Non-Target Effects of Soybean Aphid, Aphis Glycinces Matsumura (Hemiptera: Aphididae), Management in Iowa Wayne J

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2008 Non-target effects of soybean aphid, Aphis glycinces Matsumura (Hemiptera: Aphididae), management in Iowa Wayne J. Ohnesorg Iowa State University

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This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Non-target effects of soybean aphid, Aphis glycinces Matsumura (Hemiptera: Aphididae), management in Iowa

Wayne J. Ohnesorg

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Entomology

Program of Study Committee: Matthew E. O’Neal, Major Professor Joel R. Coats Diane M. Debinski

Iowa State University

Ames, Iowa

2008

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Table of Contents

Chapter 1. General Introduction and Literature Review 1 Thesis Organization 1 Introduction 1 Aphis glycines biology and ecology 1 Aphis glycines natural enemies 2 Conservation biological control 3 Aphis glycines management 4 Objectives 5 Citations 6

Chapter 2. Impact of reduced-risk insecticides on soybean aphid and their natural enemies 12 Abstract 12 Introduction 13 Materials and Method 15 Results 21 Discussion 26 Acknowledgements 29 Citations 30 Tables 37 Figure Legend 47 Figures 49

Chapter 3. Community assessment of aphids in Iowa prairies prior to the release of an exotic parasitoid 55 Abstract 55 Introduction 56 Materials and Method 60 Results 65 Discussion 70 Acknowledgements 72 Citations 73 Tables 79 Figure Legend 98 Figures 100

Chapter 4. Are prairies a source of Aphis glycines (Hemiptera: Aphididae) predators? 111 Abstract 111 Introduction 112 iii

Materials and Method 114 Results 118 Discussion 122 Acknowledgements 125 Citations 126 Tables 132 Figure Legend 143 Figures 145

Chapter 5. General Conclusions 157

Acknowledgements 159

Chapter 1

General Introduction and Literature Review

Thesis Organization

This thesis has been organized into 5 chapters. Chapter 1 contains the general introduction and literature review. Chapter 2 contains the experiment conducted with

reduced-risk insecticides and their efficacy for soybean aphid management and

conservation of natural enemies. Chapter 3 is part of a community assessment of aphids

in Iowa prairies prior to the release of an Asian parasitoid. Chapter 4 includes the study

of prairies as a source of natural enemies for soybean biological control. Chapter 5 is the

general conclusions and summary.

Introduction and Literature Review

Aphis glycines biology and ecology

Aphis glycines Matsumura (Hemiptera: Aphididae) has become a pest of soybean

(Glycine max) in North America. First discovered in Wisconsin in 2000 (Wedburg 2000,

Alleman et al. 2002), it has since spread across the Midwest and several Canadian provinces and by 2003 could be found across Iowa (O’Neal 2006). Prior to the invasion of A. glycines soybean in the central region of the United States, there was limited need 2

for pest management strategies (Fernandez-Cornejo and Jans 1999). In 2003, more than

1.6 million ha of soybean received insecticide applications for control of A. glycines populations that reached several thousand per plant (Pilcher and Rice 2005). Soybean yield losses from A. glycines herbivory of 15-40% have been recorded (Ragsdale et al.

2007).

Aphis glycines has a heterecious holocyclic life cycle typical of many aphids.

Eggs can be found on Rhamnus spp. (Rhamnaceae) and hatch in the spring. Each egg that hatches produces a fundatrix. A fundatrix will reproduce asexually giving rise to apterous viviparous females. Asexual reproduction will continue to take place for 3-4 generations.

During the third and fourth generations winged adult females (alate viviparous females) are produced. These winged females will emigrate in search of a secondary host, which for A. glycines is typically soybean. Once on soybean, A. glycines are capable of doubling its population every 1.5 days resulting in 15-18 overlapping generations, under favorable conditions (McCornack et al. 2004, Myers et al. 2005). Given their ability to migrate great distances on weather fronts, it is possible for soybean fields to be at risk in areas where A. glycines does not overwinter. Later in the growing season, the senescing soybean plants and reduction in photoperiod cause the production of gynopara. The gynopara are winged females that migrate back to Rhamnus spp. Also produced are winged males, which migrate to Rhamnus spp. The gynopara produce ovipara that mate with the winged males to produce the overwintering eggs. Eggs are typically deposited at the base of buds (Ragsdale et al. 2004, Wu et al. 2004, McCornack et al. 2005, Voegtlin et al. 2005). 3

Aphis glycines natural enemies

Competitors, pathogens, predators, and parasitoids are all types of natural enemies

of A. glycines. In North America, A. glycines is the only aphid to successfully colonize soybean and produce economically important populations. Entomopathogenic fungi have

been identified to play a role in A. glycines population regulation in New York State

(Nielson and Hajek 2005). However, there is little evidence that pathogens play a role in

population regulation in the north central region of the United States. Endemic predators

can regulate populations of A. glycines (Fox et al. 2004, Rutledge et al. 2004, Fox et al.

2005, Rutledge and O’Neil 2005, Costamagna and Landis 2006, Desneux et al. 2006,

Mignault et al. 2006, Schmidt et al. 2007). Two key predators of A. glycines have been identified, Harmonia axyridis Pallas (Coleoptera: Coccinellidae) (Rutledge et al. 2004) and Orius insidiosus (Say) (Hemiptera: Anthocoridae) (Rutledge et al. 2004, Rutledge and O’Neil 2005). However, A. glycines outbreaks still occur despite the mortality produced by predators.

Conservation biological control

Conservation biological control has been defined as modifying the environment or management practices to enhance natural enemies (e.g., aphid predators) and their deleterious effects on pests (Hajek 2004). Habitat management is one method of

conservation biological control that has been successful (Landis et al. 2000) in reducing

the impact of agricultural insect pest. Non-crop area provided for habitat management 4

can enhance natural enemies by providing alternate hosts (DeBach and Rosen 1991,

Menalled et al. 1999), shelter (Gurr et al. 1998), and non-host food (Baggen et al. 1999,

Wilkinson and Landis 2005). Cereal crops bordered with Phacelia tanacetifolia

(Hydrophyllaceae) had higher rates of syrphid-derived aphid predation, attributed to adult flies utilizing this floral resource (Sengonça and Frings 1988, Hickman and Wratten

1996). Recent evidence suggests that natural enemies that require a floral resource may benefit more from native, perennial plants than other commonly used exotic plant species

(Fiedler and Landis 2007a,b). Of the 43 native species examined, 35 occur in prairies. In

Iowa and other parts of the Midwest, prairies may act as a refuge for conservation of A. glycines predators. Prairies are grassland communities consisting of primarily grasses and forbs. This community was once a major component of the landscape of the Midwest, prior to European settlement. In Iowa, prairie now covers less than 0.1% of the area it once occupied (Smith 1998). This reduced habitat may be able to provide a valuable ecosystem service in serving as a source of predators and other natural enemies for the biological control of A. glycines.

Aphis glycines management

Despite the natural control endemic natural enemies provide, there is still a need for foliar insecticides when soybean aphid populations reach economically damaging levels (Ragsdale et al. 2007). Current economic thresholds for A. glycines are ~250 aphids per plant, with growing populations (Ragsdale et al. 2007). Furthermore, in North

America there may be a need for soybean growers to apply an insecticide to manage 5

additional arthropod pests before the occurrence of A. glycines outbreaks, such as the

bean leaf beetle (Coleoptera: Chrysomelida Ceratoma trifurcata) and two-spotted spider

mite (Acari: Tetranychus urticae Koch). Many A. glycines predators are present in fields

before the soybean aphid arrives (e.g. O. insidiosus) (Rutledge et al. 2004), the

application of a broad-spectrum insecticide may disrupt the natural control they provide

(Johnson et al. in press).

In light of the role predators play in delaying and suppressing soybean aphids,

growers may need to replace broad-spectrum insecticides for those that have a limited

impact on natural enemies, i.e. reduced-risk insecticides. The environmental protection

agency (EPA) defines reduced-risk insecticides as “insecticides that may reasonably be

expected to accomplish one or more of the four following objectives: 1) Reduce the risks

of pesticides to human health, 2) Reduce the risks of pesticides to non-target organisms,

3) Reduce the potential for contamination of groundwater, surface water or other valued

environmental resources, and 4) Broaden the adoption of integrated pest management

strategies, or make such strategies more available or more effective” (Anon 1997).

Insecticides that accomplish the first, second, and fourth objectives would be valuable for the management of A. glycines. To date, the potential for reduced-risk insecticides to manage A. glycines has not been explored.

Objectives

During the summers of 2005 and 2006 I studied Aphis glycines and the effects of

reduced-risk insecticides on the foliar natural enemies in soybean (Glycine max (L.)). In 6

the summers of 2006 and 2007 I studied the native aphid community in central Iowa prairies along with the aphidophagous and generalist predatory arthropod community.

The objectives were:

Chapter 2 objective:

1) Determine whether the impact of neonicotinoid insecticides is indistinguishable

from a commonly used, broad-spectrum insecticide (-cyhalothrin) and a reduced-

risk insecticide (pymetrozine).

Chapter 3 objectives:

1) Identify aphids, chiefly in the genus Aphis, which may experience deleterious

effects from Binodoxys communis (Gahan) (Hymenoptera: Braconidae).

2) Determine whether the aphid community in central Iowa will differ between the

three prairie types of remnant, reconstructed, and integrated.

3) Determine whether ant tending of the aphid genus Aphis and specifically Aphis

monardae differs between the three prairie types: remnant, reconstructed, and

integrated.

