting the number of eggs and nymphs in the container after
48 h. The number of nymphs (nymphs at the start plus hatched eggs minus nymphs at the end) and adults consumed was also recorded. The fate of an egg or nymph was considered to have a binary response: consumed or not consumed, and analyzed using logistic regression while accounting for the number of eggs presented to each S. camptodromus adult, given that these counts were not always equal among predators (although we made every effort to use approximately the same number of eggs). We chose this statistical approach over ordinary least squares (OLS) because the number of cases of consumed eggs were often very small for some prey species (i.e., <10), which is known to bias results obtained from OLS (Coxe et al. 2009).
The significance of the main effect of prey was based on the likelihood ratio chi-squared (G2), and odds ratios were computed after decomposing G2 into non-significant components (Agresti
1990). Statistical analyses were conducted in R (R Development Core Team 2011).
2.3.4 Three-way choice tests
Additional choice tests with S. camptodromus adults were conducted in comparison with
A. tsugae by giving each predator a choice of three adelgid species at a time using A. laricis, P. strobi, and A. cooleyi. During the four-way tests, the predator did not show a preference for mature adelgids, so they were removed from the foliage samples to prevent further oviposition.
However, since all four adelgid prey items were not available simultaneously, three-way tests were performed. To obtain sufficient sample sizes, both male and female adults of the MNP
32 strain were used in these tests. The tests were run for 72 h (rather than the 48 h for the four-way tests) to provide more opportunity for the S. camptodromus females to lay eggs. Infested foliage samples were 3-15 cm in length (total length of all pieces of each foliage type in the dish was similar) with the ends of each wrapped in damp cotton with parafilm over it to preserve moisture and prevent the predators from drowning. The number of adelgid eggs present in each sample was kept as equal from sample to sample as possible, averaging 50-80 eggs per sample. The number of eggs of each prey item on their respective foliage at the start of each test was recorded, and all other adelgid life stages were removed. Each test arena contained A. tsugae and two other adelgid species. Tests were categorized as Set A, B or C, depending on the composition of adelgids (Set A = A. tsugae, A. laricis, and A. cooleyi; Set B = A. tsugae, P. strobi, and A. cooleyi; and Set C = A. tsugae, P. strobi, and A. laricis). There were four replicates of Sets A and C and
11 replicates of Set B, based on availability of each adelgid species. Crawlers present at the start of the test were removed, and any crawlers present at the end of the test were subtracted from the number of eggs left after 72 h.
The statistical analyses of these data was conceptually the same as in the four-way choice tests with the exception of the inclusion of the main effect of prey set (A, B, or C), and the interaction of the two main effects (prey and prey set). The specific hypotheses tested in our logistic regression analysis were (1) predation does not vary among hosts, and (2) predation on
HWA does not vary by the set composition of hosts. When appropriate, odds ratios were estimated, and post-hoc tests were conducted by partitioning G2 into non-significant components
(Agresti 1990).
33 2.3.5 Paired choice tests
To further examine the prey preference of S. camptodromus, we also conducted paired choice tests when given a choice of A. tsugae and F. externa, or A. tsugae and E. lanigerum.
Because F. externa and A. tsugae can coexist in hemlock trees, an arboreal population of E. lanigerum and the progredien generation of A. tsugae can co-occur. Thus, these prey species were tested in a paired choice test. A mixture of males and females from the MNP strain of S. camptodromus was used for these tests. The experimental set-up was the same as described above for the three-way choice tests, but only A. tsugae and one other prey type was offered using
10 replicates of each combination. The eggs of F. externa and nymphs of E. lanigerum were counted at the beginning and end of the 72 h test. Data were analyzed using a paired Student’s t- test using JMP 9 (SAS Institute 2012).
2.3.6 Prey Acceptance (No-Choice Tests)
No choice feeding tests were also performed using adelgid prey items (A. cooleyi, P. strobi, A. laricis, and A. tsugae) and non-adelgid prey items (A. gossypii, P. tessellatus, F. externa, and E. lanigerum). We used 55 × 15 mm petri dishes with filter paper placed in the bottom and sealed with parafilm, except for the test with A. gossypii where we used 50 x 9 mm petri dish with a tight fitting lid and a layer of damp cotton in the bottom so the prey could not escape. Each of these tests was run for 48 h.
To test the adelgid species, S. camptodromus adults were presented with either 40 adelgid eggs alone spread out on the filter paper or 40 eggs with intact ovipositing adelgid(s) on their host material. The adelgid tests used the over-wintering generation and second generation (as a separate treatment) of A. laricis and A. tsugae, the second generation of A. cooleyii, and the over-
34 wintering generation of P. strobi. Each adelgid species and generation was tested with and without host plant material. Adelgid-infested host material consisted of 1-2 cm of a T. canadensis branch with needles, a 0.5 cm length of P. menzesii with 4 needles, 5-7 needles
(second generation) or 1-3 whorls with needles (over-wintering generation) of L. kaempferi, and either a 1 cm2 piece of excised bark or a 7 cm × 3 cm bolt of P. strobus (the latter being contained in a 7 cm high-by-7 cm diameter plastic box with a single screen vent instead of the petri dish).
Five adults from Sichuan Province (DGS or NBG strain) and five from Yunnan Province (MNP or LJS strain) were used in each test with the second generation of adelgids. All but one or two of the adults used in tests of the over-wintering generation were from Sichuan (LP), and the rest were from Yunnan (DGS or NBG strain).
The no-choice tests with the non-adelgid prey were only performed with host-plant material present and with females from either the Sichuan (DGS) or Yunnan Province (MNP).
Different strains of the predator were used at various times depending on availability at the time of the test. To accommodate the size differences between the predator and various non-adelgid prey items, we used different life stages (early instars, late instars and adults) of P. tessellatus and
E. lanigerum and adults only of F. externa. There were 10 replicates for each of these types of prey. The tests on A. gossipii were also performed with 2 cm diameter disks of either melon or hibiscus host-plant material placed on moist cotton. Twenty-five replicates were performed:
Fifteen with melon and 10 with hibiscus plants, each containing 40-45 first instar A. gossipii nymphs. The number of prey fed upon or consumed was recorded.
The effect of adelgid prey items on feeding preference of the predator was evaluated using the same method as used for the four-way choice tests. Since tests were done with and without host material, the main effects of the host material and prey item, and their interaction were analyzed using logistic regression analysis in which significance was based on G2. Control
35 for egg laying was done in the same manner as the four-way choice test because ovipositing adelgids were present.
2.3.7 Prey suitability
We evaluated the ability of S. camptodromus to complete larval development by feeding only on P. strobi (needles and bark of P. strobus) or A. laricis (on L. kaempferi foliage). The P. strobi and A. laricis were chosen because they were the most and least preferred prey items in the four-way choice test, respectively. Newly hatched S. camptodromus larvae from the DGS or
MNP strains (depending on availability) were immediately transferred individually to a rearing container containing prey items and observed until they died or reached the adult stage. Different rearing containers were used for the two prey types because of differences in needle size of the host-plant. A petri-dish (150 × 15 mm sterile petri dishes; www.daigger.com, EF1630C) was used for P. strobi and a clear 59 ml soufflé cup with a clear 2.5 cm diameter lid (Solo, Eastern
Bag and Paper, CT) for A. laricis. Each container consisted of filter paper placed at the bottom and the adelgid infested host material was placed on top of the filter paper. Foliage that contained sufficient adelgid eggs (based on choice and no-choice tests described above) was used and checked thoroughly for other predators before presenting to the S. camptodromus larvae. The larvae were reared in an environmental chamber at 20 C, with 12:12 h (L:D) and an average humidity of 80 ± 5%. The foliage was checked every other day for prey items and predator condition and changed when needed. Due to limited availability of P. strobi and A. laricis during peak larval hatch of S. camptodromus, the sample size was 10 beetle larvae on each of the prey items. Survival, the number of days to reach adulthood and size of any eclosed predator adults, were recorded. Means and standard deviations were calculated for days to adult and adult sizes.
Also, chi-square statistics was performed to determine if survival to adulthood on both prey items
36 was different at P ≤ 0.05 from the 67% survival that was reported on A. tsugae (Limbu et al.
2015).
2.4 Results
2.4.1 Choice test with four adelgids
Since ovipositing adelgids were present during this experiment, the effects of prey species and prey generation on adelgid oviposition were examined. We found that the mean oviposition by adelgids was 2.62 ± 0.42 which was not different by prey species (F = 1.64; df = 3,
45; P = 0.19) or generation (F = 1.37; df = 1,45; P = 0.25).
In this experiment, predation differed significantly among adelgid species (Figure 2-1).
Scymnus camptodromus adults were 4.0 times (95% CI: 2.7, 5.8) more likely to consume A. tsugae than P. strobi, and 251 (95% CI: 34.9, 999.9) times more likely to consume A. tsugae than the combined group of A. cooleyi and A. laricis. Similarly, the predator adults were 62.9 (95%
CI: 95% CI: 8.6, 460.4) times more likely to consume P. strobi than the combined group of A. cooleyi and A. laricis. Consumption of A. cooleyi and A. laricis did not differ from each other
(G2 = 1.8, P = 0.17), which as a group differed from P. strobi (G2 = 34.5; P < 0.01) and A. tsugae
(G2 = 40.1, P < 0.01). Among alternative adelgid hosts, P. strobi was most preferred, but the predator still strongly preferred A. tsugae over P. strobi (G2 = 5.2, P = 0.022).
2.4.2 Choice test with three adelgids
There was a significant interaction of prey-by-set composition, indicating that predation of A. tsugae in each set depended on the combination of prey items present. Consumption of A.
37 tsugae eggs was 1.9 (95% CI = 1.2, 2.8) times more likely to occur when A. tsugae was exposed to the predator in combination with P. strobi and A. cooleyi (Set B), or with P. strobi and A. laricis (Set C), than when combined with A. laricis and A. cooleyi (Set A). There was no significant difference in egg consumption between sets B and C (G2 = 0.65, df = 1, P = 0.4178), which both experienced significantly lower predation than Set A (G2 = 9.9, df = 1, P = 0.002)
(Figure 2-2).
2.4.3 Paired choice tests
When presented with the choice of F. externa and A. tsugae, the mean number of A. tsugae eggs consumed by adult S. camptodromus was 15.1 ± 2.9 (mean ± SEM) over 72 h, which was 151 times greater relative to the mean number of F. externa consumed (0.1 ± 0.1). Also, the mean number of A. tsugae eggs consumed was 19 times higher (19.3 ± 2.9) than the mean number of E. lanigerum consumed (1.0 ± 0.44).
2.4.4 No choice test with adelgid prey items
There was a significant effect of the presence of host plant material with adelgid prey (G2
= 26.9, P < 0.01), and a significant effect of adelgid species (G2 = 7.8, P = 0.04) on predation, but no significant host plant material by prey interaction (G2 = 4.7, P = 0.19).
Holding host plant material as a main effect, S. camptodromus adults were 3.3 (95% CI:
2.9-3.7) times more likely to consume adelgid prey (including alternate prey) in the absence of host material than with host material present. Considering aldegid species as a main effect, S. camptodromus was equally likely to consume A. cooleyi, P. strobi, and A. laricis (G2 = 1.2, P =
0.28), which as a group differed significantly from A. tsugae (G2 = 6.2, P = 0.013). Predators
38 were 1.9 (95% CI: 1.7, 2.1) times more likely to consume A. tsugae than the combined group of
A. laricis, A. cooleyi, and P. strobi.
The effect of generation of A. tsugae was explored in two separate cases: with and without host material present. There was a significant generation effect with (G2 = 14.6, P <
0.01) or without (G2 = 18.5, P < 0.01) host material present; the predators were 7.6 (5.6, 10.2) times more likely to eat the over-wintering generation than the 2nd generation. With host material absent, there was also a significant generation effect (G2 = 18.5, P < 0.01), with predators 25.9
(16.0, 41.9) times more likely to eat the over-wintering generation than the 2nd generation.
For A. laricis, there was no significant generation effect in the presence (G2 = 0.02, P =
0.88) or absence (G2 = 1.05, P = 0.31) of the host material.
2.4.5 No choice test with non-adelgid prey items
When P. tessellatus or E. lanigerum were presented as the only prey species, adult S. camptodromus exhibited no evidence of feeding. However, when F. externa was presented as the only choice, one prey item was chewed upon in one of the tests but was not consumed entirely.
On average, with host material present, 0.32 ± 0.031 nymphs of A. gossypii were consumed per predator adult over 48 h.
2.4.6 Prey suitability
Of the 10 S. camptodromus neonate larvae reared exclusively on P. strobi, only two
(observed less than expected, Pearson χ2 = 44.94, df = 1, P < 0.0001) pupated and eclosed to adult, which required an average of 28 ± 1.0 days. On larch, only one larva out of 10 survived
(observed less than expected, Pearson χ2 = 68.61, df = 1, P < 0.0001) and required 44 days to
39 reach adulthood. The size of the two adults reared on P. strobi was 2.48 ± 0.2 mm in length and
1.37 ± 0.1 mm in width, whereas the size of the one adult reared on A. laricis was 2.19 mm in length and 1.07 mm in width.
2.5 Discussion
Non-target testing indicated that S. camptodromus preferred A. tsugae over the alternative adelgid and non-adelgid prey items evaluated in this study. Although there was evidence that this predator will feed on other adelgid species to some extent, the predators consistently ate significantly more A. tsugae than any other adelgid species offered. We conclude, therefore, that release of S. camptodromus would not present a significant risk to non-target species. This is in contrast to the predators Scymnus ningshanensis (Yu & Yao) from China and S. tsugae from
Japan, which were released against A. tsugae in the Eastern U.S.; these Scymnus species were shown to feed equally on A. tsugae and P. strobi or A. cooleyi eggs in paired choice tests performed in a laboratory (Butin et al. 2004).
S. camptodromus larvae have a limited ability to complete development when fed only on P. strobi or A. laricis. The development time for the predator larvae to reach adulthood was
15 days longer on A. laricis at 20 °C than larvae reared on A. tsugae at the same temperature
(Limbu et al. 2015). Also, the adults reared on P. strobi or A. laricis were smaller compared to the adults reared only on A. tsugae (Limbu et al. 2015). These findings suggest that the majority of S. camptodromus larvae would not reach adulthood in the field feeding exclusively on these alternative adelgid species, and those that eclosed to an adult would likely experience fitness costs.
A small degree of feeding on P. strobi was observed, indicating the potential for S. camptodromus to survive during the summer months in the absence of A. tsugae. Using this prey
40 source, S. camptodromus adults might be able to survive the summer until A. tsugae begins to lay eggs again in the fall. Pineus strobi is also considered a pest in most of its current range, so predation by S. camptodromus on this adelgid species may be of benefit by helping to manage P. strobi as well as A. tsugae (Raske & Hudson, 1964). An additional consideration is that the likelihood of S. camptodromus larvae encountering prey on trees other than hemlock is very small since this predator exclusively chose hemlock infested foliage for oviposition in our trials. Also, during the no-choice tests on aphids, the predators were observed to avoid the host plant materials
(hibiscus and melon) on which the aphids were feeding, suggesting that S. camptodromus is not likely to encounter non-target pests such as aphids. These behaviors explain in part why this predator was never recovered from alternative tree species other than hemlocks in its native range
(Montgomery and Keena 2011).
In similar studies on Laricobius species, both larvae and adults of L. nigrinus and L. osakensis (Montgomery and Shiyake) consumed P. strobi eggs to some degree. In contrast to our results with S. camptodromus, L. nigrinus females were observed to oviposit on P. strobi in paired and no-choice tests, while L. osakensis oviposited only under no-choice conditions (Zilahi-
Balogh et al. 2002; Viera et al. 2011). However, unlike S. camptodromus both the Laricobius species larvae failed to develop to adult on this prey item alone (Zilahi-Balogh et al. 2002; Viera et al. 2011), suggesting that in the event that Laricobius larvae eclosed on P. strobi, they would eventually die without reaching maturity unless they could find A. tsugae.
When presented with F. externa, E. lanigerum, or P. tessellatus as the only food source,
S. camptodromus adults did not feed. This is in contrast to other predators of A. tsugae such as S. ningshanensis, S. tsugae, and L. osakensis, which were found to feed on P. tessellatus to some extent (Butin et al. 2004; Viera et al. 2011). This is important to note due to the significance of P. tessellatus as the primary food source for F. tarquinius, which is the only carnivorous butterfly in the continental U. S. (Butin et al., 2004; Scott, 1986). As expected, S. camptodromus is more
41 specialized on A. tsugae than the generalist lady beetle Harmonia axyridis (Pallas) that prefers P. tessellatus over A. tsugae (Butin et al. 2004).
Our findings indicate that S. camptodromus is a specialist on A. tsugae, particularly on the egg stage. Given the cold tolerance of S. camptodromus and its closely synchronized phenology with that of A. tsugae (Montgomery & Keena, 2011), the absence of meaningful non- target effects should make it an attractive candidate for biological control of A. tsugae in the
Northeastern U.S.
Figure 2-1. Reduction in mean number of adelgid eggs (± SE) after 48 h when four adelgid species were provided simultaneously to S. camptodromus. Different letters represent significant differences in mean egg reduction (G2 = 44.5; P < 0.01).
42
Figure 2-2. Influence of prey combination on the mean number (± SE) of A. tsugae eggs consumed by S. camptodromus. Prey combination presented as Set A (A. tsugae, A. laricis and A. cooleyi), Set B (A. tsugae, P. strobi and A. cooleyi) and Set C (A. tsugae, P. strobi and A. laricis). Different letters represent a significant difference in mean number of A. tsugae eggs consumed by S. camptodromus within each set (G2 = 14.1, df = 3, P = 0.003).
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44 Mausel, D. L., S. M. Salom, L. T. Kok, and G. A. Davis. 2010. Establishment of the hemlock woolly adelgid predator, Laricobius nigrinus (Coleoptera: Derodontidae), in the eastern United States. Environ. Entomol. 39: 440-448. McClure, M. S. 1991. Density-dependent feedback and population-cycles in Adelges tsugae (Homoptera, Adelgidae) on Tsuga canadensis. Environ. Entomol. 20: 258-264. Montgomery, M. E., and M. A. Keena. 2011. Chapter 5: Scymnus (Neopullus) lady beetles from China, pp. 53-76. In R. Reardon and B. onken (eds.), Implementation and status of biological control of the hemlock woolly adelgid. FHTET 2011-04. USDA Forest Service, Morgantown, WV. Montgomery, M. E., and S. M. Lyon. 1996. Natural enemies of adelgids in North America: their prospect for biological control of Adelges tsugae (Homoptera: Adelgidae), pp.89-102. In S. M. Salom, T. C. Tigner, and R. C. Reardon (eds.), Proceedings of the first hemlock woolly adelgid review, FHTET 96-10. USDA Forest Service, Charlottesville, VA. Montgomery, M. E., S. Shiyake, N. P. Havill, and R. A. B. Leschen. 2011. A new species of Laricobius (Coleoptera: Derodontidae) from Japan with phylogeny and a key for native and introduced congeners in North America. Ann. Entomol. Soc. Am. 104: 389-401. Onken, B., and R. C. Reardon. 2011. Implementation and status of biological control of the hemlock woolly adelgid. USDAFHTET-2011-04). Forest Health Technology, Morgantown, WV. Orwig, D. A., and D. R. Foster. 1998. Forest response to the introduced hemlock woolly adelgid in southern New England, U.S.A. J. Torr. Bot. Soc. 125: 60-73. Parker, B.L., M. Skinner, S. Gouli, T. Ashikaga, and H. B. Teillon. 1999. Low lethal temperature for hemlock woolly adelgid (Homoptera: Adelgidae). Environ. Entomol. 28: 1085-1091. Preisser, E. L., K. L. Oten, and F. P. Hain. 2014. Hemlock woolly adelgid in the Eastern United States: what have we learned? Southeast. Nat. 13: 1-15. Raske, A.G., and A. C. Hudson. 1964. The development of Pineus strobi (Hartig) (Adelginae, Phylloxeridae) on white pine and black spruce. Can. Entomol. 96: 599-616. Scott JA .1986. Feniseca tarquinius harvester, pp. 356-357. The butterflies of North America. A natural history and field guide. Stanford University Press, Stanford, CA. SAS Institute Inc. 2012. JMP ® 10. Cary, NC: SAS Institute Inc. Vieira, L. C., T. J. McAvoy, J. Chantos, A. B. Lamb, S. M. Salom, and L. T. Kok. 2011. Host range of Laricobius osakensis (Coleoptera: Derodontidae), a new biological control agent of hemlock woolly adelgid (Hemiptera: Adelgidae). Environ. Entomol. 40: 324-332. Zilahi-Balogh, G. M. G., L. T. Kok, and S. M. Salom. 2002. Host specificity of Laricobius nigrinus Fender (Coleoptera: Derodontidae), a potential biological control agent of the hemlock woolly adelgid, Adelges tsugae Annand (Homoptera: Adelgidae). Biol. Control. 24: 192-198.
45
Chapter 3
Scymnus camptodromus (Coleoptera: Coccinellidae) Larval Development and Predation of Hemlock Woolly Adelgid
Limbu, S., M. Keena, D. Long, N. Ostiguy, and K. Hoover. 2015. Environ. Entomol. 44: 81-89.
3.1 Abstract
Development time and prey consumption of Scymnus (Neopullus) camptodromus Yu and
Liu (Coleoptera: Coccinellidae) larvae by instar, strain and temperature were evaluated. S. camptodromus, a specialist predator of hemlock woolly adelgid, Adelges tsugae (Annand)
(Hemiptera: Adelgidae), was brought to the U.S. from China as a potential biological control agent for A. tsugae. This beetle has been approved for removal from quarantine but has not yet been field released. We observed that temperature had significant effects on the predator's life history. The larvae tended to develop faster and consume more eggs of A. tsugae per day as rearing temperature increased. Mean egg consumption per day of A. tsugae was less at 15 C than at 20 C. However, since larvae took longer to develop at the lower temperature, the total number of eggs consumed per instar during larval development did not differ significantly between the two temperatures. The lower temperature threshold for predator larval development was estimated to be 5 C, which closely matches the developmental threshold of A. tsugae progrediens. Accumulated degree-days for 50% of the predator neonates to reach adulthood were estimated to be 424. Although temperature had a significant effect on larval development and
46 predation, it did not impact survival, size or sex ratio of the predator at 15 and 20 C.
Furthermore, no remarkable distinctions were observed among different geographical populations of the predator.
3.2 Introduction
The insect pest hemlock woolly adelgid, Adelges tsugae (Annand) (Hemiptera:
Adelgidae), causes extensive decline of hemlock trees and threatens the sustainability of hemlock forests in the Eastern U.S. Native to Western North America and Asia, it was introduced in the
Eastern U.S. from Japan (Havill & Montgomery, 2008; Havill et al., 2006) and is currently endemic to 19 eastern states (USFS 2012; Preisser et al., 2014). Although A. tsugae is known to infest all hemlock species, it is more damaging to eastern hemlock, Tsuga canadensis (L.)
Carriere and Carolina hemlock, Tsuga caroliniana Engelmann than other hemlock species.
Hemlocks are important foundation tree species, dominating 1 million hectares of Eastern U.S. forests (Domec et al., 2013). Adelges tsugae can multiply rapidly when hemlocks are healthy and producing new growth (McClure, 1991). The insect injures trees by inserting their stylet bundle into plant tissue near where hemlock needle are attached and depleting nutrients from the xylem ray parenchyma cells (Young et al., 1995). Feeding damage results in yellowing and desiccation of hemlock needles, which can kill a tree in as little as 1-3 years in its southern range and 5-15 years in its northern range (Ellison et al., 2010).
Management approaches for A. tsugae include both chemical and biological control, but currently, neither approach provides the level of population suppression needed (Onken and
Reardon 2008, 2010). Biological control using a complex of natural enemies is thought to be a more sustainable, long-term solution for controlling this pest. There are no known parasitoids of
A. tsugae and native natural enemies are generalists, which are unable to reduce pest populations
47 to tolerable levels (Cheah et al., 2004). However, natural enemies of A. tsugae appear to contribute to natural control of this pest in its native range in Japan, China and Western North
America (Lu et al., 2002; Montgomery & Keena, 2011; Vieira et al., 2013; Zilahi-Balogh et al.,
2003). These include two species of Laricobius beetles and several species of coccinelids. Seven species of Scymnus (Neopullus) were found associated with hemlocks in China (Montgomery &
Keena, 2011; Yu et al., 2000); three of these, Scymnus (Neopullus) camptodromus Yu and Liu
(Sc), Scymnus (N.) sinuanodulus Yu & Yao (Ss) and Scymnus (N.) ningshanensis Yu & Yao (Sn), were collected from the Yunnan, Shaanxi and Sichuan provinces of Southwestern China and brought to the U.S. in 1995. Scymnus sinuanodulus and S. ningshanensis were released in the
U.S. in 2004 and 2007, respectively, but do not appear to have established (Montgomery &
Keena, 2011).
The third predator from this collection, S. camptodromus, differs from the other two
Scymnus species in that its life cycle is strongly synchronized with that of A. tsugae. This predator has an unusual egg diapause that coincides with the summer aestivation of A. tsugae.
Following egg diapause, S. camptodromus eggs hatch in the spring, coinciding with oviposition by their prey; the predator larvae begin feeding on A. tsugae eggs early in the season. In its native range, this species is abundant over a broad geographic area and habitats and thrives at variable prey densities. Unlike S. sinuanodulus, S. camptodromus was not found associated with any host plant other than hemlock in its native range (Montgomery & Keena, 2011). As a first step to exploring the possibility of developing S. camptodromus as a biological control agent in the U.S., non-target testing was completed using choice, no choice, and predator development studies, showing that S. camptodromus prefers A. tsugae, although it will feed on other adelgids in no-choice situations, but not aphids (Montgomery & Keena, 2011)
Larvae of Scymnus (Neopullus) species from China are voracious and, although observed to sometimes consume young crawlers or feed on other stages when they get larger, they prefer A.