Chapter 4 objectives:

1) Describe the diversity and abundance of the foliar active aphidophagous arthropod

community found in prairies in central Iowa. 7

2) Determine whether the natural enemy community in central Iowa prairies differs

between the three prairie types: remnant, reconstructed, and integrated.

Citations

Anonymous, EPA. 1997. Pesticide Registration (PR) Notice 97-3. United States

Environmental Protection Agency, September 4, 1997, Accessed March 19, 2008.

http://www.epa.gov/PR_Notices/pr97-3.html.

Baggen, L.R., G.M. Gurr, and A. Meats. 1999. Flowers in tri-trophic systems:

mechanisms allowing selective exploitation by insect natural enemies for

conservation biological control. Entomologia experimentalis et applicata 91: 155-

161.

Costamagna, Alejandro C., and Douglas A. Landis. 2006. Predators exert top-down

control of soybean aphid across a gradient of agricultural management systems.

Ecological Applications 16(4):1619-1628.

DeBach, P., and D. Rosen. 1991. Biological control by natural enemies. Cambridge

University Press, Cambridge, UK.

Desneux, Nicolas, Robert J. O’Niel, and Ho Jung S. Yoo. 2006. Suppression of the

soybean aphid, Aphis glycines Matsumura, by predators: The identification of a 8

key predator and the effects of prey dispersion, Predator Abundance, and

Temperature. Environmental Entomology 35(5): 1342-1349.

Fernandez-Cornejo, J., and S. Jans. 1999. Pest management in U.S. Agriculture.

Resource Economics Division, Economic Research Service, U.S. Department of

Agriculture. Agriculture Handbook No 717.

Fiedler, A.K. and D.A. Landis. 2007a. Attractiveness of Michigan native plants to

arthropod natural enemies and herbivores. Environmental Entomology 36(4): 751-

765.

Fiedler, A.K. and D.A. Landis. 2007b. Plant characteristics associated with natural

enemy abundance at Michigan native plants. Environmental Entomology 36(4):

878-886.

Fox, T. B., D.A. Landis, F. F. Cardoso, and C.D. Difonzo. 2004. Predators Suppress

Aphis glycines Matsumura Population Growth in Soybean. Environmental

Entomology 33(3): 608-618.

Fox, T. B., D. A. Landis, F. F. Cardoso, and C.D. Difonzo. 2005. Impact of predation

on establishment of the soybean aphid, Aphis glycines in soybean, Glycine max.

BioControl 50:545-563.

Gurr, G.M., H.F. van Emden and S.D. Wratten. 1998. Habitat manipulation and

natural enemy and natural enemy efficiency: implications for the control of pests,

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Diego, CA. 9

Hajek, Ann. 2004. Natural Enemies: An Introduction to Biological Control. pp. 80-96.

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Hickman, Janice M. and Stephen D. Wratten. 1996. Use of Phacelia tanacetifolia

Strips to Enhance Biological Control of Aphids by Hoverfly Larvae in Cereal

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Johnson, K. D., M. E. O'Neal, J. Bradshaw, M. Rice. in press. Is preventative,

concurrent-management of the soybean aphid (Hemiptera: Aphididae) and bean

leaf beetle (Coleoptera: Chrysomelidae) possible? Journal of Economic

Entomology in press.

Landis, Douglas A., Stephen D. Wratten, and Geoff M. Gurr. 2000. Habitat

management to conserve natural enemies of arthropod pests in agriculture.

Annual Review of Entomology 45:175-201.

McCornack, B.P., D.W. Ragsdale, and R.C. Vennette. 2004. Demography of soybean

aphid (Homoptera: Aphididae) at summer temperatures. Journal of Economic

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McCornack, B.P., M.A. Carrillo, R.C. Vennette, and D.W. Ragsdale. 2005.

Physiological constraints on the overwintering potential of the soybean aphid

(Homoptera: Aphididae). Environmental Entomology 34: 235-240.

Menalled, F.D., P.C. Marino, S.H. Gage, and D.A. Landis. 1999. Does agricultural

landscape structure affect parasitism and parasitoid diversity? Ecological

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Mignault, Marie-Pierre, Michéle Roy, and Jacques Brodeur. 2006. Soybean aphid

predators in Quebec and the suitability of Aphis glycines as prey for three

Coccinellidae. BioControl 51:89-106.

Myers, S.W., and C. Gratton. 2006. Influence of potassium fertility on soybean aphid,

Aphis glycines Matsumura (Hemiptera: Aphididae), population dynamics at a

field and regional scale. Environmental Entomology 35(2): 219-227.

Nielsen, C., and A.E. Hajek. 2005. Control of invasive soybean aphid, Aphis glycines

(Hemiptera: Aphididae), populations by existing natural enemies in New York

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34(5): 1036-1047.

Ragsdale, D.W., D.J. Voegtlin, and R.J. O’Neil. 2004. Soybean aphid biology in North

America. Annals of the Entomological Society of America 97: 204-208.

Ragsdale, D. W., B. P. McCornack, R. C. Venette, D. A. Potter, E. W. MacRae, E. W.

Hodgson, M. E. O'Neal, K. D. Johnson, R. J. O'Neil, C. D. Di Fonzo, T. E.

Hunt, P. A. Glogoza, and E. M. Cullen. 2007. Economic threshold for soybean

aphid (Homoptera: Aphididae). Journal of Economic Entomology 100(4): 1258-

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predators and their use in integrated pest management. Annals of the Entomological

Society of America 97(2): 240-248.

Rutledge, C. E. and R. J. O’Neil. 2005. Orius insidiosus (Say) as a predator of the

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Schmidt, Nicholas P., Matthew E. O’Neal, and Jeremy W. Singer. 2007. Alfalfa

living mulch advances biological control of soybean aphid. Environmental

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Sengonça, C. and B. Frings. 1988. Eifluss von Phacelia tanacetifolia auf Schadlings

und Nutzlingspopulationen in Zuckerrubenfeldern. Pedobiologia 32: 311-316.

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attempts. Journal of the Iowa Academy of Science 105: 275-284.

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the role of plant resource, pp. 305-325. In F.L. Wackers, P.C.J. van Rijn, and J.

Bruin (eds.), Plant-provided food for carnivorous insects. Cambridge University

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Society of America 97: 209-218. 12

Chapter 2

Impact of reduced-risk insecticides on soybean aphid and associated natural

enemies

A paper to be submitted to Journal of Economic Entomology

Wayne J. Ohnesorg, Kevin D. Johnson, and Matthew E. O’Neal

Abstract

Since the arrival of the soybean aphid, Aphis glycines Matsumura (Hemiptera:

Aphididae), in Iowa, insecticide usage has increased from fewer than 10,000 acres to as

much as 3 million acres. In field experiments conducted over two years (2005 and 2006)

three reduced-risk insecticides (imidacloprid, pymetrozine, and thiamethoxam) were

compared to a broad-spectrum insecticide (-cyhalothrin) and evaluated on three

criterion; 1) effectiveness in reducing A. glycines populations, 2) yield protection, 3) and

impact to natural enemies. In both 2005 and 2006, the foliar insecticide -cyhalothrin

provided the greatest reduction in A. glycines exposure, and the greatest level of yield

protection. The seed treatments imidacloprid and thiamethoxam provided the lowest level of A. glycines control and the lowest yield protection compared to the control.

Pymetrozine and the foliar application of imidacloprid provided an intermediate level of protection both in terms of soybean exposure to A. glycines, and yield protection when compared to -cyhalothrin. Seed treatments had no observable effect on the abundance of natural enemies. All foliar applied insecticides resulted in a reduction of the abundance of natural enemies compared to the untreated control. Pymetrozine and the foliar application 13

of imidacloprid were intermediates in the abundance of natural enemies compared to the untreated control and -cyhalothrin.

Introduction

In North America natural enemies, particularly foliage inhabiting predators, can suppress

Aphis glycines Matsumura (Hemiptera: Aphididae) populations (Fox et al. 2004, 2005,

Rutledge and O'Neil 2005, Mignault et al. 2006, Schmidt et al. 2007). Of the several species of predators that can be found in soybeans (Schmidt et al. 2008, Bechinski and

Pedigo 1981), two have been identified as key predators of A. glycines in North America,

Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) (Rutledge et al. 2004, Rutledge and O'Neil 2005) and Orius insidiosus (Say) (Hemiptera: Anthocoridae)(Rutledge and

O'Neil 2005, Desneux et al. 2006). Despite the natural control these predators provide, there is still a need for foliar insecticides when A. glycines populations reach economically damaging levels (Ragsdale et al. 2007). Furthermore, in North America there may be a need for soybean growers to apply an insecticide to manage additional arthropod pests before the occurrence of A. glycines outbreaks, such as the bean leaf beetle (Coleoptera: Chrysomelidae Ceratoma trifurcata) and two-spotted spider mite

(Acari: Tetranychus urticae Koch). Many A. glycines predators are present in fields before arrival of A. glycines (e.g. O. insidiosus) (Rutledge et al. 2004); the application of a broad-spectrum insecticide may disrupt the natural control they provide (Johnson et al. in press). 14

In light of the role predators play in delaying and suppressing A. glycines, growers

may need to replace broad-spectrum insecticides for ones that have a limited impact on

natural enemies (i.e. reduced-risk insecticides). The Environmental Protection Agency

(EPA) defines reduced-risk insecticides as “insecticides that may reasonably be expected

to accomplish one or more of the four following objectives: 1) Reduce the risks of

pesticides to human health, 2) Reduce the risks of pesticides to non-target organisms, 3)

Reduce the potential for contamination of groundwater, surface water or other valued

environmental resources, and 4) Broaden the adoption of integrated pest management

strategies, or make such strategies more available or more effective” (Anon 1997).