48 tsugae eggs (Montgomery & Keena, 2011; Lu et al., 2002). Larval feeding behavior is distinct from that of adults. The beetle larvae feed by extra-oral digestion (Delucchi, 1954; Lu et al.,
2002), while the adults chew on their prey, consuming the entire organism.
We evaluated S. camptodromus larval development and predation as a function of temperature among different beetle strains collected from two different regions in China. One of our goals was to contribute to a phenological model by establishing degree-day requirements for larval development. In addition, strain differences as a function of temperature in either developmental rate or number of prey consumed were evaluated to ensure close phenological and environmental matching for future field releases of this predator.
3.3 Materials and Methods
3.3.1 Source of predators and rearing
Different strains of S. camptodromus distinguished by geographic origin in China were collected and transported from China to the USDA Forest Service quarantine facility in Ansonia,
CT under permit. Voucher specimens of adults were deposited at the Entomology Division, Yale
Peabody Museum of Natural History, New Haven, CT. Currently, these predators are being reared at the USDA Forest Service quarantine facility in Ansonia, CT and at Pennsylvania State
University, PA after being released from quarantine. Three strains (geographic populations) of the predator, DGS (CHINA:Sichuan: 5-X-06, 5-11-XI-06, 26-IV-07), MNP (CHINA:Yunnan:
25-28-IX-05, 20-IV-07, 13-VI-07, 23-XI-07), and LJS (CHINA:Yunnan: 23-IX-05, 21-22-IV-07,
25-V-07, 11-VI-07, 21-IX-07) were used in this study (Keena et al., 2012).
This study began with eggs from the fifth generation of S. camptodromus reared in the laboratory. To simulate the conditions that initiate and then break diapause, each egg was held
49 individually in a 0.5 ml clear micro-centrifuge tube at 20 °C for 29.5 days, 15 °C for 52.5 days,
10 °C for 50 days, 5 °C for 116 days, and then transferred to 10 °C for hatching (Keena et al.,
2012). The predator eggs were observed daily at 10 °C and newly hatched larvae were immediately transferred individually to a 59 ml soufflé cup with a clear 2.5 cm diameter lid (Solo,
Eastern Bag and Paper, CT) for rearing. The opening of the cup was covered with a fine mesh cloth and was held in place by the lid with a 1 cm2 “X” cut in the center to provide ventilation. A
2.4 cm diameter piece of filter paper was placed in the bottom of the rearing containers.
3.3.2 Prey provisioning
Hemlock twigs infested with A. tsugae were collected from two locations in central
Pennsylvania: Bear Meadows (40.73 N, 77.75 W) and Penn State University's Russell E. Larson
Agricultural Research Center (40.69 N, 77.96 W). Infested twigs were stored at 5 °C until needed to delay egg hatch and prolong the period of A. tsugae oviposition. A. tsugae mothers were removed and the number of eggs counted on each twig before presenting it to the neonate beetles. As the predator larvae got older it became obvious which eggs had been fed upon; the eggs consumed by predator larvae were distinctly flat with all the egg contents sucked out, leaving an intact, yellowish orange chorion, whereas the hatched chorions were transparent and ruptured so pre-counts were not necessary. Each twig in the larval rearing cup was checked every day to ensure sufficient eggs were still available and changed every three days, or after the predator molted to the next instar. The number of A. tsugae ovisacs and fresh eggs provided per day was increased with each successive instar.
50 3.3.3 Development time of S. camptodromus larvae by temperature and strain
To assess predation by S. camptodromus, numbers of A. tsugae eggs consumed by larvae from each strain and instar at 15 and 20 °C were counted and recorded. Each time the A. tsugae- infested twigs were changed, the rearing container was examined for any dislodged A. tsugae eggs or hatched nymphs and then the twigs were observed under a dissecting microscope and all consumed A. tsugae eggs were counted and recorded. When the larva stopped feeding and transitioned to the pre-pupa, infested twigs were removed and replaced by fresh uninfested twigs to maintain humidity inside the cup and to provide a place for pupation. The non-feeding pre- pupal stage wandered around searching for a suitable place to pupate so it was difficult to distinguish the exact day the insect entered the pre-pupal stage; therefore, this stage was designated as part of the fourth instar.
3.3.4 Statistical Analysis
All statistical analyses were conducted using SAS (SAS Institute 1999, 2012). The influence of temperature and strain on survival of S. camptodromus from neonate to adult as well as sex ratio were determined using contingency analysis and Fisher Exact Tests. The number of
A. tsugae eggs consumed per S. camptodromus instar, days spent in each instar, and eggs consumed per day within each instar were evaluated with the PROC UNIVARIATE procedure with the histogram option to assess the distributional fit of each response variable. Statistically, the Shapiro-Wilk and the Anderson-Darling test were used to assess normality. However, in cases where no distributions met the normality assumption, histogram output was assessed visually to see which distribution most closely emulated the data.
51 Eggs consumed per instar, days spent in each instar, and eggs consumed per day within each instar were analyzed with a repeated measures completely randomized design.
푌푖푗푘 = 휇 + 푇푖 + 푆푗 + 퐼푘 + (푇푆)푖푗 + (푇퐼)푖푘 + (푆퐼)푗푘 + (푇푆퐼)푖푗푘 + 휀푖푗푘 (Eqn 1)
Where:
Ti = Temperature main effect
Sj = Strain main effect
Ik = Instar main effect
(TS)ij = Temperature and strain interaction
(TI)ik = Temperature and instar interaction
(SI)jk = Strain and instar interaction
(TSI)ijk =Temperature times strain times instar interaction
Number of eggs consumed per instar (note an egg was considered consumed even if it was not totally consumed by a first instar since it would have rendered it unable to hatch) and days spent in each instar were discrete variables, while eggs consumed per day within each instar was a continuous variable. The fixed effects in each model were temperature, strain, and instar.
We used a generalized linear model via PROC GLIMMIX using a pseudo-likelihood estimation technique. To account for repeated measurements of organisms over instars, we used a repeated measures statement via the random residual option in PROC GLIMMIX. We assessed several covariance structures and selected the AR (1) (or ARH (1) when needed) because it provided the most random residuals since fit statistics were not available. The response variables all had long right tails because of over-dispersion (the mean was several times smaller than the variance).
Thus, we used a negative binomial distribution with a log link function for the discrete variables, eggs consumed per instar, and days spent in each instar. The lognormal distribution with a log link function was used for the eggs consumed per day since it was the best fit for this continuous
52 variable. The auto-regressive order 1 (AR (1)) covariance structure was used to account for the multiple measurements over instar for the number of days spent in an instar. However, after running the original AR(1) model for eggs consumed per instar and eggs consumed per day, there was a homogeneity of variance violation detected via the Levene’s test, so the first-order auto- regressive heterogeneous co-variance structure (ARH (1)) model was used instead for these two variables. Residuals were evaluated for normality and the homogeneity of variance assumption.
The Kenward Rogers denominator degrees of freedom adjustment option was also used in the days in instar analysis only, and differences among means were determined by the LSMEANS option via the Tukey-Kramer post hoc analysis. An alpha level of 0.05 was used in all analyses to assess significance.
The fixed effects of temperature, sex, strain and their interactions on length and width of predators when they reached the adult stage were analyzed using PROC MIXED and the REML.
Mean differences were determined following each analysis using the least squared means test with α = 0.05 and the Bonferroni correction.
3.3.4.1 Estimating degree-day requirements (DD) for larvae
Development time to adulthood at 10, 15, 20 and 25 °C was used to estimate the relationship between temperature and developmental rate (1/day). In a separate experiment, larval development of S. camptodromus fed A. tsugae prey from neonate to adult (time in instar was not documented) was evaluated at 10 and 25 °C at the USDA Forest Service Northern
Research Station in Ansonia, CT using the same beetle strains and the same methods described above. These data were combined with the data from the developmental time experiment conducted at 15 and 20 °C for degree-day (DD) estimates. The relationship between
53 developmental rate (YDR) and temperature (T) was estimated using linear regression and is represented by the equation, YDR = β0 + β1 xT.
Development time to adulthood at 15, 20 and 25 °C was used to calculate the degree-day requirement for S. camptodromus. The degree-day calculation did not include 10 °C because responses to temperatures near a threshold often are more heterogeneous (Keena, 2006). From the relationship between temperature and development rate, the lower temperature threshold (TL) for development was calculated by setting the development rate to zero. Using the calculated TL the number of degree-days required for each individual to reach adulthood was calculated. The relationship used to calculate degree day was as follows:
DD = [constant holding temperature (15, 20, 25 °C) – TL] ×Dt, where Dt is the total development time by each individual predator to reach adulthood at a constant temperature. The cumulative proportions of predators reaching adulthood after accumulation of a specific number of degree days were used to determine the relationship between DD and the proportion that became adults using non-linear regression and the Gompertz function, P=exp [-exp(-bDD +a]
(Brown and Mayer 1988) with the Marquardt convergence method. Accumulated degree-days required for 10, 50, 90 and 99 % of the predator population to reach adulthood were calculated.
3.4 Results
3.4.1 Effect of temperature on predator survival and development time
Out of the 150 S. camptodromus larvae reared, 101 survived to adult. We did not observe a significant difference in larval survival (Fisher Exact Test; P = 1.0) and sex ratio (Fisher Exact
Test; P = 0.53) between 15 and 20 °C, nor was there an effect of strain on survivorship (Fisher
54 Exact Test; P = 0.60). However, sex ratio differed by strain (Fisher Exact Test; P= 0.002); the ratio of females to males for DGS, LJS, and MNP were 1:1, 5:1, and 1:1, respectively.
The mean number of days spent in each life-stage did not differ by strain as an overall effect, and there was no temperature*strain interaction (Table 3-1a). However, as main effects temperature and instar had significant impacts, development time was slower at 15 C than at 20
C, and time spent in each life stage significantly increased with each subsequent life stage
(Tables 1a and b). For example, the predator spent significantly more time in the fourth instar compared to the other instars regardless of strain. The 3-way interaction term was not significant, but there were significant temperature*instar and strain*instar interactions (Table 3-1a). For example, S. camptodromus larvae spent significantly more time in all life stages at 15 °C than 20
°C, except the second instar, which did not differ at the two temperatures (Table 3-1b). The strain*instar interaction indicated that no single strain consistently spent the longest or shortest amount of time in each life stage (Table 3-1b).
The total mean development time to adulthood was calculated at each temperature and for each strain. DGS, LJS and MNP took 43.2 ± 0.83, 40.5 ± 0.39, 42.5 ± 0.54 days, respectively, to reach adulthood at 15 °C and 29.5 ± 0.37, 28.8 ± 0.19, 29.7 ± 0.54 days, respectively, at 20 °C.
The average time to adult among all strains was 12 days longer at 15 °C than at 20 °C.
3.4.2 Predation by S. camptodromus larvae
The mean number of A. tsugae eggs consumed by S. camptodromus by instar did not differ among strains or between temperatures as an overall effect and also there was no significant temperature*strain interaction (Table 3-2a). As the main effect, egg consumption significantly increased with each subsequent instar from 31.9 ± 1.3 eggs in the first instar to 171
55 ± 7.0 eggs in the fourth instar, with no significant difference between the third and fourth instars
(Table 3-2b).
There was no significant 3-way interaction effect on mean egg consumption by instar, but the temperature*instar and strain*instar interactions were significant (Table 3-2a). For example, the mean number of eggs consumed within the first three instars did not differ between rearing temperatures; however, more eggs were consumed during the fourth instar at 15 °C than at 20 °C by strains LJS and MNP. Although there was a significant strain*instar interaction, the only discernable differences in mean egg consumption among strains across instars were that different strains showed slightly different trends in how much egg consumption increased through the predators' life cycle (Table 3-2a). Most differences were, however, not statistically significant.
The mean number of A. tsugae eggs consumed from neonate to pupation by strain (DGS,
LJS, and MNP) and temperature were 458 ± 17.9, 533 ± 25.6, and 535 ± 34.6, respectively, at 15
°C, and 449 ± 22.5, 440 ± 14.8 and 494 ± 26.7, respectively, at 20 °C. Because, the predators spent less time as larvae at 20 °C (see above), we compared total predation per larva per day as a function of temperature, instar, strain and their interactions (Table 3-3 and Fig. 3-1). We found that predation per day was also not different among strains and there was no 3-way interaction.
As a main effect temperature had a significant impact, the mean number of A. tsugae eggs consumed per day was significantly less at 15 °C (22.9 ± 0.8 eggs) than at 20 °C (31.4 ± 0.9 eggs)
(Fig. 3-1). Although, larvae ate less per day and took longer to develop at the cooler temperature, the mean number of eggs consumed per instar generally did not differ between these two temperatures (no significant temperature main effect). There were also significant instar*temperature and strain*instar interactions on mean egg consumption per day by instar
(Table 3-3). For example, egg predation per day was not different within the first, second or fourth instars, but DGS consumed significantly fewer eggs per day in the third instar than other strains during the same life stage regardless of temperature.
56 3.4.3 Estimation of degree-day requirement
Development time (in days) was inversely related to temperature; development rate
(1/days) increased as the temperature increased (F = 1526; df = 1, 154; P < 0.0001) (Fig. 3-2).
The relationship between temperature and development rate to adulthood can be described as,
YDR = (0.0022 ± 0.0001) T- 0.0116 ± 0.0009, where YDR is development rate and T is the corresponding temperature in degrees Celsius. The rate of development for S. camptodromus pupae was faster (steeper slope of the regression line) than the rate of development for larvae
(Fig. 3-3). The lower development temperature threshold (TL) for development to adult was calculated to be 5.18 C. The degree-day requirement for development from neonate to adult for
10, 50, 90 and 99 % of the S. camptodromus population was predicted as 397 ± 1.0, 424 ± 0.2,
2 467 ± 2.4 and 520 ± 5.0 DD, respectively (Fig. 4; R adj =0.99, F = 3308; df = 2, 18; P < 0.0001).
3.4.4 Effects of temperature, sex, and strain on adult beetle size
The length and width of S. camptodromus adults were unaffected by temperature (length:
F = 0.1; df = 1, 99; P = 0.96; width: F = 0.90; df = 1, 99; P = 0.34), sex (length: F = 0.88; df = 1,
95; P = 0.35; width: F = 2.46; df = 1, 95; P = 0.12), or their interactions (Table 3-4). However, there was a significant difference by strain on the length (F = 15.6; df = 2, 95; P < 0.001) and width (F = 19.5, df = 2, 95; P < 0.0002) of the predators, with LJS being significantly larger
(longer and wider) than DGS or MNP. To determine if this difference could be due to females in the population, which tend to be larger than males, we compared length and width of only females among strains, and obtained the same result (length: F = 7.75; df = 2, 63; P = 0.001; width: F = 14.3; df = 2, 63; P <0.0001). There was also a significant relationship between length
2 and width of the individual predators (R adj =0.26, F = 35.3; df = 1, 99; P < 0.0001).
57 3.5 Discussion
As expected, development rate (1/days) of S. camptodromus increased with increasing temperature and followed a linear relationship (Fig. 3-2). While temperature did not affect size or sex ratio of the predators at the temperatures tested, it did have significant effects on development and predation, but the impacts varied depending upon predator strain and instar. For example, on average strains, LJS and MNP consumed more A. tsugae eggs in the fourth instar at 15 C than at
20 C, while this did not occur for DGS. It is clear, however, that there was no consistent effect of strain on development or predation. LJS were bigger as adults than the other strains regardless of temperature, yet the larvae of this strain ate the same number of eggs during development as did the MNP strain. Studies are underway to determine if there are any differences among strains in fecundity. Given that S. camptodromus adults reared in the lab were bigger than the same species reared under natural conditions (Yu et al., 1996), this may not hold in the field. In our study, we also found that S. camptodromus adults were larger on average than was reported for S. sinuanodulus and S. ningshanensis (Montgomery & Keena, 2011).
The lower temperature threshold for S. camptodromus development was close to 5 °C, which is comparable to the lower threshold limit of 3.9 °C for the development of A. tsugae progrediens (Salom et al., 2002). At 25 °C, however, very few S. camptodromus larvae survived to adulthood, suggesting that the upper temperature threshold for larval development is near 25
°C, which is comparable to the upper temperature threshold of 22-27 °C for development of A. tsugae progrediens. Thus, the temperature thresholds for S. camptodromus are closely matched with those of its prey, suggesting that the predator may be able to survive where A. tsugae can thrive. At 20 °C, development time of predator larvae from hatch to adult was comparable to that of the other two predators from this genus collected in China, i.e., S. sinuanodulus and S. ningshanensis. However, under the conditions of our study at 15 °C, S. camptodromus developed
58 faster compared to S. sinuanodulus (Lu & Montgomery, 2001). S. camptodromus also developed faster than was reported for Laricobius nigrinus Fender (Coleoptera: Derodontidae). L. nigrinus was introduced as a biological control agent for A. tsugae and appears to have established in some parts of the eastern U.S. (Zilahi-Balogh et al., 2003).
Unlike other predators from China, S. camptodromus has a true aestival diapause, which is synchronous with the summer diapause of the A. tsugae neosistentes (Keena et al., 2012).
Scymnus camptodromus eggs do not hatch until A. tsugae oviposition begins in the late winter/early spring, ensuring that the developing larvae have sufficient food to reach adulthood.
This strategy may explain, at least in part, why S. camptodromus was found in greater abundance in broader geographic regions and at variable A. tsugae densities in its native range compared to other native predators (Montgomery & Keena, 2011).
Although S. camptodromus consumed greater numbers of A. tsugae eggs per day at 20 than at 15 °C, overall egg consumption per instar was not different between the two temperatures.
This can be explained by the fact that although larvae ate more A. tsugae eggs per day at 20°C, they took less time to develop at this temperature, thus ultimately requiring fewer eggs to complete development at the warmer temperature. In two predator strains at 20 C, fewer HWA eggs were consumed in the fourth than the third instar (Table 2b); however, this may have been an artifact of the experimental design. Because it was difficult to determine the specific day fourth instars entered the non-feeding pre-pupal stage, all larvae in the fourth instar were combined for analysis without separating out the pre-pupal stage. On average predator larvae consumed 31.4 ± 0.91 A. tsugae eggs per day at 20 °C, which is comparable to predation by the adults of this species reported previously (mean of 31 A. tsugae eggs per day) at 19 °C (Zhao et. al 1998). The total average consumption by S. camptodromus larvae to complete development to adult at 15 and 20 °C was 512 ± 16.0 and 454 ± 11.5 eggs, respectively, which is more than the reported 226 ± 18 and 252 ± 18 eggs consumed by L. nigrinus larvae at 12 and 18 °C,
59 respectively (Zilahi-Balogh et al., 2003), indicating that S. camptodromus has considerable potential for biological control of A. tsugae.
Field studies are needed to determine the upper temperature threshold for development of this predator since laboratory conditions at a constant 25 °C may have underestimated the upper threshold for S. camptodromus development and/or survival. It is expected that S. camptodromus will benefit from its ability to develop at very low temperatures if released in the northeastern part of the A. tsugae range. These predators are also known to survive for more than one year in a laboratory setting (Keena unpublished data). Further studies under field conditions are needed to determine the lower and higher temperature threshold for the adults in the eastern U.S., which has implications for adult survival and reproduction over multiple years. Field studies to determine when S. camptodromus eggs will hatch under natural conditions (fluctuating temperatures and weather extremes) are also planned.
These findings on developmental rates, degree-day requirements, and predator consumption will inform confined release studies on predator/prey interactions and provide baseline data for developing mass rearing procedures and planning field releases. Moreover, these results provide needed information to help improve laboratory rearing. This study, although done under constant temperatures and humidity in a laboratory setting, shows that S. camptodromus can be an effective predator of A. tsugae; further studies are needed to verify these results under field conditions.
60 Table 3-1a. GLIMMIX model for mean development time of S. camptodromus after eclosion.
Effect F Df P > F
Temp 424 1 < 0.0001
Strain 1.7 2 0.1835
Instar 1361 4 < 0.0001
Temp*Instar 19 4 < 0.0001
Temp*Strain 1.62 2 0.2014
Strain*Instar 2.65 8 0.0077
Temp*Strain*Instar 0.92 8 0.5009
See methods for details of analysis.
Table 3-2b. Mean development time [mean days ± SE (n)] by life stage of S. camptodromus reared at 15 or 20 °C after eclosion
Temp/ Instar Pupal stage strain I II III IV
15 C
DGS 6.1 ± 1.03e 3.4 ± 1.04ghij 4.2 ± 1.04f 15.6 ± 1.05a 14.0 ± 1.05ab
(21) (19) (18) (18) (18)
LJS 5.3 ± 1.03e 3.6 ± 1.03fgh 3.9 ± 1.03fg 13.9 ± 1.03ab 13.7 ± 1.04ab
(28) (28) (28) (28) (28)
MNP 5.3 ± 1.04e 3.8 ± 1.04fgh 3.8 ± 1.04fgh 15.1 ± 1.05a 14.6 ± 1.05a
(12) (12) (12) (12) (12)
20 C
DGS 3.6 ± 1.04fghi 2.8 ± 1.04j 3.1 ± 1.04hij 8.9 ± 1.04cd 11.1 ± 1.04bc
61 (17) (16) (15) (15) (15)
LJS 3.6 ± 1.03fgh 3.2 ± 1.03ghij 3.0 ± 1.03ij 8.8 ± 1.03d 10.4 ± 1.04cd
(26) (25) (24) (23) (23)
MNP 3.8 ± 1.05fgh 3.2 ± 1.05ghij 3.1 ± 1.05hij 9.0 ± 1.05cd 10.7 ± 1.06cd
(11) (10) (10) (10) (9)
Sample size (n) is based on number of survivors. Because there were significant temperature*instar and strain*instar interactions (Table 1a), all means were compared among each other. Means followed by a different letter within the table are significantly different from each other at P < 0.05 using Tukey-Kramer post hoc test.
Table 3-2a. GLIMMIX model for mean number of hemlock woolly adelgid eggs consumed by S. camptodromus after eclosion.
Effect F value df P > F
Temp 5.07 1 0.1380
Strain 2.22 2 0.5310
Instar 392 3 < 0.0001
Temp*Instar 19.4 3 < 0.0001
Temp*Strain 1.14 2 0.2020
Strain*Instar 4.95 6 < 0.0001
Temp*Strain*Instar 1.46 6 0.0956
62 Table 3-2b. Mean number [mean ± SE (n)] of hemlock woolly adelgid eggs consumed by S. camptodromus by instar reared at 15 °C or 20 °C after larval eclosion. Temp/strain I II III IV
15 C
DGS 36.0 ± 3.2g 67.8 ± 6.0f 144 ± 12.6abcd 208 ± 18.0ab
(23) (19) (18) (18)
LJS 32.1 ± 2.4g 81.2 ± 5.7ef 178 ± 12.4ab 220 ± 15.2ab
(30) (29) (28) (28)
MNP 31.2 ± 3.7g 81.6 ± 8.9ef 187 ± 19.8ab 236 ± 24.9a
20 C (12) (12) (12) (12)
DGS 36.1 ± 3.6g 81.4 ± 7.7ef 164 ± 15.6abc 168 ± 16.0abc
(19) (16) (15) (15)
LJS 29.2 ± 2.4g 104 ± 8.0cdef 211 ± 16.1ab 96.4 ± 7.7def
(28) (25) (24) (23)
MNP 28.4 ± 3.6g 102 ± 12.1cdef 223 ± 25.7ab 127 ± 15.0bcde
(11) (10) (10) (10)
Sample size (n) is based on number of survivors within each column. GLIMMIX was used to determine the effects of instar, strain, temperature and their interactions on mean eggs consumption by the predator larvae. Because there were significant temperature*instar and strain*instar interactions on mean egg consumption (Table 2a), all means were compared among each other. Means followed by a different letter within the table are significantly different from each other at P < 0.05 using Tukey-Kramer post hoc test.
63 Table 3-3. GLIMMIX model for mean number of hemlock woolly adelgid egg consumed per day by S. camptodromus after eclosion.
Effect F value df P > F
Temp 46.5 1 < 0.0001
Strain 1.31 2 0.2729
Instar 594 3 < 0.0001
Temp*Instar 7.1 3 0.0002
Temp*Strain 3.49 2 0.0329
Strain*Instar 5.66 6 < 0.0001
Temp*Strain*Instar 2.04 6 0.0609
Table 3-4. Mean size [mean ± SE (n)] of S. camptodromus following adult eclosion reared at 15 °C or 20 °C.
Temp/strain Average length (mm) Average width (mm)
15 ⁰C
DGS 2.68 ± 0.06a (15) 1.34 ± 0.02a (15)
LJS 2.83 ± 0.03b (27) 1.44 ± 0.02b (27)
MNP 2.58 ± 0.07a (12) 1.39 ± 0.02a (12)
20 ⁰C
DGS 2.59 ± 0.07a (15) 1.36 ± 0.01a (15)
LJS 2.84 ± 0.02b (23) 1.45 ± 0.01b (23)
MNP 2.70 ± 0.05a (9) 1.41 ± 0.01a (9)
Sample size (n) is based on number of survivors to adult. PROC MIXED was used to determine the fix effects of temperature, sex, strain and their interactions on length and width of predators.
64 Length and width of predator was not affected by temperature. Neither sex nor interaction between sex and strain affected predator size. In contrast, strain differences in size were apparent. Within a column, means followed by a different letter were significantly different at P < 0.05 with Bonferroni correction method.
Figure 3-1. Average eggs consumed per day among different S. camptodromus strains and instars. The solid line represents egg consumption per day at 20 ºC and the dashed line represents egg consumption per day at 15 ºC.
65
Figure 3-2. Relationship between temperature and development rate (1/d) of S. camptodromus from neonate to adult. The line represents the simple linear regression combining all predator strains; some circles represent multiple data points at a given temperature due to overlap.
Figure 3-3. Relationships between temperature and S. camptodromus larval (DTP) and pupal (DP) rates (1/days) of development. Lines represent the estimated linear relationships combining all the predator strains (DGS, LJS, and MNP). Each diamond represents mean days to pupation and each triangle represents days as pupa at each temperature.
66
Figure 3-4. Cumulative proportion of S. camptodromus neonates to reach adulthood over accumulated degree-days. Open circles represent individuals reared at 15, 20, and 25 °C; there is considerable overlap of circles.
67 References
Cheah, C. A. S.-J., M. E. Montgomery, S. Salom, B. L. Parker, S. Costa, and M. Skinner, 2004. Biological control of hemlock woolly adelgid. Reardon, R. and B. Onken (Tech. Coordinators), USDA Forest Service, Morgantown, WV, FHTET-2004-04. Delucchi, V. 1954. Pullus impexus (Muls.)(Coleoptera, Coccinellidae), a predator of Adelges piceae (Ratz.)(Hemiptera, Adelgidae), with notes on its parasites. Bull. Entomol. Res. 45: 243-278. Domec, J. C., L. N. Rivera, J. S. King, I. Peszlen, F. Hain, B. Smith, and J. Frampton. 2013. Hemlock woolly adelgid (Adelges tsugae) infestation affects water and carbon relations of eastern hemlock (Tsuga canadensis) and Carolina hemlock (Tsuga caroliniana). New Phytol. 199: 452-463. Ellison, A. M., A. A. Barker‐Plotkin, D. R. Foster, and D. A. Orwig. 2010. Experimentally testing the role of foundation species in forests: the Harvard Forest Hemlock Removal Experiment. Meth. Ecol. Evol. 1: 168-179. Havill, N. P., and M. E. Montgomery. 2008. The role of arboreta in studying the evolution of host resistance to the hemlock woolly adelgid. Arnoldia 65: 1-9. Havill, N. P., M. E. Montgomery, G. Yu, S. Shiyake, and A. Caccone. 2006. Mitochondrial DNA from hemlock woolly adelgid (Hemiptera: Adelgidae) suggests cryptic speciation and pinpoints the source of the introduction to eastern North America. Ann. Entomol. Soc. Am. 99: 195-203. Keena, M. A., R. T. Trotter III, C. Cheah, and M. E. Montgomery. 2012. Effects of temperature and photoperiod on the aestivo-hibernal egg diapause of Scymnus camptodromus (Coleoptera: Coccinellidae). Environ. Entomol. 41: 1662-1671. Lu, W., and M. E. Montgomery. 2001. Oviposition, development, and feeding of Scymnus (Neopullus) sinuanodulus (Coleoptera: Coccinellidae): a predator of Adelges tsugae (Homoptera: Adelgidae). Ann. Entomol. Soc. Am. 94: 64-70. Lu, W., P. Souphanya, and M. E. Montgomery. 2002. Descriptions of immature stages of Scymnus (Neopullus) sinuanodulus Yu and Yao (Coleoptera: Coccinellidae) with notes on life history. Coleop. Bull. 56: 127-141. McClure, M. 1991. Density-dependent feedback and population cycles in Adelges tsugae (Homoptera: Adelgidae) on Tsuga canadensis. Environ. Entomol. 20: 258-264. McClure, M. 1995. Using natural enemies from Japan to control hemlock woolly adelgid. Front. Plant. Sci. 47: 5-7. Montgomery, M. E., and M. A. Keena. 2011. Chapter 5: Scymnus (Neopullus) Lady Beetles from China, pp. 53-76. In R. Reardon and B. Onken (eds.), Proceedings, Symposium: Implementation and status of biological control of the hemlock woolly adelgid. FHTET 2011-04. USDA Forest Service, Morgantown, WV. Onken, B. and R. Reardon (compilers). 2008. Fourth symposium on hemlock woolly adelgid in the Eastern United States. FHTET-08-01. U.S. Department of Agriculture, Forest Service, Morgantown, WV, 299 pp. Onken, B. and R. Reardon (compilers). 2010. Fifth symposium on hemlock woolly adelgid in the Eastern United States. FHTET-10-07. U.S. Department of Agriculture, Forest Service, Morgantown, WV. 222 pp. Preisser, E.L., K.L. Oten, and F. P. Hain. 2014. Hemlock woolly adelgid in the Eastern United States: What have we learned? Southwest. Nat. 13:1-15.
68 Salom, S. M., A. S. Sharov, W. T. Mays, and D. R. Gray. 2002. Influence of temperature on development of hemlock woolly adelgid (Homoptera: Adelgidae) progrediens. J. Entomol. Sci. 37:166-176. SAS Institute. 2012. SAS® 9.3 In-Database Products: User’s Guide, version 4th ed. SAS Institute, Cary, NC. US Department of Agriculture Forest Service (USFS). 2012. Forest Health Protection: Hemlock Woolly Adelgid Distribution Map. Forest Health Protection Service, Newtown Square PA. Vieira, L., A. Lamb, S. Shiyake, S. Salom, and L. Kok. 2013. Seasonal abundance and synchrony between Laricobius osakensis (Coleoptera: Derodontidae) and its prey, Adelges tsugae (Hemiptera: Adelgidae). Japan. Ann. Entomol. Soc. Am. 106: 249-257. Young, R. F., K. S. Shields, and G. P. Berlyn. 1995. Hemlock woolly adelgid (Homoptera: Adelgidae): stylet bundle insertion and feeding sites. Ann. Entomol. Soc. Am. 88: 827- 835. Yu, G., Yao, D., & Liu, H. 1996. The coccinellidae collected from Tsuga with Adelges tsuga Annand (Homoptera: Adelgidae). Sci. Silvae Sin. 33: 432-440. Yu, G., M. E. Montgomery, and D. Yao. 2000. Lady beetles (Coleoptera: Coccinellidae) from Chinese hemlocks infested with the hemlock woolly adelgid, Adelges tsugae Annand (Homoptera: Adelgidae). Coleopt. Bull. 54: 154-199. Zilahi-Balogh, G. M., S. M. Salom, and L. T. Kok. 2003. Development and reproductive biology of Laricobius nigrinus, a potential biological control agent of Adelges tsugae. Biocontrol 48: 293-306.
69
Chapter 4
Effects of Temperature on Development of Lymantria dispar asiatica and Lymantria dispar japonica (Lepidoptera: Erebidae)
4.1 Abstract
Periodic introductions of the Asian subspecies of gypsy moth Lymantria dispar asiatica
Vnukovskij and L. dispar japonica Motschulsky in North America are not only threatening commercial and urban forests but also interrupting foreign trade. Although Asian gypsy moth has similar morphology to that of European and North American gypsy moth, several traits make it a greater threat, the most important being the flight capability of females. Asian gypsy moth is not yet an established pest in North America; however, infestations have been detected multiple times in Canada and the US. To facilitate detection and eradication efforts, we evaluated the effects of a range of temperatures on development time, survivorship, and fertility of eight populations of
Asian gypsy moth. Larval developmental rate increased with temperature until it reached an optimum at 29 °C. Larvae displayed significant molting problems at the highest and lowest temperatures tested (10 and 30 °C), and at 30 °C female fitness was markedly compromised, as evidenced by reduced fecundity and fertility. These findings suggest that development and survival of Asian gypsy moth may be limited by summer temperature extremes in the southern
US. We also determined the degree-day requirements for two critical life stages (from egg hatch to second instar and egg hatch to adult) and two populations of Asian gypsy moth, which represent the extremes in latitude, to predict the timing for both bio-pesticide application and adult trapping. Our data will benefit pest managers in developing management strategies, pest risk assessments, and timing for implementation of management tactics.
70 4.2 Introduction
The gypsy moth (Lymantria dispar L.) is a serious forest pest capable of completely defoliating numerous tree species throughout the northern hemisphere (Elkinton and Liebhold
1990). This polyphagous pest can consume more than 300 host species (Liebhold et al. 1995), although it prefers oaks (Quercus spp.) (Raupp et al. 1988, Shields et al. 2003, Foss and Rieske
2003). The gypsy moth found in North America is an exotic species that originated from Western
Europe (Wu et al. 2015). This subspecies of gypsy moth, introduced into Medford, MA in 1869, is expanding its range at the rate of 6-18 km/year (Tobin et al. 2007). It is currently found as far south as North Carolina, as far west as Wisconsin (USDA APHIS 2015), and has established populations in the southern parts of Canada east to Lake Superior (Régnière et al. 2009). Since
1970, gypsy moth has defoliated 81 million acres of forest (Gypsy Moth Digest 2013). The more recent incursion of two other subspecies of L. dispar, collectively known as Asian gypsy moth L. dispar asiatica Vnukovskij and L. dispar japonica Motschulsky, threatens establishment of these subspecies in North America and many other parts of the world (Matsuki et al. 2001, Pitt et al.
2007).
L. dispar asiatica is found east of the Ural Mountains in Russia, throughout China and
Korea, and in parts of Japan, whereas L. dispar japonica occurs in patches on all islands in Japan
(Pogue and Schaefer 2007). In North America, an infestation of an Asian subspecies was first detected in British Columbia in 1991 (Bogdanowicz et al. 1993, Savotikov et al. 1995), but an immediate eradication effort removed the pest successfully. Since then, Asian gypsy moth has been detected and eradicated on multiple occasions and is thus far not known to have established in North America.
Asian gypsy moth is considered a greater threat to forest and agricultural resources than their European counterparts because of distinct behavioral and ecological characteristics. Unlike
71 the European subspecies, Asian gypsy moth females are capable of sustained flight (Wallner et al.
1995, Keena et al. 2008). They have a broader reported host range, including more coniferous species (Baranchikov and Sukachev 1989) than the European gypsy moth. Asian gypsy moths are attracted to lights, including well-lit port areas where they oviposit on cargo and ships
(Wallner et al. 1995), thus causing delays at ports of entry for regulatory disinfestation and affect international trade. Eggs of the Asian subspecies from some geographic areas also require a shorter chilling period to be ready to hatch (Keena 2015). These characteristics may increase their potential to establish in new areas, increase their dispersal capability, and result in extensive damage to multiple forest types. Also, the Asian and European subspecies can successfully hybridize and the initial hybrids of European and Asian gypsy moth are able to glide varying distances, indicating better dispersal capabilities in hybrid offspring than the non-flying European parent (Keena et al. 2001). Therefore, the introduction of the Asian subspecies in areas where the
European subspecies is already present could make limiting the spread of gypsy moth difficult, requiring more aggressive management strategies than currently employed.
Despite efforts to regulate Asian gypsy moth, the pest has been introduced into new areas through international trade (Gibbons 1992, USDA 2016, Matsuki et al. 2001). A recent study on potential global distribution using ecological niche modeling suggests that Asian gypsy moth is capable of occupying almost all temperate regions of the world (Peterson et al. 2007). However, survival and establishment of the Asian subspecies in a new area depends on several factors. For example, larval development is influenced by temperature, foliage suitability, geographical origin, larval density, and sex (Casagrande et al. 1987, Hough and Pimentel 1978, Honěk 1996,
Campbell 1978, Jarošík and Honěk 2007). Because insects are ectothermic, temperature is likely the primary driving factor influencing larval gypsy moth development rate (Casagrande et al.
1987, Johnson et al. 1983). Studies on the effect of temperature on European subspecies exist, but none on Asian subspecies. Predicting phenology of the Asian gypsy moth is critical for
72 monitoring and management. For instance, Bacillus thuringiensis kurstaki (Btk), a preferred treatment for Asian gypsy moth control, is most effective if applied to the early second instar
(Reardon et al. 1994). Currently, phenology of the gypsy moth is predicted based on the
European subspecies and by climate matching, which may not be accurate for the Asian subspecies.
In the laboratory, we reared eight geographical populations of Asian gypsy moth at five constant temperatures to quantify the effects of temperature on the development rate of the larvae and determine degree-day requirements for each instar and stage. Because Asian gypsy moth is not an established pest in North America, major efforts are made to prevent its introduction through international trade, and, if an infestation is detected using pheromone traps, it is delimited and triggers immediate eradication measures. Temperature-dependent effects on development of this pest can be used to develop phenology models to predict timing for bio-pesticide application and trap deployment. Further, this study will assist development of improved risk assessments for this pest around the world and help predict the potential distribution of this pest should it become established in new areas.
4.3 Materials and methods
4.3.1 Gypsy moth populations
Six geographical populations of L. d. asiatica (C1, C2, C3, R1, R2, and Sk), and two populations of L. d. japonica (J1 and J2) from a broad range of latitudes were selected for this study to capture potential variation in development rates between these two subspecies and among populations (Table 4-1). Voucher specimens for each population were deposited at the
Entomology Division, Yale Peabody Museum of Natural History, New Haven, CT.
73 4.3.2 Larval rearing at different temperatures
Ten egg masses from each population were placed individually in 150 × 15 mm sterile
Petri dishes (model 351058, Falcon®, Tewksbury MA) and held inside a water box at 25 °C and
60 % RH with a photoperiod of 16:8 h (L: D) to initiate hatch. The eggs were placed in environmental chambers at five constant temperatures, 10, 15, 20, 25, 30 °C, with a photoperiod of 16:8 h (L: D); humidity in these chambers was 70 ± 5, 70 ± 10, 80 ± 5, 60 ± 5, and 45 ± 5 %
RH, respectively. Once more than 80% of larvae hatched from each egg mass, individual larvae were transferred to 30 ml clear plastic cups (model 9051, Frontier Agricultural Sciences, Newark,
DE) with paper tab lids (model 9049, Frontier Agricultural Sciences, Newark, DE); each cup contained 15 ml of high wheat germ artificial diet (Bell et al. 1981). The diet was made with
Wesson salt mix without iron and with 2 g of amorphous ferric phosphate added per liter of prepared diet (Riedel-deHaen AG, Germany) to provide a source of iron that is bioavailable to gypsy moth (Keena 2005). Rearing was done on artificial diet instead of host plant material to avoid confounding effects of variations in plant quality on larval development.
Ten larvae from each of the 10 egg masses per population were selected randomly and assigned to one of the temperature treatments, ensuring a sample size of 100 larvae per population per temperature. However, at 30 °C, populations J2 and C2 had unexpectedly poor survival, so we added some larval replicates with neonates 21-25 days later. Egg masses used for the additional larvae originated from the same sources as the failed replicates, held at 10 °C until use.
Manipulations of the insects were always carried out under sterile conditions in a laminar flow hood to prevent contamination. Individual larvae were reared in the same 30 ml container until they reached the fourth instar when they were transferred to 118 ml clear plastic containers
(Model ME4, Solo® Cup Company, Lake Forest, Illinois) with a paper tab lid to provide more space and were supplied with 40 ml of artificial diet. Larvae were provided with fresh diet as
74 needed until they pupated. Molting, survival, diet condition, disease, and problems with molting were recorded daily. If a larva was unable to shed its head capsule or exuviae within 2-4 days after molting began, it was declared dead. Larvae with this condition always abstained from feeding and died of apparent starvation.
Once fully sclerotized, pupae were sexed and weighed, then transferred individually to
236 ml paper cups (U508N, Solo® Cup Company, Lake Forest, Illinois) covered with a plastic bag (15 × 15 cm) held in place with a rubber band. Pupae were held until adult eclosion at the temperature at which they were reared and checked daily for adult emergence. Once adults eclosed, they were weighed within the first 24 h and any abnormalities were noted.
The 10 °C treatment was ended after 120 days because a large number of larvae died as early instars and development was so slow that they would not have reached adulthood in nature before lethal winter temperatures. To obtain the sex of the surviving larvae, therefore, these insects were transferred to 25 °C to complete development.
4.3.3 Statistical Analyses
Statistical analyses were performed using SAS 9.3 (SAS Institute Inc., Cary, NC). PROC
UNIVARIATE was used to assess the distributional fit of the data. Normality of the data was evaluated using the Shapiro-Wilk and the Anderson-Darling tests. Graphical plots were also examined to evaluate the distribution of the data.
Temperature, population, and their interactions were examined for effects on survivorship of L. d. asiatica and L. d. japonica to the pupal and adult stages. The proportion of larval survival was calculated for each egg mass within each population at each temperature (15, 20, 25, and 30
°C). Survival at 10 °C was excluded from this analysis because, as explained earlier, a large number of larvae died as early instars and larval development rate was too slow to expect
75 pupation and survival in nature before the onset of winter. The fixed effects of temperature, population, and their interactions on survival from one life stage to the next were evaluated using a generalized linear model via PROC GLIMMIX with maximum likelihood technique. The model was fitted using a beta distribution with logit link function. Because the limits of the beta distribution are zero and one, 0% survival was coded as 0.000001, and 100% survival as
0.999999. The Tukey-Kramer post-hoc analysis was used to determine differences among means at = 0.05. Residuals were also evaluated for normality and homogeneity of variance.
To determine if the responses of the Asian populations to temperature could be categorized into distinct groups, the HPSPLIT procedure was used to build a separate classification tree for each tested temperature using individuals from all populations. Duration of the first instar was used as the predictor variable at 10 °C, and total duration of the larval and pupal stages was used at the remaining temperatures. Entropy was used in the GROW statement to set the splitting criteria, cost-complexity in the PRUNE statement to specify the pruning method, and the SEED value was set at 100. A maximum of 2 branches per node was allowed and the number of nodes was set at the value that minimized the average squared error in the cost- complexity cross validation analysis in an initial unlimited run. This provided the smallest tree that adequately categorized the data. Based on this analysis the two populations that represented the extremes of variation in response to temperature across all temperatures were used to determine the upper and lower developmental temperature thresholds and degree-day requirements.
PROC GLIMMIX was used to evaluate the fixed effects of temperature, population, sex and their interactions on duration of each instar and development time from hatch to pupa. To evaluate effects on pupal and adult body weight (g) the fixed effect of ultimate instar was added to the model. Duration of development to adult was evaluated with the fixed effects of temperature, population, ultimate instars and their interactions. The model was fitted to a negative
76 binomial distribution with a log link function because the response variables had long right tails.
Differences among means were determined by the Tukey-Kramer post hoc analysis at = 0.05.
For each model, residuals were evaluated for normality and the homogeneity of variance was assessed by Levene’s test.
Only the developmental time from egg to second instar and from egg to adult are presented in the results section because these are the critical stages of the gypsy moth life cycle when treatment and monitoring should be initiated, respectively. Information on the other instars and stages is provided in supplemental tables. The relationship between temperature and pupal weight by population (only for the two populations that represented the extremes), sex, and ultimate instar was fitted to the Lysyk model using PROC NLIN and the Marquardt convergence method (Table 4-2).
We determined the degree-day requirements for onset of the second instar and adult eclosion. The relationship between temperature and rate of development from hatch to the second instar by population (R1 and C2) was estimated using a linear model. Two non-linear
(Shi/performance and Logan) and a linear model were used to evaluate the relationship between temperature (excluding 10 °C) and development rate to reach the adult stage by population (R1 and C2) and by sex. Mathematical models used to fit the relationship between developmental rate and temperature are presented in Table 4-2. PROC REG was used to examine the linear model in
SAS, and the two non-linear models were fitted using MATLAB R2015a (Mathworks 2004).
These relationships were used to calculate the lower temperature thresholds (Tmin) for the development of R1 and C2 larvae to the second instar, and the lower (Tmin), upper (Tmax) and optimum temperature (To) for development of the R1 and C2 populations to adulthood by sex.
Degree-days (DD) required for development from egg hatch to second instar by population and to adult by population and sex were estimated using the function: DD = [constant temperature – Tmin] × Dt, where Dt is the total number of days required by individual larvae to
77 reach the second instar or to reach adulthood. The relationships between accumulated degree- days and cumulative proportion of individuals to reach second instar or adult were estimated using the Gompertz function, P = exp [- exp (- b * DD + a] with the Marquardt convergence method. Accumulated degree-days required by 10, 50, 90 and 95 % of the R1 and C2 larvae to reach the second instar and to reach the adult stage were calculated.
To evaluate the effect of temperature and population on Asian gypsy moth fitness, we first examined the proportion of insects that was unable to eclose normally, as well as proportion with adult deformity, by egg mass for each population at each temperature (15, 20, 25, and
30 °C). We also examined effects of temperature (25 and 30 °C) and population on fertility.
Fertility was determined by counting the number of embryonated and unembryonated eggs produced by each female. Because survival of larvae at 30 °C was poor and the majority of adults reared at this temperature was deformed, few matings of moths reared at this temperature were possible. We were unable to rear sufficient larvae to adult at 30 °C and to evaluate the effects of 30 °C on fertility; thus, we compared moths reared and mated at 25 °C with those reared at 25 °C until pupation and transferred to 30 °C for the duration of the pupal stage, adult emergence and mating. A minimum of 20 single pair matings per population per temperature was included in the fertility test.
We estimated overall performance of moths reared from each egg mass by population at each temperature. The estimated overall performance was defined as mean weight gain per day multiplied by survival, and calculated for each population and temperature as: Psurv × rate × Pwt, where Psurv is the proportion of larvae reared from each egg mass surviving to the pupal stage, rate is their mean developmental rate (1/days) to pupation, and Pwt is their mean pupal weight (g) (Lee et al. 2006). We used PROC GLIMMIX with maximum likelihood estimation to evaluate fixed effects of temperature, population, and their interactions on estimated performance of Asian gypsy moth populations at 15, 20, 25 and 30 °C. The model was fitted to a beta distribution with
78 a logit link function. The Tukey-Kramer post-hoc analysis (at = 0.05) was used to determine differences among means. Residuals were also evaluated for normality and the homogeneity of variance.
4.4 Results
4.4.1 Survival
The mean percentage of larvae that survived to pupa (F = 3.27; df = 21, 288; P < 0.0001;
Figure 4-1) and adult stages (F = 4.74; df = 21, 288; P < 0.0001; Figure 4-1) were significantly affected by the temperature × population interaction. Mean percentage survivorship until pupation for the C1, C2, J1, J2, and SK populations was significantly higher at 15, 20, and 25 °C, than at 30 °C. There were no significant differences among populations at 15, 20, and 25 °C except for the Japanese populations at 15 °C, which had significantly lower survival to pupation than the R2 population at 15 and 20 °C.
Similarly, no significant differences in mean proportion of survivors to adult were evident within a population at 20 and 25 °C, but there was significantly higher mean percentage survivorship at both of these temperatures than at 30 °C. Some populations were more impaired at 30 °C than others. Survival to adult at 15 °C was low in some populations, as well. For example, ≤ 13 % (mean 9.89 ± 2.9 %) of J2 survived to adulthood at 15 °C, which was significantly lower than survival of larvae from the remaining populations at this temperature.
However, the larvae of some Chinese populations (C1 and C3) survived well at the lower temperature and their survival at 15 °C was not significantly different from that at 20 and 25 °C.
79 4.4.2 Development Time from Hatch to Second Instar and to Adult Emergence
The criteria used, the developmental time (D1 and DA) criteria for splitting at each node, and the resulting percentage of individuals from each population in each node of a classification tree at four different temperatures are given in Table 4-3. Each temperature classification tree had 3-4 nodes, with the first node representing the fastest development and the last node the slowest, for example < 44 days and ≥ 73 days to complete the first instar at 10 °C. Overall, at five temperatures, a greater proportion of R1 individuals consistently completed development earlier than the other populations, while a greater proportion of C2 individuals completed development more slowly than the other populations. Therefore, R1 and C2 were selected to represent the extremes of development time for the tested populations and most of the results presented and discussed below will focus on these two populations. Information for the other populations is provided in Tables SI1- SI8.
As a main effect, the mean number of days spent in each life stage was significantly different among instars (F = 14451; df = 9, 12073; P < 0.001), i.e., the number of days spent in the first instar was significantly lower than the number of days spent in the ultimate larval instar.
Therefore, development time was compared only within each instar/stage. The mean number of days from larval hatch to second instar was affected by the interactions of temperature, population, and sex (F = 1.90; df = 66, 2569; P < 0.0001) (Table 4-4). There was no significant difference in development time between populations R1 and C2 and between sexes within a temperature. However, when compared across temperatures, both populations developed significantly faster to the second instar at temperatures above 10 °C. There was no significant difference in development time to the second instar between populations or sexes among the other temperatures (15, 20, 25 and 30 °C), except for R1 males, which when reared at 30 °C developed significantly faster than all other populations at 15 and 20 °C of both sexes.
80 Similarly, the mean number of days from egg hatch to adult was affected by the interaction of temperature, population, and ultimate instar attained (F = 1.98; df = 66, 2019; P <
0.0001; Table 4-5). Larvae completed development to adult significantly faster as temperature increased. At 15 °C, larvae completed development to adult more slowly than at higher temperatures regardless of population and ultimate instar attained. However, development time at
25 and 30 °C was not significantly different between populations. Larvae pupated after various numbers of instars, ranging from four to eight, but most C1 underwent six instars, while R1 most commonly reached only five instars. Seven and eight instars occurred rarely and only at 15 and
30 °C, temperatures at which survival was lower. R1 was the only population to pupate after four instars. R1 completed development faster than C2.
4.4.3 Rate of Larval Development and Estimated Degree-Day Requirements
A linear relationship was evident between temperature and development rate for larvae from hatch to second instar in both R1 (F = 1862; df = 1, 455; P < 0.0001; Table 4-6; Figure 4-2) and C2 (F = 6716; df = 1, 479; P < 0.0001; Table 4-6; Figure 4-2); in general, larvae developed faster as temperature increased. Lower temperature thresholds calculated from this relationship indicated that the first instar of C2 has a slightly lower temperature threshold (7.77 °C) than that of R1 (8.51 °C). A significant relationship between development rate to adult eclosion and temperature was seen in all three models (Linear, Shi/performance, and Logan). Lower and upper developmental temperature thresholds obtained from different models were comparable.
As an example, the lower temperature thresholds for male C2 estimated from linear and
Shi/performance models were 7.10 and 7.24 °C, respectively. The models also indicated that C2 larvae had a lower temperature threshold than R1. However, the upper temperature thresholds for
81 larvae of these two populations were comparable. Also, the larval temperature thresholds for both sexes were similar.