Insecticides that accomplish the first, second, and fourth objectives would be valuable for the management of A. glycines. To date the potential for reduced-risk insecticides to manage A. glycines has not been explored. For such potential to be realized, the impact of putative reduced-risk insecticides on natural enemies should be assessed.

Our goal is to determine whether currently available insecticides considered reduced-risk by the EPA could be use to protect soybean yield from A. glycines herbivory while having a limited impact on the natural enemies. To accomplish this goal we tested several insecticides to determine whether they fit the definition of a reduced-risk insecticide within a soybean production system. Our objective is to determine if the impacts of neonicotinoid insecticides are indistinguishable from a commonly used, broad-spectrum insecticide (-cyhalothrin) and a reduced-risk insecticide (pymetrozine).

The former product has been shown to be effective in reducing the impact of soybean aphids on soybean yield (Ragsdale et al. 2007). The latter is a Hemipteran-specific 15

insecticide that is considered a reduced-risk insecticide and used in cropping systems

(Kayser et al. 1994, Harrewijn and Kayser 1997, Wyss and Bolsinger 1997b, Sechser et

al. 2002, Torres et al. 2003, Banks and Stark 2004). These two extremes were evaluated

with currently available systemic (apoplastic plant mobile) neonicotinoid insecticides

(imidacloprid and thiomethoxam) applied to either the seed or foliage. The impact of

these insecticides was measured on both A. glycines and foliar dwelling natural enemies during the 2005 and 2006 growing seasons.

Materials and Methods

Field site. The field site for this experiment was located at the Iowa State University

North East Research Farm in Floyd County, Iowa. In both 2005 and 2006 no-till production practices were used, with commercially available (NK S24-K4 RR in 2005 and NK S23-Z3 RR in 2006; NK brand Syngenta Seeds, Golden Valley, MN) varieties of

soybean considered susceptible to A. glycines. In 2005, soybeans were planted on 22 May

in 5-m by 30-m plots. In 2006, soybeans were planted on 6 May in 10-m by 15-m plots.

In both years soybeans were planted at 76-cm row spacing and 470,000 seeds per hectare.

Experimental design. To evaluate the impact of reduced-risk insecticides on A. glycines

and its associated natural enemies, we employed a randomized complete block design

with six insecticide treatments (Table 1) along with an untreated control; each treatment

was replicated six times. In 2006, a “zero aphid” treatment was added. The zero aphid

treatment received an insecticide application whenever aphids were detected, and this 16

treatment was meant to represent the maximum yield possible under existing field

conditions.

Insecticides. Putative reduced-risk insecticide treatments were selected based on known

specificity to the target pest (like pymetrozine) or systemic activity and thus encountered only when ingested by the herbivore (thiamethoxam, imidacloprid); the experiment was designed to determine if they will have reduced environmental exposure and limited impact on the natural enemy community within soybeans. Pymetrozine (Fulfill™,

Syngenta Crop Protection, Inc., Greensboro, NC) was included based on its novel mode of action in that it causes paralysis of the cibarial muscle, which subsequently prevents feeding in the Hemipteran suborder Sternorrhyncha (Kayser et al. 1994, Harrewijn and

Kayser 1997, Wyss and Bolsinger 1997b, Sechser et al. 2002, Torres et al. 2003, Banks and Stark 2004). In addition to its selective mode of action, pymetrozine is plant systemic (symplasticly mobile) which results in a further reduction in exposure to non- target organisms (Kayser et al. 1994, Harrewijn and Kayser 1997, Wyss and Bolsinger

1997a, 1997b, Sechser et al. 2002, Torres et al. 2003, Banks and Stark 2004). The neonicotinoid seed treatments, thiamethoxam (Cruiser™ 5FS, Syngenta Crop Protection,

Inc., Greensboro, NC) and imidacloprid (Gaucho™ 480FS Bayer Crop Science) are plant systemic (apoplastically mobile) insecticides that should result in reduced environmental exposure to non-target organisms. A foliar formulation of imidacloprid (Trimax™, Bayer

Crop Science) was chosen as well for comparison. The pyrethroid insecticide - 17

cyhalothrin (Warrior™, Syngenta Crop Protection, Inc., Greensboro, NC) was chosen to represent a foliar-applied, broad-spectrum insecticide that is commonly used in soybeans to manage the A. glycines.

Foliar insecticides were to be applied at the economic threshold (250 A. glycines per plant; Ragsdale et al. 2007). However due to low populations of A. glycines applications during the 2006 growing season, foliar insecticides were applied during the same calendar period as in 2005. A backpack sprayer and hand boom were used to apply insecticides with TeeJet 11002 Twin Jet nozzles, 38.1-cm nozzle spacing, 275 kPa pressure, and 187 liters hectare-1 of carrier (water). Seed treatments were applied to soybean seeds prior to planting. All insecticide rates can be found in Table 1. Due to the lack of current soybean registration, the foliar formulations of imidacloprid (Trimax™) and pymetrozine (Fulfill™) were applied based on recommendations from the respective commercial sources (Bayer Crop Science, J. Cantwell pers comm., and Syngenta Crop

Protection A. Moses pers comm.).

Estimating soybean exposure to A. glycines. In both years, populations of A. glycines were estimated every 7 days beginning in June (20 June 2005; 1 June 2006), with more frequent estimates made before and after foliar insecticides were applied. The estimates were based on the total number (whole plant counts) of apterous adults, alate adults, and nymphs on consecutive plants within each plot. Previous research has shown that as populations of A. glycines increase, the variability in number of A. glycines per plant decreases (Hodgson et al. 2004). Therefore, the number of consecutive plants counted 18

ranged from five to twenty, with the number of plants counted being determined by the

percentage infested with aphids during the previous sampling date. When 0% to 80% of

plants were infested with A. glycines, twenty plants were counted; when 81% to 99% of plants were infested, ten plants were counted; at 100% infestation, five plants were counted.

The seasonal exposure of soybean to A. glycines was estimated by calculating

‘aphid days’ which are based on the number of aphids per plant counted on each sampling date. The seasonal exposure of soybean plants to A. glycines is then calculated with the following equation:

x + x = i1 i t, n=1 2

where x is the mean number of aphids on sample day i, xi-1 is the mean number of aphids

on the previous sample day, and t is the number of days between samples i - 1 and i.

Summing the aphid days accumulated during the growing season (cumulative aphid days)

provides a measure of the seasonal aphid exposure that a soybean plant experienced

(Hodgson et al. 2004).

Yield. In 2005 and 2006 yields were determined by weighing grain with a grain hopper,

which rested on a digital scale sensor custom designed for the harvester. The entire plot

was used to measure yield in 2005 and the center six rows out of twelve in 2006. Yields

were corrected to 13% moisture and reported as kilograms per ha.

Natural enemy sampling. To determine the effect of each insecticide treatment on the

abundance of the foliar-based natural enemy community, individual plots were monitored

with three methods: In situ field counts, sweep nets, and yellow sticky cards (YSCs; unbaited Pherecon AM Traps, Great Lakes IPM Inc, Vestaburg, MI). These methods were selected based on the portion of the total natural enemy community each method will sample (Schmidt et al. 2008). After the emergence of soybean plants, each sampling method was employed twice each month beginning on 13 June in 2005 and 12 June in

2006. In this manner, we could account for treatment differences that may have occurred early in the season due to the seed-applied insecticides. To account for the impact of foliar insecticides later in the season, these sampling methods were employed every 3-7 days after foliar insecticides were applied. Specifically, all sampling methods were conducted at two and three days post application, 2005 and 2006 respectively, of foliar insecticides to determine what short-term impacts foliar insecticides may have on the natural enemy community.

In situ counts were conducted by visual inspection of 5-10 consecutive, undisturbed plants with identification and recording of natural enemies in the field.

Sweep net samples were collected using a 38-cm diameter net and consisted of 20 pendulum sweeps per plot running in the direction of the row. Yellow sticky cards were placed four per plot and suspended on wooden stakes such that the base of the card was slightly higher than the plant canopy. Yellow sticky cards were deployed in the field for

6-8 days. 20

Sweep net samples and YSCs were stored in a -20º C freezer in the laboratory prior to sorting and identification of natural enemies in the laboratory. Natural enemies from both methods were identified to several levels, with spiders identified to order and all insects to at least family. Damsel bugs (Hemiptera: Nabidae) were identified to genus, while lady beetles (Coleoptera: Coccinellidae) and insidious flower bugs (Hemiptera:

Anthocoridae) were identified to species. Lady beetle larvae were identified to family when early instars were collected and to species when later instars were collected.

Analysis. To determine the impact of the various insecticides on the plant exposure to A. glycines, we reported the mean aphid days accumulated each week and calculated for each treatment throughout the growing season. The impact of treatments on the accumulation of aphid days was determined using natural log-transformed data to meet the assumptions of a one-way analysis of variance (ANOVA) using PROC MIXED (SAS

2004).