R1 required fewer degree-days than C2 to reach the second instar and the difference in
DD requirements increased with the proportion of the population reaching that stage (Table 4-7;
Figure 4-3).To determine accumulated degree-days to reach adulthood by population and sex, we used the lower thresholds produced by the Shi/performance model (Table 4-6, Figure 4-4). R1 reached the adult stage with fewer degree-days than C2 (Table 4-7; Figure 4-5). Also, females had higher degree-day requirements than males (Table 4-7).
4.4.4 Pupal and Adult Body Weight of L. d. asiatica
Mean pupal weights were significantly affected by interaction of temperature, population, sex, and ultimate instar attained (F = 3.73; df = 120, 2379; P < 0.0001; Figure 4-6). Mean pupal weights of males were not significantly different from each other as a function of temperature, population and ultimate instar; however, variations in pupal weights were observed among females. Within each temperature, pupal weight was not significantly affected by population or by number of instars attained. However, differences were observed within populations when female weights were compared across temperatures. For example, female C2 reared at 25 °C that attained six instars were significantly heavier than C2 females reared at 15 °C that attained seven instars and C2 females reared at 30 °C that attained eight instars (Table 4-8). Females were considerably heavier than males; however, at 15 and 30 °C female weights were more comparable to males. The relationship between temperature and pupal weight for each sex and population is shown in Figure 4-6 (see Table 4-9 for model fit). The Tmin and Tmax temperatures for the C2 and R1 populations range between 9-11 °C and 34-36 °C, respectively. Temperature
82 had a stronger effect on female than male pupal weights and the C1 population tended to be heavier near the optimum temperature for both sexes.
The interaction between populations, temperature and sex was not significant (F = 0.57; df = 51, 1943; P < 0.99) but an interaction between temperature and sex did significantly affect mean adult weight (F = 116; df = 7, 1999; P < 0.0001; Figure 4-7). Adult females were significantly heavier than males. Male weight was not affected by temperature but females reared at 20 or 25 °C were significantly heavier than females reared at 30 or 15 °C.
4.4.5 Effect of Temperature and Population on Pupal and Adult Fitness
Both pupal mortality and proportions of deformed adults were affected by temperature, especially at 15 °C but sometimes also at 30 °C. Pupae that failed to eclose often appeared normal but some had attached exuvia or had very short or missing wing pads. Adult abnormalities included wings that failed to expand properly or stuck exuvia. Pupal mortality was significantly affected by the interaction between temperature and population (F = 4.16; df = 21, 280; P <
0.0001; Figure 4-8). Within the C1, C2, C3, and R2 populations the proportion of pupae that eclosed was similar across temperatures (15, 20, 25, and 30 °C). However, temperature significantly affected pupal mortality within the J1, J2, R1, and Sk populations. Within temperature, no difference in the proportion of pupae that died was evident among populations at
20 and 25 °C. However, at 15 °C, significantly more J2 pupae died than did pupae from the C1,
C3, Sk, and R2 populations. At 30 °C, J1, J2, and Sk also suffered significantly more pupal mortality than C1, C2, and R1.
The proportion of deformed adults was also significantly affected the interaction between temperature and population (F = 1.81; df = 21, 271; P < 0.0181; Figure 4-8). All populations had a significantly higher proportion of deformed adults when reared at 15 °C compared with 25 °C
83 except R2. R2 deformity was not affected by rearing temperature. The proportions of deformed adults was similar across populations at 20, 25 and 30 °C.
An interaction of temperature and population affected the mean proportion of embryonated eggs produced by females at 25 and 30 °C (F = 2.61; df = 6, 280; P < 0.0177; Table
4-10). When moths were reared at 25 °C until pupation and transferred to 30 °C for emergence and mating, the mean percentage of their embryonated eggs was significantly lower compared with eggs produced by females reared only at 25 °C. The proportion of embryonated eggs produced by females reared at 25 °C did not differ across populations. However, R1 produced a significantly greater proportion of embryonated eggs compared with other populations held at 25 then at 30 °C. In contrast, no eggs became embryonated when females were reared and mated at
30 °C. Females produced an average of 303.8 ± 71.96 eggs; 61.8 % of these eggs remained unembryonated while the remaining were only partially embryonated.
The estimated overall performance of Asian gypsy moth was significantly affected by an interaction of temperature and population (F = 4.50; df = 21, 280; P < 0.0001; Figure 4-9). In general, the estimated performance at 20 and 25 °C was not different among populations, except
R1, R2, and J2 had a significantly better performance at 25 °C than at 20 °C. Performance at 15 and 30 °C was consistently poorer than at 20 and 25 °C across all populations. Although most populations performed equally poorly at the highest and coldest temperatures, J2 had significantly reduced performance at the highest temperature.
4.5 Discussion
Temperature had a significant effect on the life history of Asian gypsy moth regardless of geographical origin. As expected, temperature significantly affected survivorship, development
84 rate, and fitness as evidenced by effects on moth fertility. Developmental rate increased with temperature from 10 to 25 °C for most populations and instar/stages, and declined at 30 °C.
Survival to adult declined at 15 and 30 °C compared to 20 and 25 °C. At 30 °C, fitness and fertility were also negatively affected. As expected, different populations responded differently to temperature. Populations from colder plant hardiness zones (2-5) tended to develop faster than those from warmer zones (7-8). However, the population from the coldest zone, R1, tolerated both the lower and upper temperatures evaluated better than the other populations.
Larvae experienced problems during molting at the highest and lowest temperatures tested (10 and 30 °C) leading to high mortality at these temperatures. Overall survivorship to pupa and adult stages was greatest at 20 and 25 °C and lowest at 30 °C. At 15 °C, larvae often successfully reached the pupal stage, however, a significant proportion of pupae failed to eclose to adult. Of those that did eclose, a large number were deformed, which indicates that although the moth can complete development at lower temperatures, higher temperatures are required for normal adult emergence. This finding suggests that Asian gypsy moth populations may not thrive in regions where temperatures stay above 30 or below 15 °C after egg hatch, but the degree of the impact will depend on the population. Based on our results on survivorship and deformity, the
Japanese population appeared to struggle more at extreme temperatures (15 and 30 °C) than the
Chinese and Russian populations.
Across the populations tested, Asian gypsy moth caterpillars required 55 – 85 days at
10 °C and 4 – 6 days at 30 °C to reach second instar. Similarly, the larvae completed development to adult after a significantly longer period at 15 °C than at higher temperatures.
Some variation across populations was evident when developmental times to adult were compared. The Russian R1 population developed the fastest and the Chinese C2 population the slowest. Also, cold hardiness zones would suggest that R1 would be more cold tolerant than C2 and should survive minimum temperatures throughout North America except certain locations in
85 Alaska and Northern Canada, whereas C2 may be limited in its ability to tolerate minimum temperatures across the same range (Table 4-1). Surprisingly, the Russian population also tolerated the highest temperature better than the Chinese population. Females from this Russian population (R1) also have the highest proportion of strong fliers (Keena et al. 2008), a portion of their eggs have lower thermal requirements for egg hatch (Keena 2015), and females produced a significantly greater proportion of embryonated eggs compared with other populations held at 25 then at 30 °C. Thus, the R1 population may be resilient enough to establish outside its native range, spread rapidly, and potentially cause greater damage than the other Asian populations evaluated.
The Asian gypsy moth populations developed to the second instar at a rate similar to the
North American gypsy moth population at 15, 20, 25 and 30 °C (Casagrande et al. 1987).
However, at lower temperatures the North American population appears to develop faster to second instar than the Asian gypsy moth. At 10 °C the fastest growing Asian gypsy moths (R1) required more than 50 days to reach second instar, while the North American population took only 38.06 ± 3.43 (mean ± SD) days. However, at 20, 25 and 30 °C, the R1 Russian population that pupated after four instars completed development to adult faster than is reported for the
North American population, while the C2 Chinese population developed slower. Thus, it appears that detection of Asian populations may require expanding the normal trapping season used for the North American populations to both earlier and later dates than the current standard.
However, care should be taken when extrapolating laboratory data based on constant temperatures and artificial diet to natural environments since many other factors can influence development, such as host foliage and topography. Also, the larvae from the North American population used in this comparison were reared on white oaks instead of artificial diet, which would also affect developmental rates.
86 As expected, the rate of development of Asian gypsy moth larvae increased with rearing temperature until it reached the upper temperature threshold for development. The upper and lower temperature thresholds of Asian gypsy moth are predicted to be 30–31 °C and 7–9 °C, respectively. Variations in lower and upper temperature thresholds by population were minor. All of the models used in this study predicted similar temperature thresholds for Asian gypsy moth by life-stage and population.
The second instar and adult are critical stages of the gypsy moth life cycle, as monitoring and control treatments must coincide with them. Therefore, degree-day requirements to reach these stages were determined to predict the timing for control and monitoring activities. The fastest and slowest developing populations were selected for degree-day calculation to represent the range of variation evident in the tested populations. Degree-day estimations were determined for each sex separately, even though we did not observe significant differences in development time by sex within any temperature and population. However, despite insignificant differences, we acknowledge that male Asian gypsy moth usually completed development a few days ahead of females. For example, after egg hatch, at 30 °C, the first male and female R1 emerged after 29 and 32 days while C2 emerged after 45 and 48 days, respectively. The pattern of early male emergence was consistent across all holding temperatures and the sex differences between first emergence dates increased at lower temperatures. Therefore, during monitoring for flight and application of pheromone traps, the degree-day model for males should be used to ensure capture of early emerging male moths.
Asian gypsy moth populations had a variable number of molts when reared at different temperatures, and the effects also varied by sex. In general, females pupated at the sixth instar and males pupated at the fifth; however, males had 4–7 instars and females 5–8 instars. The variation in the total number of molts by sex is in alignment with North American gypsy moths
(Casagrande et al. 1987). The only population with individuals that could pupate at the fourth
87 instar was the R1 from Russia. The moths tended to complete their development faster when they pupated at earlier rather than later instars. This suggests that the ability to pupate in an earlier instar may be an adaptation that enabled R1 to develop faster than the other populations.
Additionally, during the study supernumerary molts (7 or 8) were observed only at 15 and 30 °C, which could indicate thermal stress at those temperatures (Neven and Rehfield 1995, Neven
2000). Generally, the larvae that underwent the unusual seventh or eighth molt before pupation tended to be smaller and darker in color, which has been reported for other insects that are experiencing thermal stress.
Female Asian gypsy moth fitness was compromised when rearing was at 15 and 30 °C.
These females had reduced pupal and adult weights compared with those reared at 20 and 25 °C.
The lower weights correlate with lower fecundity and reduced fitness at these temperatures.
Female Asian gypsy moths produced fewer eggs when reared at 30 °C compared with 25 °C; these eggs were also unembryonated or only partially embryonated with little chance for eclosion.
These findings suggest that Asian gypsy moth development and survival may be limited by hot, prolonged summer temperatures in the southern United States. Also, at lower temperatures
(10 °C), Asian gypsy moth populations developed very slowly, indicating these subspecies have a lower probability of establishing in areas where the temperature stays below 10 °C during the gypsy moth larval development time frame.
Asian gypsy moth is considered a high risk pest in North America. To prevent its establishment, aggressive control and eradication treatments are employed following detection.
Timing is critical for monitoring and eradication of this pest and so far it has been based on North
American population developmental data or trapping data in the native range, which likely does not provide adequate estimates. Our data can provide better estimates for these critical time points and be used to develop an Asian gypsy moth-specific phenology model, which is in progress. Such a model would benefit pest managers in modifying management strategies and
88 developing risk assessments. Also, these data can be combined with climate data to predict the current and future (under different climate change scenarios) potential distributions of Asian gypsy moth populations throughout the world.
89
Table 4-1. Approximate location (latitude and longitude), designation of the source population, collection date, and laboratory generation of gypsy moth evaluated in this study, arranged by latitude from north to south.
Collection Collection Laboratory Plant hardiness
Population Country location datea generation Latitude Longitude zoneb
R1 Russia Shira, Khakassi Aug-1994 30 54.41ºN 90.00 ºE 2
C1 China Harbin, Aug-2012 3 45.78ºN 126.61 ºE 4
Heilongjiang (Oct- 2012)
R2 Russia Mineralni, Aug-1992 30 44.10ºN 133.15 ºE 5
Primorski
C3 China Sandeli, Sep-2011 4 41.51ºN 122.37 ºE 6
Liaoning (Oct-2012)
C2 China Yanzikou, Aug-2011 4 40.32ºN 116.15 ºE 7
Beijing, (Oct-2012)
Takizawa,
Morika,
Nishine
90
J1 Japan Hachimantai Oct-2005 15 39.73ºN 141.08 ºE 7a
City, Northern (Nov-2014)
Iwate District,
Honshu
Sk Korea Samhwa-Dong, Aug-2009 9 37.49ºN 129.06 ºE 6
Gangovon-Do (Nov-2014)
J2 Japan Nagoya, Mar-1996 28 35.15ºN 137.08 ºE 8b
Honshu
a Date in parentheses indicates when the population was received at the Ansonia quarantine facility. b Plant hardiness zones according to the Davis Landscape Architecture.
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Table 4-2. Mathematical models used to describe relationships between temperature and developmental rate of Asian gypsy moths. Model Function1 Reference
Linear regression2 푟 = 푎푇 + 푏
푇 − 푇 Logan et al. 1976 Logan3 푟 = 푎(exp(푐푇) − exp (푐푇 − ( 푚푎푥 )) 푚푎푥 푏
4 Shi/ performance 푟 = 푐(1 − exp(−푎(푇 − 푇푚푖푛))) ∗ (1 − exp(푏(푇 − 푇푚푎푥))) Shi et al. 2011
5 Lysyk 푦 = 푎(푇 − 푇푚푖푛)(푇푚푎푥 − 푇) Lysyk 2001
1 For each function, r represents the rate of development and T represents holding temperature. Tmin and Tmax represent minimum and maximum temperature thresholds, respectively. 2 The parameters a and b in a linear regression represent slope and intercept of a line, respectively. 3 In the Logan model, a represents the developmental rate at a base temperature above the lower developmental threshold, b is the high temperature boundary layer, and c is the rate increase to optimum temperature. 4 In the Shi/performance model, parameters a and b represent rate of increase rate and rate of decrease compared to optimum temperature, and c is a constant. 5 In the Lysyk model, y represent pupal weight parameters and a is an empirical constant.
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Table 4-3. Proportion of each population of Asian gypsy moth categorized (using HSPLIT in SAS) into each of the nodes based on the developmental criteria listed for each temperature. Criteria for 10 °C are based on days in the first instar (D1), while the other temperatures are based on days to adult (DA).
% Individuals from each Population in the Node Temperature Nodes R1 R2 C3 Sk J1 J2 C1 C2
10 D1 < 44.1 d 28.6 29.4 4.2 0.0 1.7 9.2 0.0 0.0
51.1> D1 ≥ 44.1d 22.6 23.5 19.7 2.5 8.6 42.5 14.7 8.2
73.1> D1 ≥51.1 d 46.4 38.8 62.0 87.5 87.9 48.3 52.9 61.2
D1 ≥ 73.1 d 2.4 8.2 14.1 10.0 1.7 0.0 32.4 30.6
15 DA < 79.1 d 89.6 50.5 8.9 12.4 17.8 0.0 16.7 12.8
99.2> D1 ≥ 79.1d 10.4 46.5 76.7 75.3 76.7 41.3 76.0 80.9
DA ≥ 99.2 d 0.0 3.0 14.4 12.4 5.5 58.7 7.3 6.4
20 DA < 44.1 d 69.3 4.3 0.0 0.0 0.0 0.0 1.1 0.0
51.1> DA ≥ 44.1d 26.7 54.8 4.3 1.1 19.0 0.0 10.0 0.0
73.1> DA ≥51.1 d 1.3 33.3 34.0 37.0 53.2 8.3 38.9 24.2
DA ≥ 73.1 d 2.7 7.5 61.7 62.9 27.8 91.7 50.0 75.8
93
25 DA < 43.0 d 97.8 33.7 0.0 2.4 25.0 0.0 0.0 0.0
48.0> DA ≥ 43.0d 2.2 52.8 35.1 50.6 59.2 15.7 46.3 7.5
51.0> DA ≥48.0 d 0.0 9.0 43.6 24.1 10.5 51.7 31.6 15.0
DA ≥ 51.0 d 0.0 4.5 21.3 22.9 5.3 32.6 22.1 77.5
30 DA < 24.1 d 52.2 7.6 0.0 0.0 3.8 0.0 0.0 0.0
32.1> D1 ≥ 24.1d 39.1 71.2 16.4 69.7 80.8 0.0 23.5 6.3
DA ≥ 32.1 d 8.7 21.2 83.6 30.3 15.4 100.0 76.5 93.7
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Table 4-4. Mean [± SE (n)] time spent (d) by L. dispar asiatica in the first instar at different temperatures by sex and population.
Temperature (°C) Sex Population 10 15 20 25 30
F R1 50.9 ± 1.91a (14) 14.4 ± 0.57b (42) 7.7 ± 0.41b (46) 4.9 ± 0.31bc (49) 4.2 ± 0.57bc (14)
F C2 66.6 ± 4.73a (4) 14.7 ± 0.48b (63) 9.0 ± 0.39b (57) 6.1 ± 37bc (49) 4.9 ± 0.78bc (9)
M R1 51.7 ± 1.86a (15) 13.5 ± 0.50b (54) 7.4 ± 0.40b (46) 4.9 ± 0.34bc (48) 3.9 ± 0.40c (32)
M C2 78.33 ± 5.13a (3) 16.5 ± 0.73b (31) 10.0 ± 0.52b (37) 6.5 ± 0.42bc (36) 5.7 ± 0.51bc (23)
Means followed by a different letter are significantly different from each other at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is the number of survivors.
95
Table 4-5. Mean [± SE (n)] time spent (d) by L. dispar asiatica from egg hatch to adult at different temperatures, by population and ultimate instar attained.
Ultimate Temperature ( °C) Population Instar 15 20 25 30
C2 5 110.5 ± 3.4 ab (10) 74.1 ± 1.5 cd (36) 52.2 ± 1.3 de (35) 47.9 ± 3.1 def (5)
C2 6 120.1 ± 1.5 a (60) 78.2 ± 1.3 c (55) 54.0 ± 1.1 de (45) 52.4 ± 1.7 de (18)
C2 7 137 ± 11.7 a (1) NA NA 59.5 ± 5.5 cde (2)
C2 8 NA NA NA 71.0 ± 6.0 cd (2)
R1 4 NA 58.3 ± 5.5 cde (2) 36.3 ± 2.0 fg (9) 30.9 ± 2.8 g (4)
R1 5 98.3 ± 1.5 b (46) 61.0 ± 0.9 cd (71) 39.6 ± 0.7 fg (81) 34.7 ± 1.1 g (27)
R1 6 112.9 ± 7.5 ab (2) 72.0 ± 6.0 cd (2) 44.8 ± 4.7 defg (2) 40.8 ± 2.9 efg (5)
R1 7 NA NA NA 44.4 ± 6.6 defg (1)
Means followed by different letters are significantly different from each other at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is the number of survivors.
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Table 4-6. Parameter values for linear and non-linear models used to describe the relationship between temperature (°C) and developmental rate of Asian gypsy moth life stages by population and sex.
1 2 Development Period Temperature range Population Sex Model a b c Tmin Tmax To n AdjR
Days in 1st instar 10 to 30 C2 both Linear 0.0093 -0.072 NA 7.77 NA NA 455 0.80
10 to 30 R1 both Linear 0.0120 -0.102 NA 8.51 NA NA 479 0.93
Days to adult 15 to 25 C2 M Linear 0.0011 -0.008 NA 7.10 NA NA 89 0.96
15 to 25 C2 F Linear 0.0010 -0.007 NA 7.05 NA NA 148 0.96
15 to 25 R1 M Linear 0.0016 -0.015 NA 8.97 NA NA 107 0.97
15 to 25 R1 F Linear 0.0015 -0.013 NA 8.95 NA NA 104 0.97
15 to 30 C2 M Shi 0.002 2.33 0.558 7.24 30.7 28.9 112 0.93
15 to 30 C2 F Shi 0.0009 2.86 1.10 7.13 30.6 29.1 157 0.95
15 to 30 R1 M Shi 0.001 2.52 1.75 9.02 30.8 29.2 133 0.95
15 to 30 R1 F Shi 0.001 2.63 1.53 9.01 30.7 29.2 119 0.95
15 to 30 C2 M Logan 0.0028 0.087 0.077 NA 30.7 NA 112 0.92
15 to 30 C2 F Logan 0.0025 0.124 0.078 NA 30.14 NA 157 0.96
15 to 30 R1 M Logan 0.0027 0.145 0.091 NA 30.18 NA 133 0.96
15 to 30 R1 F Logan 0.0025 0.095 0.090 NA 30.11 NA 119 0.96
97
See methods for details of the analysis. 1 To represents the optimum temperature for development calculated from the Shi/Performance model.
Table 4-7. Estimated accumulated degree-days (± SE) required to reach second instar and adult emergence by L. d. asiatica larvae from egg hatch for 10, 50, 90 and 99% of the population. Life stage Degree-day (± SE) requirement for larvae (%) to reach different life stages after egg hatch 2 Population Sex R Adj reached 10% 50% 90% 99%
2nd instar R1 both 68.97 ± 0.47 81.49 ± 0.10 101.1 ± 1.69 125.7 ± 3.68 0.98
2nd instar C2 both 78.58 ± 1.33 106.0 ± 0.04 150.7 ± 1.96 205.4 ± 4.47 0.99
Adult R1 M 564.1 ± 2.31 619.3 ± 0.12 705.8 ± 3.95 813.8 ± 8.73 0.99
Adult R1 F 607.3 ± 1.88 657.4 ± 0.45 736.1 ± 4.13 834.2 ± 8.72 0.99
Adult C2 M 858.3 ± 4.75 936.6 ± 0.23 1060 ± 8.04 1213 ± 17.8 0.98
Adult C2 F 912.5 ± 1.98 979.6 ± 0.22 1084 ± 3.68 1216 ± 7.99 0.99
Lower temperature thresholds are presented in Table 6 for each life stage, population, and sex. The lower temperature threshold for adults 2 was obtained from Shi/performance model. R Adj value is based on the relationship between degree-days and cumulative proportion to adulthood using the Gompertz function, P = exp [- exp(- b * DD + a].
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Table 4-8. Mean [± SE (n)] pupal weight (g) of L. dispar asiatica at different temperatures, by population, ultimate instar attained and sex.
Ultimate Temperature ( °C) Population Sex instar 15 20 25 30
C2 5 M 0.9248 ± 0.05ghi 1.1852 ± 0.05defghi 1.4292 ± 0.06bcdefghi 0.8176 ± 0.08ghi
(18) (34) (32) (5)
C2 6 M 0.9561 ± 0.06fghi 1.1837 ± 0.16defghi 1.4502 ± 0.16bcdefghi 0.8729 ± 0.05ghi
(13) (3) (4) (16)
C2 7 M NA NA NA 0.9329 ± 0.22fghi (1)
C2 8 M NA NA NA 0.6531 ± 0.15ghi (1)
C2 5 F NA 2.7018 ± 0.44abcdef (2) 3.6803 ± 0.42abc (4) NA
C2 6 F 2.8048 ± 0.08abcde 3.5109 ± 0.11abc 4.1027 ± 0.14a 2.1987 ± 0.21abcdefgh
(62) (55) (45) (6)
C2 7 F 1.2615 ± 0.29cdefghi NA NA 2.1277 ± 0.35abcdefgh
(1) (2)
C2 8 F NA NA NA 1.3288 ± 0.30bcdefghi
(1)
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R1 4 M NA 0.6719 ± 0.09ghi (3) 0.8358 ± 0.06ghi (10) 0.446 ± 0.04i (6)
R1 5 M 0.7809 ± 0.02ghi (54) 0.8838 ± 0.03ghi (43) 1.1139 ± 0.04defghi (38) 0.5347 ± 0.03i (24)
R1 6 M NA NA NA 0.9778 ± 0.16fghi (2)
R1 5 F 1.8096 ± 0.07bcdefghi 2.7872 ± 0.10abcde 2.9876 ± 0.10abcde 1.5828 ± 0.12bcdefghi
(36) (42) (47) (9)
R1 6 F 2.2771 ± 0.21abcdefgh 3.1629 ± 0.37abcde 2.5983 ± 0.42abcdefg 1.6234 ± 0.19bcdefghi
(6) (4) (2) (4)
R1 7 F NA NA NA 2.4189 ± 0.56abcdefgh
(1)
Means followed by different letters are significantly different from each other at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is the number of survivors.
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Table 4-9. Parameter values for Lysyk models used to describe the relationship between temperature (°C) and pupal weight (g) of Asian gypsy moth by population and sex.
2 Temperature range Population Sex a Tmin Tmax n AdjR
15 to 30 C2 M 0.0080 9.62 35.6 126 0.43
15 to 30 C2 F 0.0214 8.98 35.9 175 0.32
15 to 30 R1 M 0.0058 8.52 34.8 177 0.26
15 to 30 R1 F 0.0220 10.6 34.1 150 0.52
See methods for details of the analysis.
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Table 4-10. Percentage (Mean + SE [n]) of embryonated eggs of L. dispar asiatica populations by temperatures.
Temperature (°C) Population 25 30 *
C1 84 ± 3.47a (20) 13 ± 2.91c (21)
C2 81 ± 4.14a (20) 11 ± 2.57c (20)
C3 85 ± 3.4a (20) 13 ± 2.81c (23)
J1 86 ± 3.15a (20) 17 ± 3.39c (25)
R1 84 ± 3.62a (20) 43 ± 6.77b (20)
R2 81 ± 4.11a (20) 16 ± 3.25c (25)
Sk 82 ± 3.93a (20) 17 ± 3.78c (20)
Means followed by different letters are significantly different at P < 0.05 (Tukey-Kramer post hoc test). Number of evaluated egg masses is indicated in parentheses. * 30 °C data are from larvae reared at 25 °C until pupation and then moved to 30 °C for adult eclosion, mating and oviposition
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SI 1. Development time [(Mean ± SEM) n] spent as first instar by L. dispar asiatica at different temperatures by sex, and population. Temperature ( °C) Sex Population 10 15 20 25 30
F C1 63.2 ± 1.70a (23) 14.7 ± 0.54b (51) 9.02 ± 0.46b (41) 5.71 ± 0.45bc (33) 6.14 ± 0.66bc (15)
F C2 66.7 ± 4.73a (4) 14.7 ± 0.48b (63) 8.98 ± 0.40b (57) 6.07 ± 0.37bc (49) 4.88 ± 0.78bc (9)
F C3 59.6 ± 2.92a (7) 15.3 ± 0.56b (49) 10.4 ± 0.50b (41) 6.63 ± 0.42b (39) 6.60 ± 0.47bc (33)
F J1 59.9 ± 2.15a (13) 15.5 ± 0.63b (39) 9.30 ± 0.47b (43) 5.47 ± 0.39bc (39) 4.25 ± 1.03bc (10)
F J2 54.0 ± 4.25a (3) 15.6 ± 0.61b (42) 9.44 ± 0.43b (50) 6.39 ± 0.37bc (43) NA
F R1 50.9 ± 1.91a (14) 14.4 ± 0.58b (42) 7.67 ± 0.41b (46) 4.86 ± 0.31bc (49) 4.15 ± 0.57bc (14)
F R2 54.5 ± 1.97a (14) 15.0 ± 0.53b (54) 9.51 ± 0.43b (51) 6.40 ± 0.39bc (47) 5.39 ± 0.55bc (25)
F Sk 63.5 ± 1.48a (29) 17.2 ± 0.66b (40) 10.1 ± 0.42b (56) 6.28 ± 0.40bc (43) 4.00 ± 2.00bc (5)
M C1 68.2 ± 2.13a (15) 14.8 ± 0.57b (45) 9.49 ± 0.42b (53) 6.72 ± 0.32b (64) 6.33 ± 0.44bc (36)
M C2 78.3 ± 5.13a (3) 16.5 ± 0.73b (31) 10.0 ± 0.52b (37) 6.47 ± 0.42bc (36) 5.72 ± 0.51bc (23)
M C3 68.6 ± 2.02a (17) 15.8 ± 0.62b (41) 10.3 ± 0.44b (53) 7.50 ± 0.37b (56) 6.15 ± 0.49bc (34)
M J1 61.3 ± 1.43a (30) 16.4 ± 0.69b (34) 9.38 ± 0.48b (40) 5.83 ± 0.38bc (42) 5.00 ± 0.79bc (16)
M J2 48.0 ± 4.91a (2) 17.3 ± 0.73b (33) 10.3 ± 0.49b (42) 6.32 ± 0.37bc (48) 5.00 ± 1.29bc (7)
M R1 51.7 ± 1.86a (15) 13.5 ± 0.50b (54) 7.39 ± 0.40b (46) 4.93 ± 0.34bc (48) 3.96 ± 0.40c (32)
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M R2 57.1 ± 2.18a (12) 14.2 ± 0.56b (45) 10.1 ± 0.45b (49) 6.85 ± 0.38b (48) 5.63 ± 0.40bc (41)
M Sk 63.8 ± 1.23a (37) 17.5 ± 0.60b (49) 9.80 ± 0.50b (40) 6.81 ± 0.40b (46) 4.88 ± 0.55bc (28)
Means followed by different letter are significantly different from each other across the table at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is based on number of survivors.
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SI 2. Development time [(Mean ± SEM) n] spent as second instar by L. dispar asiatica at different temperatures by sex, and population. Temperature (C) Sex Population 10 15 20 25 30
F C1 38.5 ± 1.60abc (16) 9.42 ± 0.43d (51) 6.04 ± 0.38d (41) 3.90 ± 0.35de (33) 2.94 ± 0.46e (15)
F C2 41.0 ± 6.40abc (2) 9.19 ± 0.38d (63) 5.93 ± 0.32d (57) 3.71 ± 0.29de (49) 3.02 ± 0.61de (9)
F C3 46.8 ± 2.79a (6) 8.85 ± 0.42d (49) 5.54 ± 0.37de (41) 3.77 ± 0.31de (39) 3.50 ± 0.34de (33)
F J1 24.4 ± 1.37cd (13) 10.4 ± 0.51d (39) 6.07 ± 0.38d (43) 3.80 ± 0.32de (39) 3.02 ± 0.87de (10)
F J2 37.5 ± 4.3abc (2) 9.83 ± 0.48d (42) 6.36 ± 0.36d (50) 3.91 ± 0.30de (43) NA
F R1 33.3 ± 1.54bcd (14) 8.86 ± 0.46d (42) 5.74 ± 0.35de (46) 3.25 ± 0.25de (49) 3.38 ± 0.51de (14)
F R2 38.9 ± 1.80abc (12) 9.27 ± 0.41d (54) 5.60 ± 0.33de (51) 3.41 ± 0.28de (47) 3.02 ± 0.41de (25)
F Sk 23.8 ± 0.92cd (28) 10.2 ± 0.50d (40) 6.15 ± 0.33d (56) 3.77 ± 0.31de (43) 3.02 ± 1.74de (5)
M C1 41.3 ± 2.43ab (7) 9.66 ± 0.46d (45) 6.20 ± 0.34d (53) 3.96 ± 0.25de (64) 3.23 ± 0.31de (36)
M C2 NA 9.32 ± 0.55d (31) 6.31 ± 0.41d (37) 3.78 ± 0.32de (36) 3.06 ± 0.37de (23)
M C3 35.2 ± 1.79abc (11) 9.28 ± 0.48d (41) 5.83 ± 0.33de (53) 3.98 ± 0.27de (56) 3.22 ± 0.35de (34)
M J1 22.5 ± 0.88cd (29) 10.5 ± 0.55d (34) 6.08 ± 0.39d (40) 3.81 ± 0.30de (42) 3.14 ± 0.62de (16)
M J2 40.5 ± 4.50abc (2) 10.2 ± 0.56d (33) 6.50 ± 0.39d (42) 3.93 ± 0.29de (48) 3.34 ± 1.05de (7)
M R1 39.2 ± 1.67abc (14) 8.71 ± 0.40d (54) 5.44 ± 0.34de (46) 3.15 ± 0.28de (48) 3.17 ± 0.36de (32)
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M R2 42.6 ± 2.06ab (10) 9.46 ± 0.46d (45) 5.81 ± 0.34de (49) 3.59 ± 0.28de (48) 2.98 ± 0.29e (41)
M Sk 24.8 ± 0.83cd (Sk) 10.52 ± 0.46d (49) 6.05 ± 0.39d (40) 3.91 ± 0.30de (46) 3.07 ± 0.42de (28)
Means followed by different letter are significantly different from each other across the table at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is based on number of survivors.
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SI 3. Development time [(Mean ± SEM) n] spent as third instar by L. dispar asiatica at different temperatures by sex, and population.
Temperature (C) Sex Population 10 15 20 25 30
F C1 NA 10.2 ± 0.44a (51) 6.22 ± 0.39a (41) 3.84 ± 0.35a (33) 3.59 ± 0.51a (15)
F C2 NA 9.70 ± 0.39a (63) 6.61 ± 0.34a (57) 3.93 ± 0.30a (49) 3.10 ± 0.62a (9)
F C3 NA 10.5 ± 0.46a (49) 6.54 ± 0.40a (41) 3.63 ± 0.31a (39) 3.85 ± 0.36a (34)
F J1 21.4 ± 1.28a (13) 10.9 ± 0.52a (39) 5.86 ± 0.37a (43) 3.75 ± 0.32a (39) 3.12 ± 0.88a (10)
F J2 NA 11.7 ± 0.53a (42) 6.56 ± 0.37a (50) 4.10 ± 0.31a (43) NA
F R1 44.1 ± 3.83a (3) 10.5 ± 0.50a (42) 6.22 ± 0.37a (46) 3.57 ± 0.27a (49) 4.14 ± 0.56a (14)
F R2 42.2 ± 3.75a (3) 11.3 ± 0.46a (54) 6.33 ± 0.35a (51) 3.77 ± 0.30a (47) 3.46 ± 0.44a (25)
F Sk 23.0 ± 1.00a (23) 10.5 ± 0.51a (40) 6.07 ± 0.33a (56) 3.67 ± 0.62a (43) 3.00 ± 0.87a (5)
M C1 NA 11.4 ± 0.50a (45) 7.23 ± 0.37a (53) 4.05 ± 0.25a (64) 3.46 ± 0.32a (36)
M C2 NA 10.8 ± 0.59a (31) 7.19 ± 0.44a (37) 4.13 ± 0.33a (36) 3.59 ± 0.40a (23)
M C3 NA 10.5 ± 0.50a (41) 7.12 ± 0.37a (53) 3.95 ± 0.27a (56) 4.31 ± 0.41a (34)
M J1 22.5 ± 0.88cd (29) 10.5 ± 0.55d (34) 6.08 ± 0.39d (40) 3.81 ± 0.30de (42) 3.14 ± 0.62de (16)
M J2 NA 12.8 ± 0.62a (33) 7.33 ± 0.42a (42) 4.19 ± 0.30a (48) 4.32 ± 1.19a (7)
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M R1 36.0 ± 6.00a (2) 9.51 ± 0.42a (54) 5.60 ± 0.34a (46) 3.60 ± 0.29a (48) 3.42 ± 0.38a (32)
M R2 41.6 ± 4.56a (2) 11.4 ± 0.50a (45) 6.49 ± 0.36a (49) 3.76 ± 0.29a (48) 3.63 ± 0.32a (41)
M Sk 22.0 ± 0.85a (30) 11.6 ± 0.49a (49) 6.56 ± 0.40a (40) 4.02 ± 0.30a (46) 3.36 ± 0.44a (28)
Means followed by different letter are significantly different from each other across the table at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is based on number of survivors.
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SI 4. Development time [(Mean ± SEM) n] spent as fourth instar by L. dispar asiatica at different temperatures by sex, and population. Temperature (C) Sex Population 10 15 20 25 30
F C1 NA 12.7 ± 0.50ab (51) 7.19 ± 0.42ab (41) 4.51 ± 0.38b (33) 6.02 ± 0.66b (15)
F C2 NA 11.8 ± 0.43ab (63) 7.14 ± 0.35ab (57) 4.89 ± 0.33b (49) 6.35 ± 0.89ab (9)
F C3 NA 12.3 ± 0.50ab (49) 7.03 ± 0.41ab (41) 4.18 ± 0.33b (39) 4.56 ± 0.39b (33)
F J1 18.0 ± 2.45a (3) 12.1 ± 0.56ab (39) 7.09 ± 0.41ab (43) 5.00 ± 0.37b (39) 3.97 ± 0.99b (10)
F J2 NA 15.0 ± 0.60ab (42) 7.54 ± 0.39ab (50) 4.34 ± 0.32b (43) NA
F R1 NA 12.8 ± 0.55ab (42) 7.05 ± 0.39ab (46) 4.51 ± 0.30b (49) 4.10 ± 0.56b (14)
F R2 NA 12.0 ± 0.47ab (54) 7.26 ± 0.38ab (51) 4.36 ± 0.31b (47) 4.92 ± 0.52b (25)
F Sk 21.2 ± 3.25a (2) 12.1 ± 0.55ab (40) 7.18 ± 0.36ab (56) 4.39 ± 0.33b (43) 6.11 ± 2.47ab (5)
M C1 NA 15.2 ± 0.58ab (45) 8.42 ± 0.40ab (53) 5.75 ± 0.30b (64) 7.16 ± 0.47ab (36)
M C2 NA 13.6 ± 0.66ab (31) 8.63 ± 0.48ab (37) 6.26 ± 0.41ab (36) 7.63 ± 0.59ab (23)
M C3 NA 12.3 ± 0.54ab (41) 8.00 ± 0.39ab (53) 5.31 ± 0.30b (56) 4.21 ± 0.40b (34)
M J1 24.0 ± 4.89a (1) 15.9 ± 0.68ab (34) 8.33 ± 0.46ab (40) 4.84 ± 0.34b (42) 4.87 ± 0.78b (16)
M J2 NA 17.7 ± 0.73a (33) 9.81 ± 0.48ab (42) 6.47 ± 0.37ab (48) 6.33 ± 1.45ab (7)
M R1 NA 11.7 ± 0.47ab (54) 6.97 ± 0.39ab (46) 5.16 ± 0.35b (48) 4.99 ± 0.46b (32)
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M R2 NA 12.2 ± 0.52ab (45) 7.08 ± 0.38ab (49) 4.29 ± 0.30b (48) 5.28 ± 0.39b (41)
M Sk 16.0 ± 3.99ab (2) 13.8 ± 0.53ab (49) 8.36 ± 0.46ab (40) 5.41 ± 0.35b (46) 5.58 ± 0.57b (28)
Means followed by different letter are significantly different from each other across the table at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is based on number of survivors.
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SI 5. Development time [(Mean ± SEM) n] spent as fifth instar by L. dispar asiatica at different temperatures by sex, and population. Temperature (C) Sex Population 15 20 25 30
F C1 14.9 ± 0.58ab (51) 9.18 ± 0.49bc (41) 6.06 ± 0.44c (33) 7.36 ± 0.72bc (15)
F C2 14.6 ± 0.54ab (63) 10.1 ± 0.43bc (57) 8.23 ± 0.43bc (49) 8.00 ± 1.00bc (9)
F C3 15.7 ± 0.61ab (49) 9.02 ± 0.47bc (41) 6.00 ± 0.40c (39) 6.07 ± 0.45c (33)
F J1 15.5 ± 0.74ab (39) 9.78 ± 0.49bc (43) 8.86 ± 0.50bc (39) 7.50 ± 1.37bc (10)
F J2 24.0 ± 2.83a (42) 9.76 ± 0.54bc (50) 6.45 ± 0.39c (43) NA
F R1 23.8 ± 1.01a (42) 16.5 ± 0.67ab (46) 10.5 ± 0.47bc (49) 7.46 ± 0.76bc (14)
F R2 16.7 ± 0.80ab (54) 14.1 ± 0.55ab (51) 7.58 ± 0.42bc (47) 6.83 ± 0.62c (25)
F Sk 14.9 ± 0.74ab (40) 9.98 ± 0.43bc (56) 7.46 ± 0.44bc (43) 11.0 ± 3.31abc (5)
M C1 19.8 ± 0.73a (45) 14.9 ± 0.54ab (53) 10.8 ± 0.41bc (64) 9.09 ± 0.52bc (36)
M C2 18.7 ± 0.94a (31) 16.9 ± 0.68ab (37) 12.5 ± 0.59ab (36) 9.59 ± 0.66bc (23)
M C3 16.5 ± 0.69ab (41) 13.6 ± 0.51ab (53) 10.3 ± 0.43bc (56) 5.35 ± 0.45c (34)
M J1 25.6 ± 1.01a (34) 15.8 ± 0.64ab (40) 11.2 ± 0.52ab (42) 10.1 ± 1.13bc (16)
M J2 25.0 ± 2.04a 9 (33) 17.6 ± 0.67a (42) 11.9 ± 0.50ab (48) 7.00 ± 1.53bc (7)
M R1 21.0 ± 0.93a (54) 14.0 ± 0.61ab (43) 10.3 ± 0.54bc (38) 7.90 ± 0.63bc (26)
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M R2 22.1 ± 0.82a (45) 14.2 ± 0.54ab (49) 10.2 ± 0.47bc (48) 8.83 ± 0.50bc (41)
M Sk 22.1 ± 0.80a (49) 16.2 ± 0.64ab (40) 10.2 ± 0.48bc (46) 9.65 ± 0.75bc (28)
Means followed by different letter are significantly different from each other across the table at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is based on number of survivors.
112
SI 6. Development time [(Mean ± SEM) n] spent as sixth instar by L. dispar asiatica at different temperatures and population Temperature (C) Population 15 20 25 30
C1 24.2 ± 0.58bc (70) 17.6 ± 0.63cd (45) 12.5 ± 0.56de (40) 9.71 ± 0.53e (34)
C2 25.6 ± 0.584b (76) 19.3 ± 0.58cd (58) 15.2 ± 0.56de (49) 11.6 ± 0.66de (27)
C3 24.5 ± 0.54bc (81) 16.8 ± 0.53cd (59) 12.0 ± 0.50de (49) 9.48 ± 0.39e (63)
J1 25.5 ± 0.83bc (37) 17.7 ± 0.72cd (34) 12.9 ± 0.78de (21) 11.8 ± 1.40de (6)
J2 33.2 ± 0.82a (49) 18.7 ± 0.60cd (52) 13.0 ± 0.52de (48) 8.66 ± 1.20e (6)
R1 23.5 ± 1.97bcd (6) 15.5 ± 1.97cde (4) 10.0 ± 2.24de (2) 8.43 ± 1.09e (7)
R2 23.7 ± 0.71bc (47) 16.1 ± 0.94cd (18) 11.5 ± 0.63de (29) 9.63 ± 0.71e (19)
Sk 25.3 ± 0.68bc (55) 17.7 ± 0.61cd (48) 13.1 ± 0.58de (39) 11.9 ± 1.15de (9)
Means followed by different letter are significantly different from each other across the table at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is based on number of survivors.
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SI 7. Development time [(Mean ± SEM) n] spent as pupa by L. dispar asiatica at different temperatures by sex, and population. Temperature ( °C) Sex Population 15 20 25 30
F C1 30.3 ± 0.83a (44) 17.9 ± 0.68a (39) 13.3 ± 0.65abc (31) 11.4 ± 0.90abc (14)
F C2 31.1 ± 0.79a (50) 19.5 ± 0.60a (55) 13.4 ± 0.55abc (45) 11.6 ± 1.30abc (7)
F C3 30.5 ± 0.85a (43) 17.8 ± 0.66a (41) 12.9 ± 0.58abc (38) 11.8 ± 0.63abc (30)
F J1 29.8 ± 1.03a (29) 17.5 ± 0.65a (41) 12.4 ± 0.60abc (35) 11.5 ± 1.70abc (4)
F J2 33.0 ± 3.32a (4) 18.8 ± 0.76a (33) 13.0 ± 0.56abc (42) NA
F R1 26.5 ± 1.07a (23) 17.0 ± 0.68a (37) 12.0 ± 0.49abc (46) 9.62 ± 0.86c (13)
F R2 30.4 ± 1.08a (26) 16.8 ± 0.60a (45) 13.0 ± 0.55abc (43) 10.7 ± 0.77bc (19)
F Sk 31.7 ± 1.08a (28) 18.0 ± 0.58a (53) 13.4 ± 0.59abc (39) 12.0 ± 0.60abc (33)
M C1 31.6 ± 0.92a (37) 15.7 ± 0.55a (51) 14.2 ± 0.47abc (64) 12.4 ± 0.79abc (20)
M C2 31.9 ± 1.23a (21) 17.5 ± 0.69a (36) 14.4 ± 0.63abc (35) 12.6 ± 0.70abc (26)
M C3 31.8 ± 0.95a (35) 15.9 ± 0.55a (53) 14.2 ± 0.50abc (56) 12.0 ± 1.22abc (8)
M J1 32.7 ± 1.14a (25) 17.0 ± 0.65a (38) 13.8 ± 0.58abc (41) 11.7 ± 1.97abc (3)
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M J2 33.3 ± 2.35a (6) 18.6 ± 0.69a (39) 14.6 ± 0.56ab (47) 10.3 ± 0.65c (24)
M R1 28.4 ± 1.09a (25) 14.1 ± 0.60abc (38) 12.0 ± 0.53abc (46) 11.6 ± 0.58abc (35)
M R2 31.5 ± 0.98a (33) 14.6 ± 0.55ab (48) 13.3 ± 0.54abc (46) 11.4 ± 0.85abc (16)
M Sk 34.1 ± 1.00a (35) 16.5 ± 0.65a (39) 14.4 ± 0.57abc (44) 12.0 ± 0.60abc (33)
Means followed by different letter are significantly different from each other across the table at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is based on number of survivors. Time spent as pre-pupae by L. dispar asiatica was consistent among populations within temperature. Mean development time spent as a pre- pupa at 15, 20, 25, and 30 °C were 3.32 ± 0.07, 2.05 ± 0.53, 1.27 ± 0.04 and 1.14 ± 0.06 days, respectively.
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SI 8. Total development time [(Mean ± SEM) n] of L. dispar asiatica from egg hatch to adult at different temperatures by population, and ultimate instar.
Ultimate Temperature (C) Population instar 15 20 25 30
C1 5 111.9 ± 2.2 cd (6) 71.1 ± 1.3 def (35) 47.4 ± 0.9 fghij (42) 43.8 ± 1.7 efghi (5)
C1 6 120.6 ± 1.5 abcd (3) 76.2 ± 1.3 def (37) 51.9 ± 1.2 fghij (47) 50.2 ± 1.4 fghij (2)
C1 7 126.6 ± 7.9 a (1) NA NA 57.9 ± 3.4 efghij (1)
C2 5 109.7 ± 3.3 d (10) 73.6 ± 1.5 efg (36) 51.9 ± 1.2 fghij (35) 47.5 ± 3.1 fghij (5)
C2 6 120.7 ± 1.4 cd (60) 78.7 ± 1.2 def (55) 54.4 ± 1.1 fghij (45) 52.3 ± 1.7 fghij (18)
C2 7 138.0 ± 11.7 abcd (1) NA NA 59.5 ± 5.5 efgh (2)
C2 8 NA NA NA 71 ± 6 efg (2)
C3 5 114.2 ± 4.1 cd (7) 71.9 ± 1.5 efg (35) 48.2 ± 1 fghij (46) 46.4 ± 3.9 fghij (3)
C3 6 121.0 ± 1.4 cd (63) 76.9 ± 1.1 def (59) 50.8 ± 1 fghij (48) 47 ± 1.1 fghij (38)
C3 7 137.7 ± 4.8 abcd (6) NA NA 54.2 ± 2 fghij (14)
C3 8 171.3 ± 9.2 ab (2) NA NA 75.5 ± 8.7 defg (1)
J1 5 118.5 ± 2.1 cd (27) 69.5 ± 1.2 efg (46) 44.7 ± 0.9 fghij (57) 39.4 ± 2.1 hij (9)
116
J1 6 120.2 ± 2.2 cd (26) 74.8 ± 1.5 efg (33) 49.1 ± 1.6 fghij (19) 43.7 ± 3.8 ghij (3)
J1 7 163.8 ± 12.8 abc (1) NA NA NA
J2 5 124.6 ± 4.6 d (23) 77.2 ± 1.5 efg (47) 49.9 ± 1.1 fghij (57) 47.8 ± 4.9 ghij (16)
J2 6 134.3 ± 6.7 cd (56) 79.2 ± 1.5 def (43) 51.8 ± 1.1 fghij (38) 56.4 ± 7.5 fghij (26)
J2 7 198.6 ± 14.1 bcd (2) NA NA NA
R1 4 NA 57.9 ± 5.4 efghi (2) 36 ± 2 j (9) 30.7 ± 2.8 j (4)
R1 5 98.2 ± 1.5 de (46) 61 ± 0.9 efg (71) 39.6 ± 0.7 hij (81) 34.6 ± 1.1 j (27)
R1 6 113.7 ± 7.5 cd (2) 72.5 ± 6 efg (2) 45.2 ± 4.7 fghij (2) 41 ± 2.9 hij (5)
R1 7 NA NA NA 44.7 ± 6.7 fghij (1)
R2 5 105.7 ± 1.7 d (39) 66.3 ± 0.9 efg (77) 43.9 ± 0.9 ghij (61) 39.2 ± 1 ij (41)
R2 6 117.4 ± 2.5 cd (19) 73.4 ± 2.1 efg (16) 47.8 ± 1.3 fghij (28) 43.9 ± 1.8 ghij (13)
R2 7 132 ± 11.5 abcd (1) NA NA NA
Sk 5 117.6 ± 2.2 cd (25) 72.8 ± 1.3 efg (46) 47.4 ± 1 fghij (47) 39.8 ± 1.7 hij (14)
Sk 6 125.7 ± 1.8 cd (37) 77.5 ± 1.3 def (46) 51 ± 1.2 fghij (36) 49.4 ± 4.1 fghij (3)
Sk 7 180.7 ± 13.4 ab (1) NA NA NA
Means followed by different letters are significantly different from each other across the table at P < 0.05 using Tukey-Kramer post hoc test. Sample size (n) is based on number of survivors.
117
Figure 4-1. Percentage (mean ± SE) survivorship of L. dispar asiatica neonates to the pupal stage (a) and to adult (b) reared at four constant temperatures. The mean percentage survivorship of larvae to pupae (F = 3.27; df = 21, 288; P < 0.0001) and adult stages (F = 4.74; df = 21, 288; P < 0.0001) were significantly affected by the temperature and population interaction. Error bars represent the 95% confidence interval.
118
Figure 4-2. Developmental rates (1/d) of L. dispar asiatica larvae from the R1 and C2 populations from neonate to the second instar in response to constant temperatures. The solid and dashed lines represent linear regression of R1 and C2, respectively. Data points at each temperature represent mean developmental rate of each sex, open circles for the R1 population, and solid triangles for the C2 population (See methods for detail).
119
Figure 4-3. Cumulative proportion of L. dispar asiatica R1 and C2 individuals to reach the second instar over accumulated degree-days. Open circles represent R1 and open triangles represent C2 individuals reared at 10, 15, 20, 25 and 30 °C. Solid lines were fitted to cumulative proportion of individuals using Gompertz function, P = exp [- exp(- bDD +a]
Figure 4-4. Developmental rates (1/d) of L. dispar asiatica larvae from the R1 and C2 populations from neonate to adulthood in response to constant temperatures. The dashed and solid lines were fitted to Shi/Performance model of R1 and C2, respectively. Data points at each temperature represent mean developmental rate by ultimate instar; open circles for the R1 and solid triangles for the C2 population. (See methods for detail).