The natural enemy community was summarized separately for each sampling method. An analysis of variance was used to determine if the abundance of all natural enemies as well as key species were affected by insecticide applications. Total natural enemy data from sweep nets and in situ counts were square root transformed to correct for heteroscadacity prior to analysis. Data for individual subsets of the total natural enemies were transformed using log base ten. A separate ANOVA was conducted for each of the following: total predators and parasitoids collected, all Coccinellidae, H. axyridis, and O. insidiosus. 21

Due to an imbalance resulting from an additional replication of the untreated

control within blocks and the addition of the zero-aphid treatment in 2006, PROC

MIXED (SAS 2004) was used for all analysis. Natural enemies were first analyzed for

interactions between year, date, and treatment variables for each sampling method. This

was accomplished using an ANOVA with PROC MIXED and repeated measures using a

REPEATED statement (SAS 2004). Differences in the abundance of natural enemies and

abundance of key natural enemies were determined using an F-protected least significant difference (LSD) test generated using the LSMEANS statement in PROC MIXED (SAS

2004). All analyses were conducted for each date individually in order to account for seasonal variation and differences in A. glycines population levels. This also allowed differences due to seed treatments to be observed when those treatments would still be active.

Yields were estimated from seed samples collected at harvest and averaged across the treatments. Yield data were analyzed using a one-way ANOVA in PROC MIXED

(SAS 2004) using students LSD test for means separation.

Results

Soybean exposure to A. glycines and yield. In 2005, foliar insecticides were applied on

2 August when A. glycines populations averaged 211 + 48 per plant in non-seed treated

plots. However, A. glycines populations quickly surpassed the economic threshold of 250

A. glycines per plant in the control treatment (266 + 54 A. glycines per plant) by 4 August

and peaked at 1,331 + 323 A. glycines per plant on 25 August. 22

Following the application of the foliar insecticide we observed mean separation

amongst all the treatments (Fig. 1a). Amongst the seven treatments there was a general

trend in which the -cyhalothrin treated soybeans had the lowest exposure to A. glycines

(>500 CAD), followed by the foliar applied reduced-risk insecticides (~2,000 CAD). The untreated and seed-treated soybeans experienced the highest exposure to A. glycines (>

10,000 CAD)(Fig. 1a). This trend was not as consistent with regard to soybean yield.

Although the greatest yield was recorded from plots treated with -cyhalothrin, this was

not significantly different from plots treated with the highest rate of thiomethoxam or the

two foliar-applied reduced risk insecticides (Fig. 2). In general the seed treatments

provided the lowest level of protection against A. glycines. This was most noticeable in

the comparison of the seed versus foliar application of imidacloprid, the later having had

a greater reduction in A. glycines exposure and yield.

In 2006, insecticides were applied on 1 August when A. glycines populations averaged 75 + 29 aphids per plant in non-seed treated plots. Unlike 2005, A. glycines

populations did not surpass the ET and peaked at 114 + 22 A. glycines per plant on 7

August. In 2006 we observed a trend in soybean exposure to A. glycines after application

of the foliar insecticides similar to that of 2005. The lowest exposure to A. glycines was

observed in plots treated with -cyhalothrin (<100 CAD), followed by the foliar applied

reduced-risk insecticide and the untreated and seed-treated soybeans experienced the

highest exposure to A. glycines (Fig. 2b). Unlike 2005, we did not observed a significant

difference between the seed-applied and foliar-applied imidacloprid in terms of soybean 23

exposure to A. glycines. However, pymetrozine performed at an intermediate level compared to -cyhalothrin and the other treatments. Although insecticide applications reduced A. glycines populations, those reductions in populations did not result in significantly different soybean yield compared to the untreated control (Fig. 2). This is not unexpected, as the density of A. glycines in the untreated plots would not be expected to have a significant impact on soybean yield (Ragsdale et al. 2007).

Natural enemies. Natural enemies collected in 2005 and 2006 are summarized in Tables

2 and 3 based on sampling method. Sweep nets collected the greatest abundance of natural enemies, with a greater abundance of natural enemies collected in 2005 than in

2006. In 2005 Coccinellidae was the most abundant natural enemy (60.7%), with H. axyridis being the single most abundant natural enemy species (55.2). In 2006 O. insidiosus was the most abundant natural enemy in sweep net samples (29.1%). In situ sampling had the lowest abundance of natural enemies of the three sampling methods.

Orius insidiosus was the most abundant natural enemy encountered in in situ samples in both years, 35.9% and 53.9% in 2005 and 2006, respectively.

For each sampling method, the data from both years were combined and analyzed for each sampling method. The difference in natural enemy abundance between the two years is reflected in the analysis, as both year and date were significant sources of variation across all three sampling methods (Table 4). The impact of the various treatments was not observed until after the foliar insecticides were applied. There were 24

significant (P < 0.05) 2-way interactions between date and treatment for both sweep net

and sampling and YSC with a marginally significant interaction (P < 0.1) for in situ

sampling. Due to these interactions, data for all three sampling methods were analyzed

separately for each sampling date in 2005 and 2006. For sweep net sampling, the

variables of date and treatment were significant (P < 0.05). Year, date, and treatment

were significant (P < 0.05) for in situ sampling. The treatment variable for YSCs was not significant when both years of data were combined. As there were no significant differences among treatments, we do not report the results of YSC sampling.

Pre-application of foliar insecticides

We report data from the sweep net sampling prior to application of foliar insecticides only (Figs. 3a, 3b), as sweep nets collected the broadest community of natural enemies within soybeans (Schmidt et al. 2008) of the three methods used. In both

2005 and 2006 we did not observe any differences (P < 0.05) in mean total abundance of natural enemies collected with sweep net (Figs. 3a, 3b), in situ (data not shown), and yellow sticky card methods (data not shown) in soybean planted with seed-applied insecticide (seed-treatment) and those left untreated. Furthermore, we observed no effect of seed treatment on sub-groups and individual members of the natural enemy community, including the Coccinellidae, O. insidiosus and H. axyridis across all three sampling methods.

Post application of foliar insecticides

Only after foliar insecticides were applied, during both 2005 and 2006, did we observe differences (P < 0.05) amongst the various treatments. The impact of the various 25

foliar insecticides was observed by sweep net and in situ sampling, but not with YSC

(Table 4). This impact was observed in total natural enemies, O. insidiosus,

Coccinellidae, and H. axyridis.

Using a sweep-net, we observed significant (P < 0.05) differences among the treatments (Table 5) in the abundance of total natural enemies, O. insidiosus,

Coccinellidae, and H. axyridis. After the foliar insecticides were applied, there were no observable differences (P < 0.05) in natural enemy abundance between seed treatments and the untreated control (Table 5). Foliar applications of imidacloprid and pymetrozine were commonly indistinguishable from each other. These insecticides were often an intermediate between the untreated control and -cyhalothrin in the abundance of all natural enemies categories analyzed, and occasionally indistinguishable from the untreated control. The foliar application of the insecticide -cyhalothrin consistently had the lowest abundance of any natural enemy category (Table 5).

Treatment differences for in situ sampling (Table 6) in 2005 were similar to those from sweep nets in 2005 after application of foliar insecticides. Seed treatments were commonly grouped with the untreated control, and the foliar applications of imidacloprid and pymetrozine typically were intermediates between the untreated control and - cyhalothrin in the abundance of total natural enemies, O. insidiosus, total Coccinellidae, and H. axyridis.

Significant differences (P < 0.05) among treatments after the application of foliar insecticides in 2006 were generally similar to those in 2005. During 2006 when we sampled plots with a sweep net, we observed treatment differences (P < 0.05) in the 26

abundance of all natural enemies as well as specific members of this community (i.e. O.

insidiosus, total Coccinellidae, and H. axyridis; Table 5). Again seed treatments were

commonly indistinguishable from the untreated control in the abundance of all natural

enemy categories. The foliar application of imidacloprid was commonly indistinguishable

from the untreated control in the abundance of all natural enemy categories analyzed for

both sweep net and in situ sampling methods. Pymetrozine once again was an

intermediate between the untreated control and -cyhalothrin in the abundance of all natural enemy categories, with -cyhalothrin having the lowest abundance. In situ sampling treatment differences in 2006 were significant (P < 0.05) for the abundance of total natural enemies, O. insidiosus, and total Coccinellidae (Table 6), however results were not significant (P < 0.05) at any DAT for the abundance of H. axyridis (Table 6).

Discussion

Stern et al. (1959) proposed four ways to reduce the impact of insecticides on non-target organisms: 1) choose a selective mode of action which is toxic only to the pest, 2) use application techniques that reduce exposure of non-target organisms, 3) apply insecticides at time when non-target organisms are protected, and 4) applying a broad- spectrum insecticide with a short half-life while leaving reservoir areas from which recolonization of non-target organisms can occur. The selective mode of action suggested by Stern et al. (1959) relies on reduced toxicity to lower the risk to non-target organisms, while the other three methods rely on reducing exposure of the non-target organism to the insecticide in order to lower the total risk. We focused our research efforts on confirmed and putative reduced-risk insecticides that either had a selective mode of action specific 27

to A. glycines and other closely related insects, and on plant systemic (both apoplasticly and symplasticly transported compounds) insecticides which reduced the environmental exposure of natural enemies to the insecticide.

From a practical standpoint no one benefits from reduced non-target impacts if the pest populations are not controlled to a satisfactory extent by the reduced-risk insecticides. The impacts of the reduced-risk insecticides in reducing A. glycines populations were mixed, with the neonicotinoid seed treatments (imidacloprid and thiamethoxam) providing some reduction (typically not significant). Reductions in A. glycines populations were small and inconsistent. This lack of reduction in A. glycines populations by seed-applied neonicotinoids is consistent with other published research

(Johnson et al. 2008, Johnson 2006, McCornack and Ragsdale 2006). Although the performance of foliar imidacloprid was inconsistent from 2005 to 2006, the foliar applications (timed with larger populations of A. glycines) of both imidacloprid and pymetrozine provided greater A. glycines population reductions (sometimes equal to - cyhalothrin) than the seed treatments (imidacloprid and thiamethoxam).

Reductions in pest populations are not sufficient alone for adoption of reduced- risk insectides. The ability of the insecticide to protect yield must also be considered.

Neonicotinoid seed treatments (imidacloprid and thiamethoxam) provided some soybean yield protection (typically not significant), however, the yield protection provided by the seed treatments were small and inconsistent. This lack of significant yield protection from seed applied neonicotinoids is consistent with other published research (Johnson et 28

al. 2008, Johnson 2006, McCornack and Ragsdale 2006). The performance of foliar imidacloprid was inconsistent from 2005 to 2006 for protection of yield. In both 2005 and 2006, foliar applied imidacloprid and pymetrozine provided soybean yield protection equal to the broad-spectrum insecticide (-cyhalothrin). The increased efficacy of the foliar insecticides illustrates the importance of insecticide timing which is again consistent with other published research (Johnson 2006, McCornack and Ragsdale 2006).

Seed treatments did have a reduced impact on the abundance of natural enemies when compared to the foliar applied insecticides. However, seed treatments did not prevent yield loss due to A. glycines herbivory. Foliar applied insecticides did reduce the abundance of all the natural enemies examined, when compared to both the seed treatments and the untreated control. The foliar application of the reduced-risk insecticide pymetrozine effectively reduced the impact on natural enemies while reducing A. glycines populations and protecting yield. A foliar application of imidacloprid could constitute a reduced-risk insecticide as it had a reduced impact on natural enemies and reduced of A. glycines populations. However, this insecticide produced inconsistent results from 2005 to 2006. The formulation of the product has been changed for both the

2006 and 2007 growing seasons from the formulation used in 2005. Consistent results will be needed to determine whether or not this product will be able to conserve natural enemies and effectively reduce A. glycines populations.

In recent laboratory bioassays performed by Kraiss and Cullen (2008), reduced- risk insecticides demonstrated differential toxicities to A. glycines and H. axyridis. While 29

their products and testing arena differed from ours, their results support that reduced-risk

insecticides are capable of reducing A. glycines populations with a reduced impact on

natural enemies (specifically H. axyridis). However, the impact of the insecticide varied

with the developmental stage of H. axyridis. We did not account for these differences.

However, our reduced-risk insecticide (pymetrozine) did have a reduced impact on all of

our natural enemy categories in the field.

The success of any reduced-risk insecticide will hinge on its effectiveness against

the target pest and protection of yield. Our data suggest that the seed treatments of thiomethoxam and imidacloprid are not effective in reducing A. glycines populations and

consequently unable to protect yield. Typically, economically damaging populations of

A. glycines occur later in the season in Iowa. Seed treatments by then have lost their

potency. In contrast, a well-timed foliar application of an insecticide provided reductions

in A. glycines populations and protected yield. The use of a foliarly applied insecticide

would have a greater potential to impact natural enemies. A reduced-risk insecticide

would reduce this potential for impacts on natural enemies. As pymetrozine is not labeled

for use in soybean, further work is needed to identify other established and potential

reduced-risk insecticides available for use in soybean for A. glycines management.

Acknowledgements

We thank the Iowa Soybean Association and North Central Soybean Research

Program for financial support of our research. We would also like to thank both Bayer 30

CropScience and Syngenta Crop Protection for supplying both insecticides and additional financial support of our research.

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Table 1.Insecticides and rates used during 2005 and 2006 field experiments

Treatment Formulation Rate 1

Control NA NA

Zero-aphid control2 -cyhalothrin Warrior 1 SC 227 ml per ha

+ chlorpyrifos Lorsban 4 E 273 ml per ha thiamethoxam Cruiser 5 FS 50g per 100 kg thiamethoxam Cruiser 5 FS 100g per 100 kg imidacloprid Gaucho 480 F 62.5g per 100 kg imidacloprid 3 Trimax 4 E 105 ml per ha

pymetrozine 3 Fulfill 50 WG 192.6g per ha

-cyhalothrin 3 Warrior 1 SC 227 ml per ha

1 Seed treatment rates are given as grams formulated product per 100 kg seed and foliar

treatment rates are given as ml formulated product per hectare.

2 Zero-aphid control was added in 2006 and was applied when aphids were detected

(three applications, 5 June, 13 July, and 1 August).

3 Foliar treatments were applied on 2 August 2005 (211 A. glycines per plant at

application) and 1 August 2006 (75 A. glycines per plant at application). 38

Table 2. Natural enemies collected using a sweep net during 2005 and 2006

Order Family Species

Coleoptera Coccinellidae Coccinella septumpunctata

Coleomegilla maculata

Harmonia axyridis

Hippodamia convergens

Hippodamia parenthesis

Diptera Syrphidae

Hemiptera Anthocoridae Orius insidiosus

Nabidae Nabis spp.

Pentatomidae Podisus maculiventris

Hymenoptera Braconidae

Ichneumonidae

Neuroptera Chrysopidae

Hemerobiidae

Araneae

Opiliones 39

Table 3. Natural enemies observed in situ during 2005 and 2006

Order Family Species

Coleoptera Coccinellidae Harmonia axyridis

Unidentified2

Diptera Syrphidae

Hemiptera Anthocoridae Orius insidiosus

Nabidae Nabis spp.

Pentatomidae Podisus maculiventris

Hymenoptera Aphelinidae1

Braconidae1

Neuroptera Chrysopidae

Hemerobiidae

Araneae

Opiliones

1 Identified only as mummies found on plants

2 Larvae in early instars that lack characteristic coloration used for identification

Table 4. ANOVA revealing the response of natural enemy abundance to foliar and seed applied insecticides to soybeans in 2005 and 2006.

Method Period1 Variable F-value DF2 P-value

Sweep net Pre Year 34.25 1, 75.3 <0.0001

Date 27.41 6, 147 <0.0001

Treatment 0.23 5, 72.9 0.9460

Date*Treatment 0.79 10, 156 0.6410

Year*Treatment 0.49 5, 75 0.7804

Post Year 25.52 1, 95.8 <0.0001

Date 54.20 3, 213 <0.0001

Treatment 20.24 5, 90.8 <0.0001

Date*Treatment 2.17 15, 200 0.0081

Year*Treatment 2.02 5, 95.8 0.0821

In situ Pre Year 0.01 1, 94.7 0.9084

Date 70.2 2, 158 <0.0001

Treatment 0.84 5, 96.3 0.5266

Date*Treatment 0.43 10, 170 0.9291

Year*Treatment 2.03 5, 94.4 0.0815

Post Year 16.11 1, 64.7 0.0002

Date 35.02 1, 81.9 <0.0001

Treatment 7.53 5, 64.2 <0.0001 41

Table 4. continued

Method Period1 Variable F-value DF2 P-value

Date*Treatment 1.93 5, 82.5 0.0981

Year*Treatment 2.03 5, 64.2 0.0864

Yellow Sticky Cards Pre Year 99.23 1, 63 <0.0001

Date 56.52 2, 124 <0.0001

Treatment 1.61 5, 70.8 0.1684

Date*Treatment 2.06 10, 144 0.0312

Year*Treatment 1.55 5, 63 0.1871

Post Year 146.67 1, 97.5 <0.0001

Date 9.91 3, 201 <0.0001

Treatment 0.96 5, 95 0.4491

Date*Treatment 0.47 15, 226 0.9529

Year*Treatment 0.42 5, 97.3 0.8307

1 Data analyzed from pre or post application of foliar insecticides

2 Degrees of freedom, numerator followed by denominator degrees of freedom 42

Table 5. Mean (+ SEM) of total natural enemies, Orius insidiosus, Coccinellidae, and

Harmonia axyridis collected in soybeans using a sweep net following application of foliar insecticides