120
Figure 4-5. Cumulative proportion of L. dispar asiatica individuals from the R1 and C2 populations to complete development to adult over accumulated degree-days. Different data points represent individuals reared at 15, 20, 25, 30 °C of two populations of L. dispar asiatica, circles for the R1 and triangles for the C2 (Open marker for females and solid for males). Solid lines were fitted to cumulative proportion of female and dotted lines to male using Gompertz function: P = exp [- exp(- bDD +a].
121
Figure 4-6. Body weight (Mean ± SEM) of male and female L. dispar asiatica pupae reared at different constant temperatures. Different data points represent the ultimate instars within each sex (open markers female and solid markers male) for two populations of L. dispar asiatica, circles for the C2 and triangles for the R1. Solid lines were fitted to female weight and dotted lines to male weight using function: y = a (T – Tmin) (Tmax – T) (Lysyk 2001).
122
Figure 4-7. Body weight (Mean ± SEM) of male and female L. dispar asiatica adults reared at different constant temperatures. Different letters above bars represent significant differences at P < 0.05 using Tukey-Kramer post hoc test.
123
Figure 4-8. Percentage of (Mean ± SE) L. dispar asiatica pupal mortality (a) and deformed adults (b) reared at five constant temperatures. Percentage of pupal mortality (F = 4.16; df = 21, 280; P < 0.0001) and deformed adults (F = 1.81; df = 21, 271; P < 0.0181) was significantly affected by the interaction of temperature and population. Error bars represent the 95% confidence interval.
124
Figure 4-9. Estimated overall performance of Asian gypsy moth by population at different temperatures. The estimated overall performance was significantly affected by an interaction of temperature and population (F = 4.50; df = 21, 280; P < 0.0001). Error bars represent the 95% confidence interval.
125
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126 Keena, M. A., M. J. Côté, P. S. Grinberg, and W. E. Wallner. 2008. World distribution of female flight and genetic variation in Lymantria dispar (Lepidoptera: Lymantriidae). Environ. Entomol. 37: 636-649. Keena, M. A., W. E. Wallner, P. S. Grinberg, and R. T. Cardé. 2001. Female flight propensity and capability in Lymantria dispar (Lepidoptera: Lymantriidae) from Russia, North America, and their reciprocal F1 hybrids. Environ. Entomol. 30: 380-387. Keena, M. A. 2015. Inheritance and World Variation in Thermal Requirements for Egg Hatch in Lymantria dispar (Lepidoptera: Erebidae). Environ. Entomol. 45: 1-10. Lee, K. P., J. S. Cory, K. Wilson, D. Raubenheimer, and S. J. Simpson. 2006. Flexible diet choice offsets protein costs of pathogen resistance in a caterpillar. Proc. Soc. B. 273:823-829. Liebhold A. M., K. W. Gottschalk, R. M. Muzika, M. E. Montgomery, R. Young, K. O’Day, and B. Kelley. 1995. Suitability of North American tree species to the gypsy moth: a summary of field and laboratory tests. USDA For. Serv. Gen. Tech. Rep. GTR-NE-211: 1-34. Logan, J. A., D. J. Wollkind, S. C. Hoyt, and L. K. Tanigoshi. 1976. An analytic model for description of temperature dependent rate phenomena in arthropods. Environ. Entomol. 5: 1133-1140. Lysyk, T. J. 2001. Relationships between temperature and life history parameters of Muscidifurax zaraptor (Hymenoptera: Pteromalidae). Environ. Entomol. 30: 147-156. Matsuki, M., M. Kay, J. Serin, R. Floyd, and J. K. Scott. 2001. Potential risk of accidental introduction of Asian gypsy moth (Lymantria dispar) to Australasia: effects of climatic conditions and suitability of native plants. Agric. For. Ent. 3: 305-320. Mathworks. 2001. Curve fitting toolbox for use with MATLAB. User’s Guide (2001). Retrieved August 1, 2016 from http://cda.psych.uiuc.edu/matlab_pdf/curvefit.pdf. Neven, L. G. 2000. Physiological responses of insects to heat. Postharvest Biol. Technol. 21: 103- 111. Neven, L. G., and L. M. Rehfield. 1995. Comparison of prestorage heat treatments on fifth-instar codling moth (Lepidoptera: Tortricidae) mortality. J. Econ. Entomol. 88: 1371-1375. Peterson, A. T., R. Williams, and G. Chen. 2007. Modeled global invasive potential of Asian gypsy moths, Lymantria dispar. Entomol. Exp. Appl. 125: 39-44. Pitt, J. P. W., J. Régnière, and S. Worner. 2007. Risk assessment of the gypsy moth, Lymantria dispar (L), in New Zealand based on phenology modelling. International journal of biometeorology, 51: 295-305. Pogue, M. G., and P. W. Schaefer. 2007. A review of selected species of Lymantria Hübner (1819) including three new species (Lepidoptera: Noctuidae: Lymantriinae) from subtropical and temperate regions of Asia, some potentially invasive to North America. FHTET-2006-07. USDA For. Serv., Forest Health Technology Enterprise Team, Morgantown, WV. Raupp, M. J., J. H. Werren, and C. S. Sadof. 1988. Effects of short-term phenological changes in leaf suitability on the survivorship, growth, and development of gypsy moth (Lepidoptera: Lymantriidae) larvae. Environ. Entomol. 17: 316-319. Reardon, R., N. R. Dubois, and W. McLane. 1994. Bacillus thuringiensis for managing gypsy moth: a review. National Center of Forest Health Management FHM-NC-01-94. Régnière, J., V. Nealis, and K. Porter. 2009. Climate suitability and management of the gypsy moth invasion into Canada. Biol. Invasions. 11: 135-148. SAS Institute. 2011. SAS/STAT® 9.3 User’s Guide. Care. SAS Institute Inc., Cary, NC. Savotikov, I. F., A. I. Smetnik, and A. D. Orlinskii. 1995. Situation of the Asian form of gypsy moth (Lymantria dispar) in Russia and in the world. EPPO Bulletin, 25: 617-622.
127 Shields, V. D., B. P. Broomell, and J. O. Salako. 2003. Host selection and acceptability of selected tree species by gypsy moth larvae, Lymantria dispar (L.). Ann. Entomol. Soc. Am. 96: 920-926. Shi, P., F. Ge, Y. Sun, and C. Chen. 2011. A simple model for describing the effect of temperature on insect developmental rate. J. Asia Pac. Entomol. 14: 15-20. Tobin, P. C., S. L. Whitmire, D. M. Johnson, O. N. Bjørnstad, and A. M. Liebhold. 2007. Invasion speed is affected by geographical variation in the strength of Allee effects. Ecol. Lett. 10: 36-43. USDA, APHIS. 2016. Pest Alert: Asian gypsy moth. Retrieved October 4, 2016 from https://www.aphis.usda.gov/publications/plant_health/content/printable_version/fs_phasi angm.pdf Wallner, W. E., L. M. Humble, R. E. Levin, Y. N. Baranchikov, and R. T. Carde. 1995. Response of adult lymantriid moths to illumination devices in the Russian Far East. J. of Econ. Entomol. 88:337-342. Wu, Yunke, J. J. Molongoski, D. F. Winograd, S. M. Bogdanowicz, A. S. Louyakis, D. R. Lance, V. C. Mastro, and R. G. Harrison. 2015. Genetic structure, admixture and invasion success in a Holarctic defoliator, the gypsy moth (Lymantria dispar, Lepidoptera: Erebidae). Mol. ecol. 24:1275-1291.
128
Appendix A
Efficacy test of Scymnus camptodromus in the field using confined releases of predator adults and larvae
A.1 Introduction
Scymnus camptodromus Yu & Liu, is a predatory lady beetle collected from China. It has several characteristics that would make it a good potential biological control agent for hemlock woolly adelgid (HWA) such as i) a true aestival diapause that coincides with the summer aestivation of HWA, ii) the predator’s eggs hatch in synchrony with HWA oviposition in Spring, and iii) it occurs abundantly over a broad geographic area and in diverse habitats at various densities of HWA in its native range. Previous studies performed in the laboratory showed that S. camptodromus is an HWA specific predator and will feed voraciously on HWA eggs. However, the predator has not been approved for field release and examined for its efficacy against HWA in field conditions. To evaluate the efficacy of S. camptodromus against HWA, we performed an experiment using confined field release of the beetle in two consecutive field seasons in
Pennsylvania. Our objectives were to examine survival and oviposition of S. camptodromus, and determine the impacts the predator had on an HWA population under field conditions.
129 A.2 Materials and Methods
A.2.1 Study sites and predator population
Confined release of S. camptodromus was performed over two consecutive seasons for the periods of 17 April – 11 June 2012 and 20 April – 25 June 2013 at a demonstration woodlot at Rock Springs (Penn State experimental farm) (40 ⁰ 41' 53.2 "N, 77 ⁰ 58' 00.3"). Fifth and sixth generations of S. camptodromus adults and larvae reared at the USDA-Forest Service Northern
Research Station Quarantine laboratory in Ansonia, CT were used for these confined releases. S. camptodromus adults were mated before release and to do that, a male and a female identified by shape of their abdomen (Yu et al. 2000), were placed inside a 59 ml cup with a clear 2.5 cm diameter lid (Solo, Eastern Bag and Paper, CT) and provided with HWA infested hemlock twigs.
These adults were held in a 20 °C chamber with a 12:12 h (L: D) and 50-60 % RH for one week.
A.2.2 Spring 2012 confined release of S. camptodromus
Twenty trees between 12-20 feet tall with 20- 50 % of the branches infested with HWA were selected for predator evaluation based on the ability of the tree to support at least one replicate of each treatment. The average number of eggs per ovisac was determined for each branch used in the experiment by counting the eggs within 30 randomly selected ovisacs from each tree. The study consisted of three treatments: a bag with adult S. camptodromus, an empty bag, and an un-bagged branch. Three branches of each tree containing about 300 ovisacs were selected and randomly assigned to one of the three treatments. The bags used for confined release were 45 × 60 cm in size and composed of fine mesh cloth (no-see-um, Seattle Fabrics,
Inc.) sewed shut on one end. In the field, the open end of the bag was secured using zip ties.
130
Temperature data loggers (EL-WIN-USB, www.lascarelectrionics.com) were placed inside and outside the bags on the experimental trees to record temperatures inside and outside the bags.
Bags with confined predators contained two adults, either two females or a male-female pair. In the bags without S. camptodromus, branches were carefully inspected, and any visible predators were removed before securing the bag to the branch. For the unbagged controls, 300 ovisacs were counted from the tip and marked. To evaluate treatment duration on survival and oviposition of the confined predators, half of the branches were collected after one month on 15
May 2012, while the remaining branches were collected after two months on 11 June 2012.
Branches were removed with bags in place and brought to the laboratory for evaluation. These branches were cut into smaller pieces of 12-13 cm in length to access and count all HWA life stages, measure any new plant growth, and record survival and presence/absence of S. camptodromus eggs.
To determine the degree of predation by S. camptodromus, we calculated the number of
HWA eggs lost for each experimental branch using the following equation:
Total eggs lost = (mean number of eggs per ovisac × number of ovisacs (pre-release)) –
(total remaining eggs + crawlers + settled 1st instar + older nymphs (post-harvest)
A.2.3 Estimation of average HWA eggs consumed by S. camptodromus adults and larvae
Prior to the 2013 field release, we performed a preliminary study to estimate the mean number of HWA eggs consumed per day by a predator adult. We used three strains of S. camptrodromus; DGS (22) [strain (number)], MNP (31) and LJS (6). The predator adults were monitored individually while confined in a 59 ml cup with a 2.5 cm diameter clear lid (Solo,
Eastern Bag and Paper, CT). A 1 cm2 “X” was cut in the center of the lid to provide ventilation and the opening was covered with a fine mesh cloth to prevent escape. A 2.4 cm diameter piece
131 of Whatman filter paper was placed at the bottom of the rearing containers. HWA eggs on each infested twig were counted prior to providing them to the predator. The predators were held in 20
°C incubator with 16:8 h (L: D) and 75 % RH. After 72 hours, the predators were removed and the number of HWA eggs remaining were counted and recorded. The mean number of eggs consumed by individual S. camptodromus larvae was based on results from our previous study
(Limbu et al. 2015).
A.2.4 Field release of S. camptodromus 2013
Confined field release of S. camptodromus adults and larvae was performed in Spring
2013. Fifth and sixth generation S. camptodromus adults and sixth generation S. camptodromus larvae (second instar) were used for field release. Details of treatments used in the study is presented in Table A-1. A 30 × 45 cm bag (no-see-um, Seattle Fabrics, Inc.) was used for the adult treatment and empty bag treatments, and a 62 x 62 cm (same material) bag was used for the larval treatment.
We selected 12 trees, 12-25 feet tall with 20 -50 % of the branches infested with HWA at
Rock Springs (same field site as 2012). The mean number of HWA eggs per ovisac was determined prior to field release as described above. The number of HWA ovisacs enclosed in each treatment bag was based on the mean number of HWA eggs per ovisac, and the estimated number of HWA eggs, an adult and larval predators were expected to consume during the field trial period. We estimated that three 2nd instar predator larvae would require nearly six weeks to reach adulthood so we provided 1500 eggs (500 per larva) in each treatment bag containing the larvae. Additionally, based on our preliminary results on HWA eggs consumed by S. camptodromus adults at 20 °C, we estimated that 1200 HWA eggs should be sufficient to feed two S. camptodromus adults for two weeks.
132 Every two weeks, bags with beetles, un-bagged branches and empty bags were collected from the field. The next day, three new branches were selected from the same tree and the harvested treatments were replaced. Any dead beetles (in bag with adult) were replaced at that time. Unlike the adult treatments, S. camptodromus larvae were left undisturbed for 6 weeks to allow them to develop to adults. The collected branches were brought to the laboratory for evaluation. From each treatment branch we removed a 15 cm piece from the tip (from 21-22 cm total length of the branch) and counted all HWA life stages (adults, settled first instar and older nymphs) remained in the piece. We also selected 30 HWA ovisacs per treatment and counted the total number of eggs present per ovisac. For the confined adult and larvae bags, S.camptodromus survival was assessed and recorded.
A.2.5 Statistical analyses
The statistical analyses were performed in SAS (SAS Institute Inc., Cary, NC). For the confined release study in 2012, the number of HWA eggs lost was compared among treatments using PROC GLM. Significant differences among means were determined using the Tukey HSD with α = 0.05. Since half of the treatments were collected at the end of one month and other half at the end of two months, only treatments left for the same amount of time were compared with each other.
For the confined release study in 2013, the mean number of HWA eggs per ovisac and mean number of other life-stages of HWA (crawler, settled first instar nymph, older nymph) remaining in each treatment over time were compared among treatments using PROC MIXED and maximum likelihood estimation method. The model used treatments (bag with beetle, empty bag and unbagged branch), time (2, 4, 6, 8 week), and their interaction as fixed effects.
Differences among treatments were determined using the LSMEANS via Tukey-Kramer post hoc
133 test with α = 0.05. Comparisons were also made between bags with S. camptodromus larvae and un-bagged branches for mean HWA life-stages (eggs, crawlers, settled first instar, older nymph) left in each treatment after six weeks using PROC ANOVA and differences among means were determined using the Tukey HSD with α = 0.05.
A.3 Results
A.3.1 Confined release 2012
The mean number of HWA eggs per ovisac at the beginning of the study was 120 ± 8.1.
After one month, there was no effect of predation on the total number of HWA eggs lost (F =
0.55; df = 2, 27, P = 0.58). However, after two months predation by S. camptodromus significantly reduced the number of HWA eggs compared to the empty bag and un-bagged branches (Figure A-1). Additionally, 90 % of S. camptodromus adults survived and only one egg was recovered at the end of one month; 60 % of S. camptodromus survived with 26 eggs laid by one female in the two-month field trial.
A.3.2 Confined release 2013
In the lab, the mean number of HWA eggs consumed by S. camptodromus adults at 20 °C was 35 ± 1.9 eggs per day (Table A-2). In the field experiment, the mean number of HWA eggs per ovisac at the beginning of the study was 30 ± 0.37.
The effect of time (2, 4, 6, 8 weeks), treatment (bag with beetle, empty bag and un- bagged), and their interaction on the number of HWA eggs per ovisac in each treatment was evaluated. There was no significant time x treatment interaction (F = 1.90; df = 6,131, P <
134 0.0847). There was a significant effect of time on the number of HWA eggs left in each bag regardless of treatment (F = 20; df = 3,131, P < 0.0001). As expected the number of HWA eggs declined over time. For example, mean HWA eggs per ovisac left in each bag at the end of 2, 4,
6 and 8 weeks were 59 ± 3.34, 28 ± 2.09, 0.39 ± 0.07, and 2.11 ± 2 eggs per ovisac respectively.
The number of HWA eggs per ovisac also varied by treatment; there was a significant reduction in HWA eggs in bags with predator adults compared to the other two treatments after 8 weeks (Figure A-2). The number of crawlers and older nymphs of HWA did not vary among treatments, but the number of settled first instars was significantly lower in bags containing S. camptodromus adults compared to the controls (Table A-3). After 6 weeks, the number of HWA life stages (eggs/ovisac, crawlers, settled 1st instars, older nymph) was not different between bags with S. camptodromus larvae or empty branch (Table A-3), although the reduction in the number of older nymphs was close to being statistically significant (P = 0.06).
At the end of 6 weeks, when a bag containing larvae was compared with unbagged branches we did not find any difference on HWA eggs per ovisac, crawlers, settled first instars, and on older HWA nymphs (Table A-4).
A.3.2 Survival and oviposition
On average, greater than 70 % of adults survived over each 2 weeks period branches were sampled (Figure A-3). However, out of 36 larvae confined to HWA-infested bags, 19 successfully reached adulthood, 6 died as larvae, 12 died as pupae, and 5 were lost. Most of the dead pupae were found at the end of the bag near the zip tie where they had drowned in a pool of rain water that collected at this location in the bag. Only older generation females (a year older) laid eggs during the study, individuals that had just become adults right before the study started did not. In the confine release only 2 bags containing older generation females had 7 eggs.
135
A.4 Discussion
In both field seasons, S. camptodromus adult predation affected HWA populations compared to the two control treatments. In 2012, our objective was to determine the overall effect of predation by S. camptodromus adults on HWA eggs. After one month, almost all the predators were found alive but the impact of predation was not significant compared to the control treatments. However, after two months we saw a significant reduction in the number of
HWA eggs in the treatment containing predator adults. It should be noted that we did not consider predator prey ratios during this field trial. We provided 300 HWA ovisacs per treatment each containing an average of 120 eggs and the HWA females continued to produce eggs, which may explain why we did not see a difference among treatments within the one month. We also had some heavy rainfall during the field experiment (both months) which washed away more
HWA eggs in the un-bagged treatment than bagged treatments (damaged to HWA ovisacs due to heavy rain were observed in unbagged branches), which may have had a confounding effect on our results.
In 2013, we found a significant reduction in mean number of HWA eggs and young settled nymphs in bags with predator adults compared with those without predators, suggesting that the predator adults prefer eggs and the young settled stages of HWA over older stages. Also, during both 2012 and 2013, two different generations of S. camptodromus were used. The generation from the previous year oviposited in the field, suggesting that the predator may reproduce multiple years in a field provided they survive. Given the difficulty in sexing S. camptodromus adults, it is possible that the young adults released in bags did not mate.
136 We did not observe any significant predation effect by S. camptodromus larvae, however, treatment with S. camptodromus larvae was only compared with un-bagged branches, which was were not protected and suffered damage due to heavy rains. Results may have been different if we had been able to compare the larval treatment with the empty bagged branch treatment.
Furthermore, 33.3 % of predator larvae suffered mortality before or after pupation. The larvae chose to pupate at the closed end of the bag, where rain water accumulated and eventually drowned the pupae. For future studies with S. camptodromus larvae, more replications should be used, better bagging techniques should be considered, and comparison should also be done with the empty bagged branches.
Our results indicate that lab reared S.camptodromus adults can survive the field, reproduce, and impact the HWA population. Also, larvae were able to survive to adulthood despite the challenges of being enclosed in a bag. Therefore, S. camptodromus if released may have the potential to establish in a setting to contribute to the predator complex as a biological control agent of HWA in the Eastern U.S.
137
Table A-1. Four branches from each tree were selected and randomly assigned for the different treatments.
Beetle Assessment Treatments stage and Caged Estimated number of HWA eggs enclosed period(s) number Unbagged branch 2 weeks None No 1200 Empty bag 2 weeks None Yes 1200 Bag with beetle 2 weeks 2 adults Yes 1200 Bag with larvae 6 weeks 3 larvae Yes 1500
Table A-2. Mean [± SEM (n)] HWA eggs consumed by three different strains of S. camptodromus adults per day and mean eggs oviposited in 72 hours at 20 °C in the lab.
Strain HWA eggs consumed Predator eggs laid in
per day 72 hours
DGS 34.5 ± 3.5 (20)
LJS 39.3 ± 2.2 (33) 2.06 ± 0.2
MNP 32.1 ± 7.4 (6)
Grand Total 35.3 ± 1.9 (59) 2.06 ± 0.2
Sample size (n) based on number of S. camptodromus of each strain used in the study.
Table A-3. Mean (± SE) HWA life stages remaining on experimental branches containing S. camptodromus adult as measured every 2 weeks over an 8-week period in 2013.
Treatments Crawlers settled first instars Older nymphs
Bag with beetle 8.33 ± 1.90a 108 ± 17.9a 32.7 ± 8.90a
Empty bag 12.4 ± 1.90a 208 ± 17.9b 63.7 ± 8.90a
138 Un-bagged branch 13.0 ± 1.90a 146 ± 17.9b 48.7 ± 8.90a
Statistics F = 1.7; df = 2, 98, F = 8.03; df = 2, 98, F = 2.98; df = 2, 98, P = 0.18 P = 0.0006 P = 0.06 Means compared using two-way ANOVA. Due to natural development in the field, the number of each life stage of HWA differed with time. Within column means followed by a different letter indicate significant differences among treatments at P < 0.05.
Table A-4. Mean (± SE) HWA stages on bagged and unbagged experimental branches after 6 weeks of predation by S. camptodromus larvae in the field in 2013. Treatments Eggs per 30 Crawlers settled first instars Older nymphs ovisacs
Predator larvae 15 ± 4a 0a 148 ± 23a 69 ± 15a
Un-bagged branch 13 ± 4a 0.8± 0.7a 100 ± 25a 84 ± 21a
Statistics F = 0.33; F = 1.3; F = 1.94; F = 0.32;
df = 1, 22, df = 1, 22, df = 1, 22, df = 1, 22,
P = 0.57 P = 0.27 P = 0.18 P = 0.06
Means compared using two-way ANOVA. Means followed by a different letter within a column are significantly different at P < 0.05.
139
Beetle Empty Unbagged bag branch
Figure. A-1. Mean reduction in number of HWA eggs by treatment at the end of two months of predation by S. camptodromus in the field. Total HWA eggs lost was significantly greater in bags with S. camptodromus adults (F = 16.7; df = 2, 27; P < 0.0001) compared to the two controls. * indicates significant difference at P < 0.05.
Beetle Unbagged branch
140 Figure A-2. HWA eggs that remained per ovisac on each experimental branch after eight weeks of exposure to S. camptdromus adult confined to HWA-infested branches in the field. HWA eggs remaining in bags significantly differed by time (F = 201; df = 3, 131; P < 0.0001) and by treatment (F = 3.42; df = 2, 131; P = 0.04) with a significant reduction in HWA eggs in bags with beetles. There was no significant interaction between time and treatment (F = 1.9; df = 6, 131, P = 0.0847). *indicates significant difference of beetle treatment compared to controls at P < 0.05.
Figure A-3. Percentage of adult S. camptodromus that survived confined release in the field at each 2 week check in 2013.
References
Limbu, S., M. A. Keena, D. Long, N. Ostiguy, and K. Hoover. 2015. Scymnus camptodromus (Coleoptera: Coccinellidae) larval development and predation of hemlock woolly adelgid (Hemiptera: Adelgidae). Environ. Entomol. 44: 81–89. Yu, G., Montgomery, M. E., & Yao, D. 2000. Lady beetles (Coleoptera: Coccinellidae) from Chinese hemlocks infested with the hemlock woolly adelgid, Adelges tsugae Annand (Homoptera: Adelgidae). Coleopts. bull.54:154-199.
141
Appendix B
Petition for Unconfined Field Release of the Exotic Predator Scymnus camptodromus for Biological Control of Hemlock Woolly Adelgid (Adelges tsugae), in the Eastern United States
Samita Limbu,1 Melody Keena,2 and Kelli Hoover1 1Department of Entomology, Pennsylvania State University, University Park, PA 16802 2Northern Research Station, USDA – Forest Service, CT 06514
B.1. Introduction
B.1.1 Proposed Action
Release of Scymnus (Neopullus) camptodromus Yu and Liu (Coleoptera: Coccinellidae) for management of the Hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera:
Aldelgidae).
B.1.2 Proposed of release
To manage the hemlock woolly adelgid (HWA) by reducing its population densities in infested areas and slowing its spread to new areas.