Year DAT1 Treatment2, 3 Total NE O. insidiosus Coccinellidae H. axyridis

2005 2 Untreated 2.4 + 1.2 AB 0.0 + 0.0 NS 1.2 + 0.6 A 0.8 + 0.6 NS

Thiomethoxam 0.7 + 0.5 BC 0.0 + 0.0 NS 0.3 + 0.3 AB 0.3 + 0.3 NS

Imidacloprid(ST) 2.8 + 1.1 A 0.0 + 0.0 NS 0.8 + 0.4 AB 0.2 + 0.2 NS

Imidacloprid 2.0 + 1.0 AB 0.0 + 0.0 NS 1.3 + 1.0 AB 0.8 + 0.8 NS

Pymetrozine 1.5 + 0.6 ABC 0.2 + 0.2 NS 0.7 + 0.2 AB 0.5 + 0.2 NS

-cyhalothrin 0.2 + 0.2 C 0.0 + 0.0 NS 0.0 + 0.0 B 0.0 + 0.0 NS

8 Untreated4 ------

Thiomethoxam 10.2+1.5 A 3.4 + 1.2 A 3.6 + 0.5 A 2.8 + 0.6 A

Imidacloprid(ST) 4.3 + 2.1 BC 1.5 + 0.8 B 1.8 + 1.0 BC 1.7 + 0.8 AB

Imidacloprid 6.8 + 1.5 ABC 2.5 + 0.9 AB 2.7 + 0.8 AB 2.3 + 0.9 A

Pymetrozine 4.7 + 1.9 BC 2.0 + 1.0 B 2.5 + 0.9 ABC 2.3 + 0.9 A

-cyhalothrin 2.0 + 0.7 C 0.3 + 0.3 C 0.8 + 0.5 C 0.3 + 0.2 B

13 Untreated 17.8+4.9A 0.5 + 0.2 AB 14.0 +4.2A 13.7+4.1A

Thiomethoxam 9.8 + 2.2 AB 1.5 + 0.6 A 5.2 + 1.7 BC 4.2 + 1.5 BC

1 DAT = Days After Treatment of foliar insecticides

2 Foliar insecticides were applied on August 2 and August 1 in 2005 and 2006, respectively

3 Seed treatments were applied at planting on May 22 and May 6 in 2005 and 2006, respectively

4 Data lost due to mechanical failure of storage equipment 43

Table 5. continued

1 2, 3 Year DAT Treatment Total NE O. insidiosus Coccinellidae H. axyridis

13 Imidacloprid(ST) 9.5 + 1.3 AB 1.3 + 0.8 A 6.0 + 0.4 B 5.3 + 0.6 B

Imidacloprid 5.7 + 1.4 BC 1.5 + 0.6 A 2.8 + 0.7 CD 2.5 + 0.6 CD

Pymetrozine 4.2 + 1.2 CD 0.0 + 0.0 B 1.3 + 0.6 DE 1.3 + 0.6 D

-cyhalothrin 1.2 + 0.5 D 0.0 + 0.0 B 0.2 + 0.2 E 0.2 + 0.2 E

22 Untreated 22.0+4.3A 0.0 + 0.0 B 19.2 +4.2A 18.8+4.1A

Thiomethoxam 12.7+2.8 B 0.0 + 0.0 B 11.8 +2.4B 11.3+2.3B

Imidacloprid(ST) 17.7+3.0 AB 1.0 + 0.5 A 14.0 +2.9AB 13.8+2.9AB

Imidacloprid 6.0 + 1.8 C 0.3 + 0.2 A 4.0 + 1.5 C 3.7 + 1.3 C

Pymetrozine 3.5 + 0.8 C 0.7 + 0.3 AB 1.8 + 0.5 CD 1.8 + 0.5 C

-cyhalothrin 2.3 + 0.5 C 0.2 + 0.2 AB 0.7 + 0.3 D 0.5 + 0.3 D

2006 3 Untreated 1.4 + 0.6 BC 0.0 + 0.0 C 0.5 + 0.3 NS 0.3 + 0.2 NS

Thiomethoxam 1.5 + 0.6 AB 0.8 + 0.5 AB 0.0 + 0.0 NS 0.0 + 0.0 NS

Imidacloprid(ST) 2.5 + 0.4 A 0.5 + 0.3 BC 0.0 + 0.0 NS 0.0 + 0.0 NS

Imidacloprid 1.7 + 0.7 AB 1.0 + 0.4 A 0.5 + 0.3 NS 0.5 + 0.3 NS

Pymetrozine 0.8 + 0.5 AB 0.2 + 0.2 BC 0.5 + 0.5 NS 0.5 + 0.5 NS

-cyhalothrin 0.3 + 0.3 C 0.0 + 0.0 BC 0.0 + 0.0 NS 0.0 + 0.0 NS

6 Untreated 2.8 + 0.5 AB 0.5 + 0.2 AB 0.7 + 0.3 A 0.5 + 0.2 NS

Thiomethoxam 1.3 + 0.8 BC 0.2 + 0.2 AB 0.7 + 0.7 AB 0.7 + 0.7 NS

Imidacloprid(ST) 3.2 + 1.0 A 1.0 + 0.6 A 0.3 + 0.3 AB 0.3 + 0.3 NS

Imidacloprid 3.0 + 1.1 A 0.7 + 0.4 AB 1.0 + 1.0 AB 1.0 + 1.0 NS 44

Table 5. continued

1 2, 3 Year DAT Treatment Total NE O. insidiosus Coccinellidae H. axyridis

6 Pymetrozine 1.5 + 0.8 BC 0.0 + 0.0 B 0.5 + 0.3 AB 0.3 + 0.2 NS

-cyhalothrin 0.2 + 0.2 C 0.0 + 0.0 B 0.0 + 0.0 B 0.0 + 0.0 NS

13 Untreated 3.7 + 1.0 A 0.0 + 0.0 NS 1.0 + 0.3 A 0.8 + 0.3 A

Thiomethoxam 1.7 + 0.6 B 0.2 + 0.2 NS 0.5 + 0.2 AB 0.5 + 0.2 AB

Imidacloprid(ST) 3.3 + 0.7 AB 0.0 + 0.0 NS 1.3 + 0.6 A 0.8 + 0.4 A

Imidacloprid 2.0 + 1.1 BC 0.0 + 0.0 NS 0.6 + 0.5 AB 0.6 + 0.5 AB

Pymetrozine 2.0 + 0.8 AB 0.0 + 0.0 NS 0.7 + 0.4 A 0.5 + 0.4 AB

-cyhalothrin 0.2 + 0.2 C 0.0 + 0.0 NS 0.0 + 0.0 B 0.0 + 0.0 B

21 Untreated 10.5+0.9 AB 4.3 + 0.8 A 3.6 + 0.8 A 3.3 + 0.7 A

Thiomethoxam 7.8 + 1.2 AB 3.5 + 0.8 A 2.8 + 0.4 A 2.7 + 0.4 A

Imidacloprid(ST) 13.7+2.2 A 6.0 + 1.8 A 4.2 + 0.3 A 3.3 + 0.3 A

Imidacloprid 11.2+2.3 AB 4.0 + 1.4 AB 3.8 + 0.8 A 3.3 + 0.9 A

Pymetrozine 6.0 + 1.0 C 1.7 + 0.5 B 0.2 + 0.1 B 0.2 + 0.1 B

-cyhalothrin 1.5 + 0.7 D 0.3 + 0.2 C 0.2 + 0.1 B 0.2 + 0.1 B 45

Table 6. Mean (+ SEM) of total natural enemies, Orius insidiosus, Coccinellidae, and

Harmonia axyridis observed in situ on soybeans following application of foliar insecticides