142 B.1.3 Need for release
A. tsugae is a major pest of Eastern hemlock, Tsuga canadensis (L.) Carriére and
Carolina hemlock, Tsuga caroliniana Engelmann in the eastern United States, threatening the sustainability of hemlock forests in the Eastern U.S. Originally introduced into the eastern United
States from southern Japan (Havill et al., 2006), A. tsugae was first reported in 1951 in
Richmond, VA. Currently, the pest is endemic to 19 states from Maine to Georgia (Preisser et al.,
2014; USFS, 2012) encompassing approximately 40% of the T. canadensis range and 100% of the T. caroliniana range (USFS, 2012). The pest has been reported to kill hemlocks of all ages and sizes; it can kill a tree in little as 1-3 years in its southern range and 5-15 years in its northern range (Ellison et al., 2010). Proliferation of A. tsugae is believed to be more rapid in the South due to the warmer climate, which allows prolonged feeding and reduced winter mortality of adelgids resulting in faster hemlock decline (Ford et al., 2012; Parker et al., 1999; Skinner et al.,
2003).
A. tsugae is causing substantial damage to hemlock resources in the eastern U.S. It is estimated that A. tsugae infestation of landscape trees is causing a loss of $2000-$7000 per homeowner (Holmes et al., 2005). Although hemlock is not valued as a commercial timber resource, these are important foundation tree species of eastern forests and the Appalachian
Mountains, dominating 1 million hectares of eastern U.S. forests (Domec et al., 2013). It is the fifth longest lived tree species (Peterson & Peterson, 2001), highlighting its importance ecologically. Moreover, hemlocks are shade tolerant trees that co-evolved with deciduous trees in mixed forest settings and are known for their ability to mediate soil moisture, stabilize stream base-flows, and regulate stream temperature. Cool, moist microclimates with a slow rate of nitrogen cycling and slow decomposing acidic litter are unique to hemlock dominated forests
(Ellison et al., 2010) and there are no ecological equivalent species to replace hemlock. Because
143 of these unique characteristics hemlocks support a distinctive group of terrestrial and aquatic organisms. Biodiversity reduction in the eastern United States has been a major concern after hemlock mortality due to A. tsugae infestation. Moreover, Carolina hemlock is a rare endemic species to the Appalachian Mountains; its loss would lead to extinction not only of this tree species, but also the extirpation of the community of organisms associated with Carolina hemlock
(Ward et al., 2004).
A. tsugae has spread rapidly since its introduction; there are no known parasitoids, insufficient generalist native predators, and an abundance of susceptible hemlock stands in the eastern U.S. (Hakeem et al., 2013; Havill et al., 2014; Montgomery & Lyon, 1995; Wallace &
Hain, 2000). Although A. tsugae can be managed chemically in urban environments, chemical control in a forest setting is not practical economically and could have non-target and broader ecological impacts. Since, natural enemies of A. tsugae appear to contribute to natural control of this pest in its native range in Japan, China and Western North America (Lu et al., 2002;
Montgomery & Keena, 2011; Vieira et al., 2013; Zilahi-Balogh et al., 2003) development of classical biological control for managing A. tsugae is considered the most promising approach for forest settings.
B.1.4 Reasons for choice of the agent
Exploration for natural enemies of A. tsugae included collection of S. camptodromus from the Sichuan and Yunnan provinces in southwestern China. S. camptodromus is an effective predator in its native ecosystem; this species was collected in abundance over broad geographic areas and habitats, and found thriving at variable prey densities (Figure B-1). The adults of the species feed on all life stages of A. tsugae throughout their development (Cheah et al., 2004) and larvae of this predator feed on multiple life stages, although they feed most voraciously on A.
144 tsugae eggs (Montgomery & Keena, 2011). This predator’s phenology is closely aligned with that of A. tsugae; S. camptodromus eggs diapause while A. tsugae are in summer aestivation, hatching in spring as A. tsugae begin laying eggs (Keena et al., 2012). Cold tolerance of this predator also makes it an attractive choice for biological control in the northeastern U.S.
Moreover, a study on non-target effects of this predator was performed and suggests that it is quite specific to A. tsugae and is not known to have a negative impact on non-target species that have been evaluated (Limbu et al., 2016).
B.1.5 Specific location of rearing/quarantine facility and name of qualified personnel operating the facility
Different strains of S. camptodromus distinguished by geographic origin in China were collected in 2006-07 and transported from China to the USDA Forest Service quarantine facility in Ansonia, CT under permit. Voucher specimens of adults were deposited at the Entomology
Division, Yale Peabody Museum of Natural History, New Haven, CT. Currently, these predators are being reared at the USDA Forest Service quarantine facility in Ansonia, CT and at
Pennsylvania State University, PA after being released from quarantine. Locations of different strains (Geographic populations) of the predator are listed in Table B-1.
B.1.6 Timing of the release
During field exploration in China, Scymnus lady beetles were found in abundance during early spring and larvae of the beetles were observed during April-May when temperature ranges between 5-15 ºC. Based on this information, spring is likely a suitable time for release of this predator in the eastern U.S. Although, release of the beetle depends on mass production beetles
145 in the laboratory, the idea is to schedule the release during spring coinciding with the beginning of A. tsugae oviposition in the field. The A. tsugae infestation where the beetle adult or its larvae are to be released should be moderate so that the hemlock tree is not heavily impacted and there is greater likelihood that the adelgid will remain active on the tree for several generations.
B.1.7 Methods to be used
Scymnus camptodromus larvae need A. tsugae eggs as a primary food source for survival.
Therefore, A. tsugae infested foliage will be collected and stored in a laboratory. If the collected foliage has mature adelgids, but no eggs, the collected foliage will be placed at 15 C to encourage oviposition by A. tsugae. When A. tsugae lay enough eggs, the infested foliage will be transferred to a 5 C chamber to slow down the rate of A. tsugae egg hatch to extend the oviposition period so food is available longer to feed predator larvae.
S. camptodromus eggs will be held individually in a 0.5 ml clear micro-centrifuge tube at different temperatures for various numbers of days to simulate the conditions that initiate and then break diapause. Newly hatched larvae will be reared individually in a 59 ml soufflé cup with a clear 2.5 cm diameter lid at 15 or 20 ºC and 12:12 h L:D photoperiod (Limbu et al.2014). The
HWA infested branches will be provided to the larvae after careful examination to remove any other natural predators from the branch. The HWA infested foliage inside the cup will be changed every three days to insure sufficient food supply to the developing larvae. After adult eclosion, they will be sexed after 2-3 days when they have completely hardened; the predators will be reared in groups of 10-15 individuals with a 1:2 male to female ratio. The adult cage will consist of a transparent plastic container with lid. The cage will be provided with A. tsugae infested foliage in a cup containing water and will be changed every two weeks or as needed. In addition to the A. tsugae infested foliage, the predator will be given a supply of honey wheast
146 mixture on filter paper as an alternate food source. Adults will be maintained at 15 or 20 ºC and a
12:12 h L:D photoperiod when there is a sufficient supply of food and transferred to 10 ºC when the food supply begins to run low.
B.1.8 Disposal of any host material and pathogens, parasites, hyperparasitoids of agent accompanying import shipment
All waste materials from these cultures and any associated shipping materials will be disposed of properly. The permit to have these insects requires that all waste materials be double bagged and prepared by one of the following methods prior to disposal: 1) frozen at -20 F for 72 hours, 2) autoclaved or 3) incinerated, before entering the municipal waste stream. All imported insects are kept in growth chambers and observed in quarantine through the first generation to ensure no infection by parasitoids. Any parasitoids that are found will be curated and identified.
Any insects that die of suspicious causes will be evaluated for pathogens. A few progeny will be tested for potential infection by microsporidia. Tools are sterilized daily and when removed from quarantine. All workers that enter quarantine wear lab suits in the containment area and follow standard operating procedures.
B.1.9 Agencies and/or individuals that will be involved in the release and monitoring
U.S. Department of Agriculture (Melody Keena, R. Talbot Trotter III) Forest Service, NRS Quarantine Facility Ansonia, CT 06401
Cornell University: Mark Whitmore Department of Natural Resources Ithaca, NY
147 Pennsylvania Department of Conservation Resources, Bureau of Forestry, Forest Pest Management Director: Don Eggen P.O. Box 8552 Harrisburg, PA 17105
Pennsylvania State University: Kelli Hoover and Samita Limbu Department of Entomology University Park, PA, 16802
B.2. Target Pest Information
B.2.1 Taxonomy
Scientific name: Adelges tsugae Annand Full classification: Animalia, Arthropoda, Insecta, Hemiptera, Adelgidae, Adelges tsugae Common name: Hemlock Woolly Adelgid
Sufficient characterization to allow unambiguous recognition
A. tsugae are minute insects and, depending on generation, the adult size ranges from 0.6 to 2 mm (Havill et al., 2006). These are piercing and sucking insect pests of conifers in the genera Tsuga (Havill & Foottit, 2007; Preisser et al., 2014) and are usually found settled at the base of the hemlock needle cushion. The A. tsugae populations in the eastern U.S. have a complex polymorphic life cycle that includes two parthenogenetic generations and one sexual generation, but due to lack of the suitable primary host, this species is not observed to have a sexual cycle in the introduced range (Jetton et al., 2014; McClure, 1989). The two parthenogenetic generations are the sistens (early spring-summer) and progredien generations
(spring), which are wingless, while the sexual generation has wings. Despite the sexual cycle not having been observed in the introduced range, A. tsugae continuously produces the winged sexual generation, which ultimately dies because it is unable to find suitable hosts. The parthenogenetic generations have six developmental stages; egg, four nymphal instars, and the adult. Both
148 generations produces wax filaments that cover the whole body and the eggs they lay. The waxy covering protects them from predation and desiccation and helps reflect UV light (Jones et al.,
2014). Sistens can lay up to 350 eggs and progrediens up to 75 eggs, but fecundity greatly depends on host plant condition (McClure 1991).
B.2.2 Economic impact of pest and benefits of the target organism
Eastern hemlock is not valued as a commercial timber species; however, because it has value as an ornamental plant, the economic impact of A. tsugae on hemlock has focused on landscape settings. A. tsugae is reported to cause an estimated loss of around $2000-$7000 per home (Holmes et al., 2005) and total household expenditure is estimated to be around $130 million annually (Aukema et al., 2011). They cause substantial loss to real estate values, which is estimated to be around $260 million per year. Additionally, HWA cost the federal government about $14 million and local government about $170 million annually (Aukema et al., 2011).
There has not been an economic analysis regarding hemlock loss in forest environments, however, the ecological value of hemlocks in the eastern U.S. forest has been acknowledged.
Hemlocks are known for creating unique microclimates in forest settings on which other species rely for their survival. Being one of the longest-lived trees of eastern North America, hemlocks have direct and indirect association with several other species, therefore mortality of this tree has a vast impact on forest ecosystems. Biodiversity reduction associated with hemlock loss has been reported extensively (Buck et al., 2005; Ellison et al., 2005; Ross et al., 2003). While A. tsugae in urban settings can be regulated by monitoring and management options, there are no practical methods available to prevent the infestation and loss in forest environments. Establishing a complex of natural enemies in the eastern U.S. is being explored as a sustainable option for
149 control of A. tsugae, as it appears that this pest is being regulated by its natural enemies in its native range.
B.2.3 Economic impact of pest and benefits of the target organism
Fourteen adelgid species are known to be associated with conifers in the United States and Canada but none of them are considered beneficial (Table B-2) (Johnson & Lyon, 1991).
B.2.4 Distribution of pest
Native Range: Japan, China, Pacific Northwest of North America.
North America: A. tsugae was first identified in Richmond, Virginia in 1951 and damage on eastern hemlock was reported starting in 1969 in Pennsylvania (Gouger 1971). The infestation currently prevails in 19 states from Maine to Georgia (See Appendix 1) comprising 40% of the eastern hemlock range and 100% of the Carolina hemlock range in the U. S. The rate of spread has been rapid since the 1970’s and has been faster in the south than in the north because of weather conditions (Evans & Gregoire, 2007). Although A. tsugae seems to prefer warmer southern weather, the pest is capable of adapting to cold climates (Butin et al., 2005), which suggests that it will continue to spread to the north and west throughout the eastern hemlock’s range.
B.2.5 Regulatory and/or pest status of the target in state, provincial or federal law
Quarantines are currently in effect for Maine, Michigan, New Hampshire, Ohio,
Vermont, and Wisconsin to prevent the movement of A. tsugae out of its infestation range. No
150 live or harvested hemlock material is allowed to be shipped into these states from any county in the U.S. in which A. tsugae is present. In 2008, Canada also established a quarantine that restricts any movement of live or harvested hemlock from all the states that have confirmed A. tsugae infestation including western states.
B.2.6 Knowledge of status of other biological control organisms (indigenous and introduced) that attack the pest.
Foreign explorations outside and within the U.S. were made in search of potential natural enemies of A. tsugae. Several predators were found in association with A. tsugae during these explorations and the ones with the most potential were imported for evaluation; several have been introduced into the eastern United States.
In Japan, several collections of predators were made, but Sasajiscymnus tsugae showed the most potential in lab and field evaluations. In Japan, it was responsible for 86% of adelgid mortality in landscape sites and 99 % adelgid mortality in forest sites. This predator prefers adelgids to aphids but was found able to complete development feeding on several other adelgid species in host range tests (McClure & Cheah, 1998). The first release of S. tsugae was in 1995 and since then over 2 million of these lady beetles have been released at more than 400 sites in 16
U.S. states. There were initial reports of establishment and impact on A. tsugae populations
(Cheah, 2011; Cheah & Mcclure, 2002; McDonald et al., 2008), however, S. tsugae has experienced difficulty in establishing and has generally shown no significant population growth
(Asaro et al., 2005; Casagrande et al., 2002).
Similarly, Laricobius osakensis (Coleoptera: Derodontidae) was discovered in Japan and imported into the U.S. in 2006. This predator was first kept in a quarantine facility in
Blacksburg, VA where the life history and biology of the insect was studied (Lamb et al. 2008).
151 Host range tests performed with this predator showed that this insect is specific to A. tsugae, prefers A. tsugae in comparison with other tested adelgid species, and is not able to develop on the alternate hosts tested (Vieira et al., 2011). Following release from quarantine, confined release of this predator was made in the field in 2010/11. Results showed that this predator has potential as biological control agent of A. tsugae in the eastern U.S. However, it was discovered that this predator colony had the cryptic co-genitor Laricobius naganoensis, which led to placement of the predator back into quarantine until the colony could be purified. This species is now being actively released and its establishment is being monitored.
Another derodontid, Laricobius nigrinus (Coleoptera: Derodontidae), was found in association with A. tsugae in western North America in 1997 and was introduced into eastern
North America for biological control of A. tsugae. The larval development of this predator is closely synchronized with oviposition by sistens and a host-range test done in quarantine showed that it is a specific predator of A. tsugae. L. nigrinus was released in 2000 and to date 200,000 beetles have been released at over 200 sites (Havill et al., 2014). However, this predator was also found to hybridize with the native eastern North American Laricobius species L. rubidus, which will feed on A. tsugae but prefers pine bark adelgid (Pineus strobi). The repercussions of this are several and difficult to predict. One possibility is that this hybridization may be beneficial by avoiding inbreeding depression and might improve the ability of L. nigrinus to survive in eastern climates relative to pure breeds. Alternatively, there could be negative effects of this biological control agent if the hybrid feeds less on the target pest. Therefore, more work has to be done to test fitness of hybrids and compare them with their parents in terms of prey preference and host location behavior (Havill et al., 2012).
Additional, foreign exploration to China during 1995 to1997 resulted in finding abundant natural enemies of A. tsugae that included 70 species of lady beetles. Among these seven were
Scymnus (Neopullus) species and three stood out as having the greatest potential as biological
152 control agents for A. tsugae in the eastern U.S; these include Scymnus (Neopullus) camptodromus
Yu and Liu (Sc), Scymnus (N.) sinuanodulus Yu & Yao (Ss) and Scymnus (N.) ningshanensis Yu
& Yao (Sn). These species were collected from the Yunnan, Shaanxi and Sichuan provinces of
Southwestern China and brought to the U.S. beginning in 1995. Limited release of S. sinuanodulus and S. ningshanensis was performed in the U.S. in 2004 and 2007, respectively, but do not appear to have established (Havill et al., 2014; Montgomery & Keena, 2011). Of the three
Scymnus species, S. camptodromus was considered to have the most potential because of several attributes. For example, in its native range, this species was found feeding on all life stages of A. tsugae throughout their development (Cheah et al., 2004). The larvae of this predator will also feed on different life stages, although they feed most voraciously on A. tsugae eggs (Montgomery
& Keena, 2011). More importantly, this predator has an unusual egg diapause, which is rare among coccinellids, allowing phenology of S. camptodromus to closely align with that of A. tsugae. Consequently, the predator eggs are in diapause when A. tsugae isn’t available due to summer aestivation; the predator eggs will not hatch until spring, which coincides with the start of A. tsugae oviposition. Additionally, the cold tolerance of this predator makes it an attractive choice for biological control in the northern part of the eastern U.S. Host-range tests performed with S. camptodromus showed that it is a specialist predator of A. tsugae and will not be a threat to non-target species (Limbu et al. 2014).
B.3. Biological Control Agent Information
B.3.1 Taxonomy
Scymnus (Neopullus) camptodromus Yu and Liu (Coleoptera: Coccinellidae). No synonymy or common names.
153
B.3.2 Methods used to identify the agent
According to Chen et al. 2014, the total length and width of S. camptodromus is 1.89-
2.14 mm and 1.24-1.36 mm, respectively, and they have an oval, moderately convex body. The elytra are reddish brown with a black stripe at the lateral margins extending to 4/7 the length of the elytra. The mouth parts, head, and antennae are brown in color. The legs are brown with femora and tibiae black. Males and females are externally similar but females have an entirely black head and legs. This particular species of Scymnus (Neopullus) is distinct from other species; it is recognizable by its entirely black pronotum and reddish brown elytra with an obtriangular black marking at its base.
B.3.3 Location of voucher specimens
Voucher specimens of adults were deposited at the Entomology Division, Yale Peabody
Museum of Natural History, New Haven, CT.
B.3.4 Natural geographic range, other areas of introduction, and expected attainable range in North America (also habitat preference and climatic requirements of the agent)
S. camptodromus was collected from Sichuan and Yunnan provinces of southwestern
China located between 26.3 º N and 33.3 º N latitude at elevations of 1990 and 3200 meters (Fig.
1). Similar to the eastern United Sates, forests in China have diverse stands with a mixture of conifers and deciduous trees (Montgomery and Keena, 2011) and hemlock stands in China are shade tolerant and drought intolerant (Montgomery 1999). Climatic conditions where these predators were collected are more southerly in latitude and higher in altitude than the targeted
154 location of release in the eastern U. S. Seasonal temperature measurements between two collection sites (Nibago and Yunnan) in China and two potential release sites in the U.S. (VA and
NC) show that they share similar winter temperatures, between -21 º C and -18 ºC (Fig. 2); however, summer temperatures in China are much cooler. The cooler temperature in summer in
China at the time of collection was associated with a monsoon that occurred during that time
(Montgomery & Keena, 2011).
B.3.5 Source of the culture/agent in nature (name of collector, name of identifier)
S. camptodromus were collected in Yunnan and Sichuan provinces of the Southwestern
China (26.3 ºN and 33.3 ºN latitudes) by Zhao Jianhua in Sichuan and Li Li in Yunnan and identified by G. Y. Yu and H. P. Liu.
B.3.6 Host/biocontrol agent interaction (e.g., parasitoid, pathogen, parasite, competitor, antagonist, etc.)
No entomopathogens or hyperparasitoids have been discovered infecting or attacking S. camptodromus in shipments received from China. A parasitoid wasp was found emerging from other Scymnus spp. adults imported from China so parasitism will need to be monitored in any new shipments. To date, only one Scymnus sinuanodulus beetle has been found to have microsporidia out of 463 that have been tested. No S. camptodromus beetles have been tested for microsporidia but some will be soon.
155
B.3.7 Life history (including dispersal capability and damage inflicted on host insect or mite)
S. camptodromus life history information was collected during monthly sampling in
China and also a study was conducted in the USDA Forest Service quarantine facility in Ansonia,
CT.
S. camptodromus is a univoltine lady beetle. In China, the adults of the predator were found in abundance during spring and fall, while the larvae of the predator were present during
April and May. Under laboratory conditions, female beetles begin to lay eggs one month after emergence and oviposit about 10-14 eggs/female/week at 20 C. This predator is unique compared with other lady beetles from China because shortly after oviposition the eggs go into diapause and will not hatch until the next spring. Diapause guarantees survival of S. camptodromus when A. tsugae are not available and additionally helps it to withstand subfreezing temperatures. This also allows the predator to have a closely aligned life history to that of A. tsugae. Larval development of the predator occurs when there are A. tsugae eggs available in nature and according to the temperature data for collection sites in China, larval development occurs between 5-15 C. The lower threshold for larval development of the predator was determined to be close to 5 C in the laboratory (Limbu et al. 2014). There are four larval instars of S. camptodromus and its development depends on temperature. It takes about 40-43 days at 15
C and about 29 days at 20 C to develop to adult after eclosion (Table B-3). The degree-day requirement for development from neonate to adult for 10, 50, 90 and 99% of the S. camptodromus population was predicted as 397 ± 1.0, 424 ± 0.2, 467 ± 2.4 and 520 ± 5.0 DD, respectively. Both adult and larvae of the predator feed on different life stages of A. tsugae, although larvae will feed voraciously on A. tsugae eggs. The mean numbers of hemlock woolly adelgid eggs consumed by S. camptodromus by instar reared at 15 °C or 20 °C are shown in the
156
Table B- 4. Per day consumption by predator larvae was about 23 eggs at 20 C and 31 eggs at
15 C (Chapter 3, Figure 3-1).
B.3.8 Known host organism based on valid literature records, host data from museum specimens, and unpublished records.
Scymnus species in China were collected from three species of hemlock (Tsuga chinensis,
T. dumosa and T. forrestii) occurring in the Sino-Himalayan region. S. camptodromus was never been collected from other tree species co-occurring with hemlock species although other Scymnus species were also discovered on white pine.
B.3.9 History of past use of the biological control agent.
To our knowledge, S. camptodromus has never been used as a biocontrol agent.
B.3.10 Pathogens, parasites, hyperparasitoids of agent and how to eliminate them from a culture of the agent
Scymnus lady beetles have few parasites or diseases (Ceryngier & Hodek, 1996; Riddick et al., 2009). However, a parasitoid of concern would be Centistes scymni Ferriere, which was observed efficiently parasitizing Scymnus (Pullus) impexus (Mulsant). The Scymnus lady beetle was brought from Europe to control balsam woolly adelgid and special care was taken to avoid importing the parasitoid along with the beetle (Delucchi, 1954). Importantly, no pathogens, parasitoids, or hyperparasitoids have been observed attacking S. camptodromus, either in observations in China or in specimens shipped to the United States.
157 B.3.11 Standard Operating Procedure stating how agent will be handled in quarantine
An SOP was approved by APHIS for our quarantine laboratory and a foreign import permit specifies how the insect will be moved and handled in quarantine. We have developed standard rearing procedures for S. camptodromus in the laboratory.
B.3.12 Other closely related genera, sibling species or closely-similar species in North America
Scymnus is the largest genus of lady beetle with more than 600 described species
(Montgomery & Keena, 2011). All the Scymnus species in the IT IS database are listed below
(Table B-5). S. camptodromus fall in the Scymnini tribe and there are 6 genera that belong to the tribe in North America: Cryptolaemus, Didion, Scymnus, Nephus and Diomus (Gordon, 1985).
However, currently, Didion is considered as subgenera of genus Scymnus (Montgomery & Keena,
2011). There are seven subgenera of genus Scymnus; (Pullus) Mulsant, (Didion) Casey,
(Neopullus) Sasaji, (Parapullus) Yang, (Mimopullus) Fursch and (Orthoscymnus) Canepari
(Montgomery & Keena, 2011). S. camptodromus belongs to subgenus Scymnus (Neopullus) and there are 29 described species of Scymnus (Neopullus) but none of them are found in the Neartic zone (Chen et al., 2014).
There is one native lady beetle belonging to subgenus Scymnus (Pullus), currently being studied for the control of hemlock woolly adelgid. Scymnus (Pullus) coniferarum is native to the western United States and is known to feed on both Pineus strobi (Hartig) and A. tsugae. There also have been 17 non- native species of Scymnus lady beetles imported to the U.S. but only two of them are known to have established. Scymnus (pullus) impexus Mulsant, native to Europe, was introduced to control balsam woolly adelgid (Adelges piceae Ratzeburg) and Scymnus (Pullus) suturalis Thunberg, also native to Europe, was introduced to control adelgids on pine. Although
158 S. impexus showed promise, it is thought to have died out since its last recovery in 1978 in British
Columbia (Harris & Dawson, 1979). However, S. suturalis is established in several northeastern
States and has been found attacking P. strobi and A. tsugae.
B.4. Environmental & Economic Impacts of the Proposed Release
B.4.1 Known impact on vertebrates including humans
Establishment of S. camptodromus is not expected to have any undesirable impacts on vertebrates, including humans. It is also unlikely that S. camptodromus would become a nuisance in residences. In contrast to other nuisance lady beetles like Harmonia axyridis, S. camptodromus is not a generalist predator, does not have an aggregation behavior, and lays about
10-14 eggs/week/female at 20 ⁰C only with an ample supply of A. tsugae eggs (Montgomery &
Keena, 2011). Moreover, immediately after oviposition the predator eggs go into diapause requiring winter temperatures and will not hatch until the following spring when A. tsugae become available.
B.4.2 Direct impact of the organism (e.g. intended effects on targets, direct effects on non- targets)
S. camptodromus has been observed to have a direct effect on the target host A. tsugae.
The adults of S. camptodromus feed on all life stages of A. tsugae and larvae of Scymnus
(Neopullus) species are voracious and, although observed to consume other life stages of A. tsugae, they prefer its eggs (Lu et al., 2002; Montgomery & Keena, 2011).
A study on the number of HWA eggs an S. camptodromus adult can consume per day was done in late winter of 2013. A total of 59 lab reared fifth generation S. camptodromus adult
159 were used for these studies. Three strains DGS (22), MNP (31), and LJS (6) were monitored individually in 59 ml soufflé cups with 2.5 cm diameter clear lid (Solo, Eastern Bag and Paper,
CT). The predator adults were left for 72 hours in a 20 °C incubator with a photoperiod 16L:8D and 75% RH. On average it was observed that the predator adult consumed a little over 100
HWA eggs in 72 hours at 20C (Table B-6).
Another study was performed to determine development time and A. tsugae eggs consumed during the larval stage of S. camptodromus. A total of 150 S. camptodromus larvae
[DGS (51), LJS (74), MNP (25)] were reared at 15 °C (81) and 20 °C (69). The insects were maintained in environmental growth chambers at 32-40% RH and a 12L:12D photoperiod. Each
S. camptodromus larva was monitored for development, A. tsugae egg consumption, and survival until they reached adult eclosion. The total average consumption by S. camptodromus larvae to complete development to adult at 15 and 20 °C was 512 ± 16.0 and 454 ± 11.5 eggs, respectively
(Limbu et al. 2014). On average predator larvae consumed 31.4 ± 0.91 A. tsugae eggs per day at
20 °C, which is comparable to predation by the adults of this species reported previously (mean of 31 A. tsugae eggs per day) at 19 °C (Zhao et. al 1998), suggesting that S. camptodromus has considerable potential for biological control of A. tsugae.
B.4.3 Alternate Hosts Tested
To evaluate the feeding preference of S. camptodromus, the predator was given the choice of native adelgids (A. laricis, P. strobi, A. tsugae, A. cooleyi) and non-adelgid prey items
(F. externa, E. lanigerum, P. tessellate, A. gossypii) in choice and no-choice tests. Non-target testing on S. camptodromus showed that it will feed to some degree on other adelgid species, but highly prefers A. tsugae (Table B-7).
160 B.4.4 Choice Tests
A series of choice tests measuring feeding on A. tsugae and on other alternate hosts by S. camptodromus were conducted at 15 °C with a photoperiod of 12:12 (L:D) for 48 hours (four- way choice test) or 72 hours (three-way choice test and paired choice test). Three-way and four- way choice tests were performed among sympatric adelgid species (A. cooleyi, P. strobi, and A. laricis). In a three-way choice arena, a single adult predator was given the choice of A. tsugae and two other adelgid species, while in four-way choice tests, all three adelgid species were provided simultaneously along with A. tsugae. Paired choice tests were performed with a non- adelgid prey item (F. externa or E. lanigerum) where a single adult predator was given a choice of A. tsugae and one alternate prey item (Table B-8). The choice tests revealed that the predator distinctly preferred A. tsugae over tested adelgid and non-adelgid prey items (Chapter 2, Figure 2-
1).
B.4.5 No-choice Tests (Feeding)
No choice feeding tests were performed using adelgid prey items (A. cooleyi, P. strobi, A. laricis, and A. tsugae) as well as non-adelgid prey items (A. gossypii, P. tessellatus, F. externa, and E. lanigerum). An adult S. camptodromus was given only one type of prey item in a choice arena at 15 C with a photoperiod of 12:12 (L:D) h for 48 h. There were no significant differences in feeding preference of S. camptodromus on A. cooleyi, P. strobi, and A. laricis
(G2=1.2, P=0.28), which as a group differed significantly from A. tsugae (G2=6.2, P=0.013).
Predators were 1.9 (95% CI: 1.7, 2.1) times more likely to consume A. tsugae than the combined group of A. laricis, A. cooleyi, and P. strobi. Additionally, when P. tessellates or E. lanigerum was presented as the only prey choice, the adult S. camptodromus exhibited no evidence of
161 feeding. However, when F. externa was presented as the only choice, one prey item was chewed upon in one of the tests but was not consumed completely. On average, 3.7 nymphs of A. gossypii were consumed per adult predator.
B.4.6 No-choice Tests (Larval development)
A development test was done on the most preferred of the alternate hosts by placing a newly hatched S. camptodromus larva in a chamber and offering only P. strobi on white pine foliage as prey. Due to limited availability of beetle larvae and P. strobi, only 2 beetle larvae were tested. Of the two, only one of the larvae pupated and eclosed to adult, but it was malformed and died within 2 days of adult eclosion.
Hence, the choice and no-choice feeding test shows that S. camptodromus distinctly preferred A. tsugae over alternate adelgid and non-adelgid prey items.
Although there was an indication that this predator will feed on other adelgid species to some extent, it fed far less on anything that was not A. tsugae. Hence, S. camptodromus prefers adelgids over non-adelgids prey items and among tested adelgids strongly prefers
A. tsugae.
B.4.7 Possible direct or indirect effects on threatened and endangered species in North America
There are no known direct or indirect effects of S. camptodromus on threatened and endangered species in North America. Moreover, it is important to note that S. camptodromus consumed no P. tessellates in no-choice tests although other Scymnus species from China were found consuming this prey item to some extent (Butin et al. 2004). This is important since P.
162 tessellates is a primary food source for F. tarquinius, the only carnivorous butterfly in the continental United States (Butin et al., 2004; Scott, 1986). Under the circumstances of the host range study, S. camptodromus appears to be quite specific to A. tsugae posing no threat to non- target species.
B.5. Post Release Monitoring (In designing monitoring plans please note that pre- release baseline measurements of targets and non-targets provide for better monitoring data and documentation of effects. Also, some effects may take years or decades to manifest while others may not be long lasting)
Prior to release, the trees at the site will be sampled using the same methods to be employed during post-release sampling. Post-release sampling will be conducted beginning the year after the release of S. camptodromus from a minimum of 60 trees from each release site.
Monitoring of the predator will be conducted moving out in all directions up to one mile from the release sites during the first 5 years by beat sampling for 2 hours using light colored umbrellas from 6 heavily infested branches chosen at random from the lower and upper canopy of each tree.
Sampling will be done every two weeks in November, April, May and June when predators are likely to be active. Additionally, six samples of HWA infested branches from 60 trees selected at random will be clipped in the spring to look for predator larvae that may not fall off during beat sampling. Sampling for the larvae will also be done at the upper and lower canopy of the sampled tree. The collected larvae will be reared in the lab until emergence for identification of the predator. The length of the clipped twigs will be kept equal and density of the predator will be calculated.
Along with the post-release sampling for the predator, the impact of the release on A. tsugae populations will be evaluated in the release sites prior to the release and every few years after each release. For the estimation of A. tsugae population levels, five 15 cm branch tips will
163 be selected randomly from the lower and upper canopy of each of 30 trees per site. We will use a
sampling scheme to determine density of live settled sistens (sistens aestivation) on growing tips,
density of live sistens during October/November on previous year's growth (sisten activation),
and fecundity (sisten ovipostion) during early spring oviposition (about March). To sample for
fecundity, 30 ovisacs will be selected randomly from each sampled branch and eggs will be
counted.
Table B-1. Location of the source populations of Scymnus camptodromus from China (Keena et al. 2012)
Province County Site name Short Latitude Longitude Altitude(m) Year: collection name (north) (east) date Sichuan Jinchuan Sesiman SFF 31.30º 101.30º 2784 2005: June 28 Sichuan Baoxing Nibagou NBG 30.68º 102.68º 2750-2790 2005: May, Sept 2007: April 1, May 6 Sichuan Danba Dingguoshan DGS 30.61º 101.75º 2890-3100 2006: Oct. 5, Nov. 5- 11 2007: April 26 Yunnan Yulong Maoniuping MNP 27.17º 100.26º 3168-3170 2005: Sept. 25-28 2007: April 20, June 13, Nov. 23 Yunnan Yulong Laojunshan LJS 26.66º 99.81º 2930-3027 2005: Sept. 23 2007: April 21-22, May 25, June 11, Nov. 21 Yunnan Lanping Shanshenmiao LP 26.45º 99.34º 2780-2956 2007: May 23, June 10, Nov. 20
164 Table B-2. Important species of conifer-feeding adelgids (Havill & Foottit, 2007)
Species Hosts Distribution Origin Eastern U.S. to Rocky Adelges laricis Larch; red, black spruce Europe Mts. A. oregonensis Larch Pacific N.W. Unknown A. piceae (Balsam Western and eastern Balsam, Fraser Fir Europe woolly adelgid) N. America A. cooleyi (Cooley Colorado Blue, Engelmann, Western N. Western U.S. spruce gall adelgid) Sitka,White Spruce; Douglas fir America A. abietis (Eastern Norway, White Spruce Eastern U.S. and B.C. Europe spruce gall adelgid) A. tsugae (Hemlock Hemlock Eastern U.S. Japan woolly adelgid) A. lariciatus (Spruce North Larch, Norway spruce Eastern U.S. gall adelgid) America Pineus strobi (Pine White, Scots, Austrian North bark Eastern U.S. and CA. Pine America adelgid) P. pinifoliae (Pine leaf Red, Black spruce; White North Transcontinental adelgid) pine America Western and eastern P. boerneri Red pine Unknown U.S. Western and North P. coloradensis Red, pitch pine eastern U.S. America Eastern N. P. floccus White pine; red, black spruce Eastern U.S. America Norway, white, red, Colorado North P. similis blue spruce America North P. sylvestris Scots pine Eastern U.S. America
165 Table B-3. Mean development time [mean days ± SE (n)] by life stage of S. camptodromus reared at 15 or 20 °C after eclosion (Limbu et al. 2015). Larval stages Temp/Strain Pupal stage I II III IV 15 ⁰C DGS 6.1 ± 1.03e 3.4 ± 1.04ghij 4.2 ± 1.04f 15.6 ± 1.05a 14.0 ± 1.05ab
LJS 5.3 ± 1.03e 3.6 ± 1.03fgh 3.9 ± 1.03fg 13.9 ± 1.03ab 13.7 ± 1.04ab
MNP 5.3 ± 1.04e 3.8 ± 1.04fgh 3.8 ± 1.04fgh 15.1 ± 1.05a 14.6 ± 1.05a
20⁰C DGS 3.6 ± 1.04fghi 2.8 ± 1.04j 3.1 ± 1.04hij 8.9 ± 1.04cd 11.1 ± 1.04bc
LJS 3.6 ± 1.03fgh 3.2 ± 1.03ghij 3.0 ± 1.03ij 8.8 ± 1.03d 10.4 ± 1.04cd
MNP 3.8 ± 1.05fgh 3.2 ± 1.05ghij 3.1 ± 1.05hij 9.0 ± 1.05cd 10.7 ± 1.06cd
Table B-4. Mean number [mean ± SE (n)] of hemlock woolly adelgid eggs consumed by S. camptodromus by instar reared at 15 °C or 20 °C after larval eclosion (Limbu et al. 2015).
Larval instars Temp/strain I II II IV 15 ⁰C DGS 36.0 ± 3.2g 67.8 ± 6.0f 144 ± 12.6abd 208 ± 18.0ab
LJS 32.1 ± 2.4g 81.2 ± 5.7ef 178 ± 12.4ab 220 ± 15.2ab
MNP 31.2 ± 3.7g 81.6 ± 8.9ef 187 ± 19.8ab 236 ± 24.9a
20⁰C DGS 36.1 ± 3.6g 81.4 ± 7.7ef 164 ± 15.6ab 168 ± 16.0ab
LJS 29.2 ± 2.4g 104 ± 8.0ef 211 ± 16.1ab 96.4 ± 7.7def
MNP 28.4 ± 3.6g 102 ± 12.1ef 223 ± 25.7ab 127 ± 15.0bde
166
Table B-5. Listing of all the Scymnus species in IT IS database.
Obs. Scymnus species Geographic division Jurisdiction/Origin 1. Scymnus abbreviatus LeConte, 1852 – valid North America Canada (native) 2. Scymnus americanus Mulsant, 1850 – valid North America Continental U.S. (native) 3. Scymnus apicanus J. Chapin, 1973 – valid North America Canada, Continental U.S. (native) 4. Scymnus apiciflavus Motschulsky, 1858 – valid Oceania Southern Asia Hawaii (introduced) 5.. Scymnus apithanus Gordon, 1976 – valid North America Continental U.S. (native) 6. Scymnus aquilonarius Gordon, 1976 – valid North America Canada (native) 7. Scymnus ardelio Horn, 1895 – valid North America Canada, Continental U.S. 8.. Scymnus aridoides Gordon, 1976 – valid North America Continental U.S. (native) 9. Scymnus aridus Casey, 1899 – valid North America Canada, Continental U.S. 10. Scymnus barberi Gordon, 1976 – valid North America Canada, Continental U.S. 11. Scymnus binotulatus Boheman, 1859 – valid Oceania, Southern Asia Hawaii (introduced) 12. Scymnus brullei Mulsant, 1850 – valid North America Canada, Continental U.S. 13. Scymnus bryanti Gordon, 1976 – valid North America Continental U.S. 14. Scymnus caffer Gordon, 1976 – valid North America Continental U.S. 15. Scymnus calaveras Casey, 1899 – valid North America Canada, Continental U.S. 16. Scymnus carri Gordon, 1976 – valid North America Canada 17. Scymnus caudalis LeConte, 1850 – valid North America Canada, Continental U.S. 18. Scymnus caurinus Horn, 1895 – valid North America Canada, Continental U.S. 19. Scymnus cervicalis Mulsant, 1850 – valid North Ameria Continental U.S. 20. Scymnus circumspectus Horn, 1895 – valid North America Continental U.S. 21. Scymnus cockerelli Casey, 1899 – valid North America Continental U.S. 22. Scymnus compar Casey, 1899 – valid North America Canada, Continental U.S. 23. Scymnus coniferarum Crotch, 1874 – valid North America Canada, Continental U.S. 24. Scymnus consobrinus LeConte, 1852 – valid North America Continental U.S. 25. Scymnus coosi Hatch, 1961 – valid North America Continental U.S. 26. Scymnus creperus Mulsant, 1850 – valid North America Continental U.S.
167
Obs. Scymnus species Geographic division Jurisdiction/Origin 27. Scymnus difficilis Casey, 1899 – valid North America Continental U.S. 28. Scymnus dorcatomoides Weise, 1879 – valid Oceania, Southern Asia Hawaii (introduced) 29. Scymnus elusivus Gordon, 1976 – valid North America Continental U.S. 30. Scymnus enochrus Gordon, 1976 – valid Middle America, North America Continental U.S., Mexico 31. Scymnus erythronotum Gordon, 1976 – valid North America Continental U.S. 32. Scymnus falli Gordon, 1976 – valid North America Continental U.S. 33. Scymnus fenderi Malkin, 1943 – valid North America Canada, Continental U.S. 34. Scymnus festatus Wingo, 1952 – valid North America Continental U.S. 35. Scymnus flavescens Casey, 1899 – valid North America Continental U.S. 36. Scymnus fraternus LeConte, 1852 – valid North America Continental U.S. 37. Scymnus garlandicus Casey, 1899 – valid North America Continental U.S. 38. Scymnus gilae Casey, 1899 – valid North America Continental U.S. 39. Scymnus guttulatus LeConte, 1852 – invalid North America Continental U.S. 40. Scymnus hesperius Gordon, 1976 – valid North America Continental U.S. 41. Middle America, North America, Continental U.S., Mexico (native) Scymnus horni Gorham, 1897 – valid Oceania Hawaii(introduced) 42. Scymnus howdeni Gordon, 1976 – valid North America Continental U.S. 43. Scymnus huachuca Gordon, 1976 – valid North America Canada, Continental U.S. 44. Scymnus hubbardi Gordon, 1976 – valid North America Continental U.S. 45. Scymnus humboldti Casey, 1899 – valid North America Canada, Continental U.S. 46. Scymnus ignarus Gordon, 1976 – valid North America Continental U.S. 47. Europe & Northern Asia (except China), Continental U.S. Scymnus impexus Mulsant, 1850 – valid North America 48. Scymnus impletus Gordon, 1976 – valid North America Canada, Continental U.S. 49. Scymnus indianensis Weise, 1929 – valid North America Continental U.S. 50. Scymnus iowensis Casey, 1899 – valid North America Continental U.S. 51. Scymnus jacobianus Casey, 1899 – valid North America Continental U.S. 52. Scymnus kansanus Casey, 1899 – valid North America Canada, Continental U.S.
168
Obs. Scymnus species Geographic division Jurisdiction/Origin 53. Scymnus lacustris LeConte, 1850 – valid North America Canada, Continental U.S. 54. Europe & Northern Asia (excluding Hawaii (introduced) Scymnus levaillanti Mulsant, 1850 – valid China) Oceania 55. Middle America, North America, Continental U.S., Mexico (Native) Hawaii Scymnus loewii Mulsant, 1850 – valid Oceania (introduced) 56. Scymnus louisianae J. Chapin, 1973 – valid North America Continental U.S. 57. Scymnus luctuosus Casey, 1899 – valid North America Continental U.S. 58. Scymnus majus Gordon, 1985 – valid North America Continental U.S. 59. Scymnus marginicollis Mannerheim, 1843 – valid North America Canada, Continental U.S. 60. Scymnus margipallens (Mulsant, 1850) – valid Middle America, South America Mexico 61. Scymnus martini Gordon, 1976 – valid North America Continental U.S. 62 Scymnus mendocino Casey, 1899 – valid North America Continental U.S. 63. Scymnus mimoides Gordon, 1976 – valid North America Continental U.S. 64. Scymnus monticola Casey, 1899 – valid North America Continental U.S. 65. Scymnus mormon Casey, 1899 – valid North America Continental U.S. 66 North America, Oceania Canada, continental U.S. (Native) Hawaii Scymnus nebulosus LeConte, 1852 – valid (introduced) 67 Scymnus nemorivagus Wingo, 1952 – valid North America Continental U.S. 68. Scymnus neomexicanus Gordon, 1976 – valid North America Continental U.S. 69. Scymnus nevadensis Weise, 1929 – valid North America Continental U.S. 70. Scymnus nigricollis Gordon, 1976 – valid North America Continental U.S. 71. Scymnus notescens (Blackburn, 1889) – valid Australia 72. Scymnus nugator Casey, 1899 – valid North America Continental U.S. 73. Scymnus nuttingi Gordon, 1976 – valid North America Continental U.S. 74. Scymnus ocellatus Sharp, 1885 – valid Oceania Hawaii 75. Scymnus opaculus Horn, 1895 – valid North America Canada, Continental U.S. 76. Scymnus pacificus Crotch, 1874 – valid North America Continental U.S. 77. Scymnus pallens LeConte, 1852 – valid North America, Oceania Continental U.S. (native) Hawaii (introduced)
169
Obs. Scymnus species Geographic division Jurisdiction/Origin 78. Scymnus papago Casey, 1899 – valid North America Continental U.S. 79. Scymnus paracanus J. Chapin, 1973 – valid North America Canada, Continental U.S. 80. Scymnus paracanus linearis Gordon, 1976 – valid North America Canada, Continental U.S. 81. Scymnus paracanus paracanus J. Chapin, 1973 – valid North America Continental U.S. 82. Scymnus pauculus Gordon, 1976 – valid North America Continental U.S. 83. Scymnus peninsularis Gordon, 1976 – valid North America Continental U.S. 84. Scymnus phelpsii Crotch, 1874 – invalid North America Continental U.S. 85. Scymnus postpictus Casey, 1899 – valid North America Canada, continental U.S. 86. Scymnus pulvinatus Wingo, 1952 – valid North America Continental U.S. 87. Scymnus puncticollis LeConte, 1852 – valid North America Canada, Continental U.S. 88. Scymnus quadrivittatus Mulsant, 1850 – valid Africa, Oceania Hawaii 89. Scymnus renoicus Casey, 1899 – valid North America Continental U.S. 90. Scymnus rubricaudus Casey, 1899 – valid North America Continental U.S. 91. Scymnus securus J. Chapin, 1973 – valid North America Canada, Continental U.S. 92. Scymnus semiruber Horn, 1895 – valid North America Continental U.S. 93. Scymnus simulans Gordon, 1976 – valid North America Continental U.S. 94. Scymnus socer LeConte, 1852 – valid North America Continental U.S. 95. Scymnus solidus Casey, 1899 – valid North America Canada, Continental U.S. 96. Europe & Northern Asia (excluding Hawaii (Introduced) Scymnus subvillosus (Goeze, 1777) – valid China), Oceania 97. Europe & Northern Asia (excluding Continental U.S. & Hawaii (introduced) Scymnus suturalis Thunberg, 1795 – valid China), Oceania 98. Scymnus tahoensis Casey, 1899 – valid North America Canada & continental U.S. (Native) 99. Scymnus tenebricus Gordon, 1976 – valid North America Continental U.S. 100. Scymnus tenebrosus Mulsant, 1850 – valid North America Canada & Continental U.S. (native) 101. Middle America, North America, Alaska & Hawaii (introduced) Mexico Scymnus uncinatus Sicard, 1924 – valid Oceania (native) 102. Scymnus uncus Wingo, 1952 – valid North America Continental U.S.
170
Obs. Scymnus species Geographic division Jurisdiction/Origin 103. Scymnus utahensis Gordon, 1976 – valid North America Continental U.S. 104. Scymnus uteanus Casey, 1899 – valid North America Continental U.S. 105. Scymnus varipes Blackburn, 1896 – valid Australia, Oceania Hawaii (introduced) 106. Scymnus vividus Sharp, 1885 – valid Oceania Hawaii (Native) 107. Scymnus weidti Casey, 1899 – valid North America Continental U.S. 108. Scymnus wickhami Gordon, 1976 – valid North America Continental U.S. 109. Scymnus wingoi Gordon, 1976 – valid North America Continental U.S.
171
Table B-6. Mean [± SEM (n)] HWA eggs consumed by three different strains of S. camptodromus adults per day in the lab. Predator strain Predator generation Average A. tsugae eggs consumed
DGS 5 34.5 ± 3.5 (20)
LJS 5 39.3 ± 2.2 (33)
MNP 5 32.1 ± 7.4 (6)
172 Table B-7. Test prey on associated host plant, native range of test prey, and rationale for selection of prey item used in host specificity tests (Limbu et al. 2016). Prey Host plant Native range Rationale for
testing
Adelges tsugae Annand(Hemlock Tsuga canadensis (L.) Japan Target pest woolly adelgid) Carrière (Eastern hemlock)
Pineus strobi (Hartig) Pinus strobus (L.) Eastern North Taxonomic America similarity
Adelges cooleyi (Gillette) (Cooley Pseudotusga menziesii Western North Taxonomic spruce gall adelgid) (Mirb.) Franco America similarity
Adelges laricis Vallot (larch Larix kaempferi Europe Taxonomic adelgid) (Lam.) Carrière similarity
Fiorinia externa Ferris (elongate T. Canadensis Japan Co-occur on same hemlock scale) host
Paraprociphilus tessallatus Fitch Alnus serrulata (Ait.) Eastern North Ecological (Woolly alder aphid) Willd America significance
Eriosoma lanigerum Hausm. Malus domestica North America Similar to woolly (Woolly apple aphid) Borkh. alder aphid
Aphis gossypii Glover (Cotton Hibiscus rosa Worldwide Another aphid aphid) species
Native range information for adelgids derived from (Havill & Foottit, 2007).
173 Table B-8. Mean (±S.E) number of non-adelgid prey items eaten in 72 h in paired choice tests. Mean A. tsugae Mean alternate host Host n consumed consumed A. tsugae vs F. externa 10 15.1 ± 2.9 0.1 ± 0.1 A. tsugae vs E. lanigerum 10 19.3 ± 2.9 1.0± 0.4
Figure B-1. Map showing the primary collection sites in China of three species of Scymnus brought to the eastern U.S. (Montgomery and Keena 2011)
174
Figure B-2 Average monthly temperatures (º C) in selected locations in China and in potential release sites in the U.S. (Mongomery and Keena 2011)
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179 VITA Samita Limbu was born on February 3, 1988, in a small city called Lalitpur in Kathmandu valley in Nepal. She completed her undergraduate degree with honors in Agriculture from Himalayan College of Agricultural and Sciences and Technology (HICAST) in Kathmandu, Nepal in 2010. During her undergraduate studies, she also got internship opportunities with Dr. Luke A. Colavito to work on Integrated Pest Management (IPM) program designed for small farmers at Winrock International and International Development Enterprises (IDE) in Nepal funded by USAID. After completing her undergraduate program she moved to the US for graduate studies in Entomology. In fall 2011, she joined the Pennsylvania State University and began her graduate research with Dr. Kelli Hoover. Her research was particularly focused on the study of effects of temperature on exotic insect’s biology and life-stage duration for estimation of degree-day requirement for key life-stage with the goal to improve management practices. She graduated with a Ph.D. in Entomology in Dec of 2016. Her publications are listed below:
1. Limbu, S., M. Keena, F. Chen, G. Cook, H. Nadel, and K. Hoover. 2017. Effects of temperature on development of Lymantria dispar asiatica and Lymantria dispar japonica (Lepidoptera: Erebidae). Environ. Entomol. Submitted 2. Limbu, S., K. Cassidy, M. Keena, P. Tobin, and K. Hoover. 2016. Host range specificity of Scymnus camptodromus (Coleoptera: Coccinellidae), a predator of hemlock woolly adelgid. Environ. Entomol. DOI: http://dx.doi.org/10.1093/ee/nvv174. 3. Limbu, S., M. Keena, D. Long, N. Ostiguy, and K. Hoover. 2015. Scymnus camptodromus (Coleoptera: Coccinellidae) larval development and predation of hemlock woolly adelgid. Environ. Entomol. 44(1): 81-89. 4. Limbu, S., M. Keena, D. Long and K. Hoover. 2014. Scymnus camptodromus Development and Predation of Hemlock Woolly Adelgid. Proceedings of the 2014 Interagency Research Forum on Invasive Species January 2014. Poster abstract. 5. Limbu, S., K. Cassidy, M. Keena, D. Long and K. Hoover. 2013. Development of Scymnus camptodromus for Biological Control of Hemlock Woolly Adelgid. In Mcmanus and Gottschalk Eds. Proceeding of the 2013 Interagency Research Forum on Invasive species January 2013. Poster abstract.