Year DAT1 Treatment2,3 Total NE O. insidiosus Coccinellidae H. axyridis

2005 8 Untreated 2.0 + 1.0 A 0.3 + 0.3 NS 1.5 + 1.0 A 1.0 + 0.6 A

Thiomethoxam 0.0 + 0.0 AB 0.2 + 0.2 NS 0.3 + 0.3 AB 0.3 + 0.3 AB

Imidacloprid(ST) 1.0 + 0.7 AB 0.2 + 0.2 NS 0.2 + 0.2 AB 0.2 + 0.2 B

Imidacloprid 0.7 + 0.4 AB 0.3 + 0.3 NS 0.2 + 0.2 AB 0.0 + 0.0 B

Pymetrozine 0.7 + 0.5 AB 0.0 + 0.0 NS 0.7 + 0.5 AB 0.2 + 0.2 AB

-cyhalothrin 0.3 + 0.2 B 0.0 + 0.0 NS 0.0 + 0.0 B 0.0 + 0.0 B

14 Untreated 4.5 + 1.8 AB 0.8 + 0.4 AB 2.5 + 1.2 A 0.5 + 0.3 AB

Thiomethoxam 1.5 + 0.6 BC 0.6 + 0.4 AB 1.0 + 0.7 ABC 0.2 + 0.2 BC

Imidacloprid(ST) 2.8 + 0.7 A 2.2 + 1.6 A 1.5 + 0.8 ABC 1.2 + 0.8 A

Imidacloprid 2.4 + 0.9 C 1.0 + 1.0 AB 0.5 + 0.4 BC 0.3 + 0.2 ABC

Pymetrozine 1.6 + 0.9 ABC 0.5 + 0.5 B 1.2 + 0.4 AB 0.3 + 0.2 ABC

-cyhalothrin 6.0 + 1.8 C 0.2 + 0.2 B 0.3 + 0.2 C 0.0 + 0.0 C

2006 3 Untreated 3.2 + 1.0 AB 1.8 + 0.5 AB 0.3 + 0.3 B 0.0 + 0.0 NS

Thiomethoxam 2.3 + 0.6 AB 1.0 + 0.5 ABC 0.0 + 0.0 B 0.0 + 0.0 NS

Imidacloprid(ST) 4.7 + 1.0 A 2.2 + 0.7 A 0.8 + 0.4 A 0.2 + 0.2 NS

1 DAT = Days After Treatment of foliar insecticides

2 Foliar insecticides were applied on August 2 and August 1 in 2005 and 2006, respectively

3 Seed treatments were applied at planting on May 22 and May 6 in 2005 and 2006, respectively 46

Table 6. continued

Year DAT1 Treatment2,3 Total NE O. insidiosus Coccinellidae H. axyridis

3 Imidacloprid 1.5 + 0.6 BC 0.7 + 0.3 BC 0.3 + 0.2 AB 0.0 + 0.0 NS

Pymetrozine 1.0 + 0.4 BC 0.2 + 0.2 C 0.3 + 0.2 AB 0.0 + 0.0 NS

-cyhalothrin 0.7 + 0.3 C 0.0 + 0.0 C 0.0 + 0.0 B 0.0 + 0.0 NS

6 Untreated 5.8 + 1.2 A 3.2 + 0.7 A 0.8 + 0.4 AB 0.1 + 0.1 NS

Thiomethoxam 1.7 + 0.6 B 1.5 + 0.6 A 0.0 + 0.0 C 0.0 + 0.0 NS

Imidacloprid(ST) 3.7 + 1.0 AB 2.3 + 0.6 A 0.2 + 0.2 BC 0.2 + 0.2 NS

Imidacloprid 3.8 + 1.4 AB 1.5 + 0.8 A 1.2 + 0.8 A 0.2 + 0.2 NS

Pymetrozine 1.8 + 0.5 B 1.8 + 0.5 A 0.0 + 0.0 C 0.0 + 0.0 NS

-cyhalothrin 0.2 + 0.2 C 0.0 + 0.0 B 0.0 + 0.0 C 0.0 + 0.0 NS

13 Untreated 6.4 + 1.4 AB 4.3 + 0.9 AB 0.2 + 0.1 NS 0.0 + 0.0 NS

Thiomethoxam 9.0 + 1.9 A 5.2 + 1.0 A 0.0 + 0.0 NS 0.0 + 0.0 NS

Imidacloprid(ST) 6.8 + 2.0 AB 2.5 + 0.6 AB 0.2 + 0.2 NS 0.2 + 0.2 NS

Imidacloprid 5.5 + 2.2 BC 4.3 + 1.7 B 0.2 + 0.2 NS 0.0 + 0.0 NS

Pymetrozine 2.8 + 0.7 C 3.2 + 1.1 B 0.0 + 0.0 NS 0.0 + 0.0 NS

-cyhalothrin 0.3 + 0.3 D 0.0 + 0.0 C 0.0 + 0.0 NS 0.0 + 0.0 NS 47

Figure 1. Comparison of the seed and foliar applied insecticides showing soybean exposure to A. glycines based on average cumulative aphid days at the Floyd County research site in 2005 (a) and 2006 (b). Soybean were planted on 22 May in 2005 and 6

May in 2006. Thiamethoxam (Cruiser) and imidacloprid (Gaucho) were applied to seeds prior to planting. Imidacloprid (Trimax), pymetrozine (Fulfill), and -cyhalothrin

(Warrior) were applied to foliage on 2 August in 2005 and August 1 in 2006 when aphid populations averaged 211 and 75 A. glycines per plant, respectively. Means labeled with a unique letter were significantly different (P < 0.05).

Figure 2. Comparison of seed and foliar applied insecticides on soybean exposure to A.

glycines based on yield (kg per hectare) at Floyd County in 2005 (a) and 2006 (b).

Soybean were planted on 22 May in 2005 and 6 May in 2006. Thiamethoxam (Cruiser)

and imidacloprid (Gaucho) were applied to seeds prior to planting. Imidacloprid

(Trimax), pymetrozine (Fulfill), and -cyhalothrin (Warrior) were applied to foliage on 2

August in 2005 and August 1 in 2006 when aphid populations averaged 211 and 75 A.

glycines per plant, respectively. Means labeled with a unique letter were significantly

different (P < 0.05).

Figure 3. Seasonal abundance of the total natural enemy community in soybean using

sweep nets in 2005 (a) and 2006 (b). Soybean were planted on 22 May in 2005 and 6

May in 2006 with seed treatments of imidacloprid and thiomethoxam were applied as

seed treatments at planting. Foliar treatments of imidacloprid, pymetrozine, and - 48

cyhalothrin were applied on 2 August in 2005 and 1 August in 2006 and are denoted by

an arrow. Significant differences (P < 0.05) are denoted by an asterisk (*) more detail is given in Table 5. 49

30000 Fig. 1a A

) A SEM + ( s y da 20000 hid p a

B cumulative

Mean 10000

C D E

50g 100g 62.5g -cyhalothrin Pymetrozine Pymetrozine Imidacloprid Imidacloprid Imidacloprid Thiamethoxam Thiamethoxam Untreated

Seed-applied insecticides Foliage-applied insecticides 50

Fig. 1b 400 A

300 A A A B

200

(SEM) cumulative aphid days Mean (SEM) 100

B C C D 0

rid p 50g 100g halothrin 62.5g y metrozine control y -c Untreated Imidaclo P Zero aphid Zero aphid Thiamethoxam Imidacloprid Thiamethoxam

Seed-applied insecticides Foliage-applied insecticides 51

Fig. 2a

4450 A AB

AB AB

4150

SEM) SEM) BC

3900 Mean yield kg per hectare (+ yield kg perMean hectare

3650

50g 100g 62.5g -cyhalothrin Pymetrozine Pymetrozine Imidacloprid Imidacloprid Imidacloprid Untreated Thiamethoxam Thiamethoxam

Seed-applied insecticides Foliage-applied insecticides

Fig. 2b 5000

SEM) SEM)

4000

3000

Mean yield kg per hectare (+ yield kg perMean hectare

2000

rid p

50g 100g control 62.5g Zero aphid Zero aphid -cyhalothrin Pymetrozine Pymetrozine Untreated Imidaclo Thiamethoxam Thiamethoxam Imidacloprid Imidacloprid

Seed-applied insecticides Foliage-applied insecticides 53

Fig. 3a

) * SEM

+ ( enemies

* natural

Mean

* 54

Fig. 3b

* ) SEM

+ ( enemies natural

* * Mean * 55

Chapter 3

Community assessment of aphids in Iowa prairies prior to the release of an exotic

aphid parasitoid

A paper to be submitted to Journal of Insect Conservation

Wayne J. Ohnesorg, Jessica M. Orlofske, and Matthew E. O’Neal

Abstract

To what extent a classical biological control program against the soybean aphid

(Aphis glycines Matsumura (Hemiptera: Aphididae)) will impact or even require as an over-wintering host the native Aphis community is not clear. Initial estimates suggest that over 350 species of aphids are found within the North Central region of the U.S. The initial screening of parasitoids for A. glycines has focused on species that utilize a limited host range. Several species of Aphis can be found on prairie host plants; a habitat that once covered the majority of the Iowan landscape but now is limited to less than 0.1% of that area. Our objectives were to 1) survey aphid and plant communities in prairies and 2) determine the effect of prairie type on these communities in 26 central Iowa prairies during 2006 and 2007. Prairies consisted of three types: remnant, isolated reconstruction, and integrated reconstruction. Sampling was conducted May-September weekly/bi- weekly on three sites while the rest were sampled once a month. Over the course of the study, we found a total of 49 species of aphids including 13 from the genus Aphis. Using non-metric multidimensional scaling analysis we observed a difference in the aphid community composition amongst the three prairie types. Hierarchical cluster analysis demonstrated that Aphis mornardae in high abundance indicated remnant prairies while 56

Microparsus variabilis in high abundance indicated reconstructed or integrated prairies.

In the genus Aphis, we observed A. monardae to have greatest percentage of parasitized aphids within genus at 3.94%. Of the 13 species of Aphis found, ants heavily tended 9 species (> 70% of colonies). There was no difference in the percentage of Aphis ant- tended colonies between the three types of prairie. Given the high rates of ant tending experienced by a number of Aphis, those species may be afforded some ecological protection from a classical biological control program regardless of how prairies are managed.

Introduction

The invasive species Aphis glycines Matsumura (Hemiptera: Aphididae) is a significant pest of soybean (Glycine max) in North America. First discovered in

Wisconsin in 2000 (Wedburg 2000, Alleman et al. 2002), it has since spread across the

Midwest and several Canadian provinces and by 2003 could be found in every County of

Iowa (O’Neal 2006). In 2003, more than 1.6 million ha of soybean received insecticide applications for control of A. glycines populations that reached several thousand per plant

(Pilcher and Rice 2005). Soybean yield losses of 15-40% from A. glycines herbivory have been recorded (Ragsdale et al. 2007). Endemic predators can regulate populations of A. glycines (Fox et al. 2004, Rutledge et al. 2004, Fox et al. 2005, Rutledge and O’Neil

2005, Costamagna and Landis 2006, Desneux et al. 2006, Mignault et al. 2006, Schmidt et al. 2007). However, A. glycines still produces outbreaks despite the mortality produced by predators. 57

Importation of exotic natural enemies into the US (i.e., classical biological control) has been proposed for managing A. glycines (Heimpel et al. 2005). In Asia, A. glycines natural enemies include predators and parasitoids which contribute significantly to their mortality on soybeans (Miao et al. 2007), likely contributing to the aphids non- pest status in most of its’ native range (Heimpel et al. 2005). Yet the current A. glycines natural enemy community in North America lacks a significant parasitoid presence

(Schmidt et al. 2008). Importation of Asian parasitoids into North America has begun with the recent release of the braconid Binodoxys communis (M. O’Neal unpublished data, Wyckhus et al. 2007)

It has been argued that exotic species proposed for biological control be assessed for the potential risk to attack non-target species not just on a physiological basis but also on an ecological basis (Simberloff and Stiling 1996, Louda et al. 2003a, b). Ecological risk can primarily take two forms. The first includes direct effects on other species.

Secondly, a biological control agent may also have unintended interactions with other species that belong to the same feeding guild (i.e., aphid parasitoids). In the case of A. glycines and B. communis, this would include native aphids and the current parasitoid community utilizing these aphids.

Investigation into the direct non-target impacts on aphids has been conducted to determine the biological and potential ecological host range of B. communis (Wyckhuys and Heimpel 2007, Wyckhuys et al. 2007). This work has been conducted primarily in laboratory settings. However, it lacks the population level data needed from the field to better estimate the ecological host range (Louda et al. 2003b). Laboratory tests of host 58

range can be inaccurate. In a post hoc analysis of two Cotesia spp. (Hymenoptera:

Braconidae), Van Drieche et al. (2003) found that both would be rejected, due to their broad physiological host range, under current biological control screening methods.

However, under field conditions these wasps demonstrated a preference for the intended target, the invasive Pieris rapae (Lepidoptera: Pieridae). Conversely, laboratory studies have indicated low risk to native, non-target organisms and after release these hosts were utilized. This has been documented for Rhinocyllus conicus (Coleoptera: Curculionidae) used for the control of invasive Eurasian Carduus spp. (Asteraceae) in North America

(Kok and Surles 1975, Rees 1977. Rhinocyllus conicus has greatly expanded its host range past the target Carduus spp. to include native Cirsium spp. This expansion of host range has had negative impacts on native Cirsium spp. (Louda et al. 2003a)

The greatest risk to native aphids in North America should be to aphids in the genus Aphis as B. communis has a limited host range (Desneux et al. submitted). Also, aphids whose phenologies may overlap with that of A. glycines could be utilized as alternate hosts by B. communis. Competition between B. communis and other parasitoids may take place for these aphids as hosts. It is unknown whether the competition will be direct or indirect and could result in displacement as was observed between an invasive and native coccinellids (Evans 2004).

Ants will often tend and defend colonies of aphids. Ants commonly tend honeydew-producing aphids. Ants will defend aphid colonies from attacks by predators and parasitoids (Pierce and Mead 1981, Völkl 1992, Heimpel et al. 1997, Hubner 2000,

Wyckhuys et al. 2007). Tending ants may directly attack the predators and parasitoids of 59

the aphids (Pierce and Mead 1981, Völkl 1992, Heimpel et al. 1997, Hubner 2000,

Wyckhuys et al. 2007). Also, ants can interfere by removing or preying upon parasitized aphids and mummies (Hill and Hoy 2003, Persad and Hoy, 2004, Wyckhuys et al. 2007).

In a laboratory study, the combination of ant tending and a physical refuge dramatically reduced the amount of parasitism experienced by a non-target aphid species by B. communis (Wyckhuys et al. 2007).

In light of the potential non-target impacts to aphids that may occur with the release of exotic parasitoids, we initiated a survey of aphids within Iowa. The habitat that we focused my survey was selected based on preliminary survey of aphids found in the

Midwest (D.J. Voegtlin pers. comm.). In order to focus our search efforts, we chose to use a series of filters to reduce the list. Only native aphids from the subfamily Aphidinae were considered. Next, in order to further focus our efforts, plant species that serve as a summer or winter host to multiple species of aphids were selected, and the plant species occurring as both summer and winter hosts were selected. The majority of these host plants occur in prairies. These filters allowed us to reduce the number of aphids on the original list to 84 (~22%) of the original 382 species.

The goal of our study is to provide background knowledge about native aphids in central-Iowa prairies. Our primary objective was to identify aphids, chiefly in the genus

Aphis, which may experience deleterious effects from B. communis. We hypothesize that reconstructed prairies may not have similar aphid communities as would occur in remnant prairies. Another objective was to determine if ant tending of Aphis spp. differed between our prairie types. 60

The information on the aphid community in prairies from this analysis is based on sampling conducted prior to the release of B. communis. Factors related to habitat quality need to be assessed for their affect on the aphid community structure. Any future post- release of B. communis analysis will need to take into account these sources of variation.

Methods and Materials

Sites. The study sites included 26 prairies (Table 1) located in central Iowa (Fig. 1) that were sampled in 2006 and 2007. Field sites were selected from three general categories of remnant, isolated reconstructions, and integrated reconstructed prairies and follow the definition from Shepherd and Debinski (2005). Our sites consisted of 11 remnants, 10 isolated reconstructions, and 5 integrated reconstructions. Sites that were classified as remnants were areas where the original seed bank has remained and has not received additional seed from other sources. Isolated reconstructed prairies were agricultural fields that had been converted into prairie plantings. Integrated reconstructed prairies were sites where reconstructed prairies were located directly adjacent to remnant prairies and other restored areas. All integrated prairie sites were located at Chichaqua Bottoms Greenbelt in Polk County, Iowa with the exception of Turtlehead Fen in Jasper County. Chichaqua

Bottoms Greenbelt is a large-scale remnant/reconstructed natural area encompassing more than 2,600 hectares.

Aphid Sampling. Aphid populations in our prairies were sampled using a transect method modified from New (1998). Three random 2-m by 25-m belt transects were 61

sampled for aphids at each sampling time. Prior to sampling a prairie, the prairie was divided roughly into quarters. Random numbers were obtained from a random number table to determine which quarter of each prairie to sample. From the center of the quarter, another random number was obtained to determine in which of four directions the transect would be laid out: right, left, forward, or back. The above-ground portion of all plants were inspected for aphids. In the field, aphids were identified to morpho-species based on aphid color and host plant. For each morpho-species, the number of colonies found on each transect was recorded. As an estimate for mean colony size and abundance, the first 10 colonies were counted and recorded for each morpho-species. If fewer than 10 colonies of a given morpho-species were discovered, all aphids within each colony were counted. For morpho-species that had more than 10 colonies, the size of the first 10 discovered colonies were measured. The number of parasitized aphids, in the form of intact mummies was counted. In addition we recorded whether or not ants were tending aphids in the first 10 colonies. Ants were not recorded unless active tending was observed. The overall percent of parasitized aphids and ant-tended colonies was estimated for each aphid species per each transect.

We used the following method to estimate the percentage of aphids within a given species that were parasitized, and the percentage of colonies tended by ants. The sum of the mummies found in the field along each transect was divided by the number of aphid counted from colonies as described above. This percentage was multiplied by the total number of aphids species found on that transect to obtain an estimate of the total number of mummies. The number of mummies was summed and divided by the total number of 62

aphids found across all transects, sites, and sampling dates. The categorization of ants tending aphid colonies was recorded as described above. From this information the percentage of the 10 colonies (or less) tended by ants was calculated and multiplied by the total number of colonies to obtain total number of ant-tended colonies. The total number of ant-tended colonies was divided by the total number of colonies found across all transects, sites, and sampling dates. These percentages were calculated for each species and we report these percentages.

The mean colony size for each species was calculated and then multiplied by the number of colonies found and summed among all aphid morpho-species to attain total aphid abundance on each transect. Total aphid abundance at a prairie was calculated to be the mean of the total aphid abundance of the three transects.

Plant stems containing morpho-species colonies were collected into 17.15 by 8.10 by 4.13 cm plastic boxes (Pioneer Plastics, Inc Dixon KY) in water pics. Aphid colonies were maintained at room temperature in the laboratory. Aphid specimens, specifically apterous and alate forms, were collected and stored in 95% ethyl alcohol and then mounted on slides for species identification. Voucher specimens of aphids have been deposited with the Illinois Natural History Survey at the University of Illinois

Champaign/Urbana. All mummies on collected plant material were reared and submitted for identification. Identification of parasitoids was not complete in time for the preparation of this manuscript. Ants from aphid collections were stored in 70% ethyl alcohol and submitted for species identification to Dr. James C. Trager of the Shaw 63

Nature Reserve, Gray Summit, MO. Voucher specimens of ants are deposited in the

Insect Museum at Iowa State University.

Vegetation Sampling. Vegetation sampling was conducted at each site along 3 representative random transects between late July and early August. Vegetation was surveyed with a 1m2 quadrat at 5-m intervals for a total of 15 quadrats per site (Haney and Apfelbaum 1994). Species composition (richness), percent cover, and frequency were calculated for each site.

Analysis. To determine the effect of prairie type on the abundance and diversity on the aphid community, aphids were analyzed utilizing a hierarchical cluster analysis. This analysis will categorize our sites by groups based on the abundance and composition of the aphid community sampled. If prairie type does have an effect on aphid abundance and diversity, we would expect three groups or clusters with a specific prairie type dominating each cluster. Similar studies have been have utilized this analysis for insect communities (Gering et al. 2003, Økland and Bjørnstad 2003). We utilized a Bray-Curtis distance metric for analysis with Ward’s linkage method. Data were analyzed using R statistical software (version 2.5.1, © The R Foundation for Statistical Computing).

An ordination analysis, non-metric multidimensional scaling (NMDS), was also used to compare the aphid and plant communities across the three prairie types. NMDS summarizes relationships between all pairs of species data in order to represent it in multiple dimensions as distances, such that the closer two points are, the more similar 64

they are (i.e., same species composition; Kenkel and Orlóci 1986). We utilized the Bray-

Curtis distance measure for analysis of both aphid and plant communities since Bray-

Curtis is considered a robust measure of distance for ecological data (Faith et al. 1984).

Data were analyzed using R statistical software (version 2.5.1, © The R Foundation for

Statistical Computing). Drawing convex hulls for each prairie type visualized differences in species composition.

In preparation for both analyses, the data were prepared as follows. All sites that were sampled monthly were included in the analysis. Data from weekly/bi-weekly sampling was taken from the sampling period closest to the middle of the month to keep samples non-biased. In 2006, only data from June through August were included in the analysis. All months were included from 2007, except September. Data from September was not included as only a sub-sample of the sites was sampled. The total abundance of each species was tallied for each site. Any species that occurred at less than 5% of the prairie sites was excluded from the analysis. By utilizing this criteria taxa that occurred at a single were not included and reduced the total number of taxa analyzed. In all, 27 aphid species were included in both analyses. For vegetation data, the importance value (Curtis and Macintosh 1951), sum of the relative percent cover and relative frequency, was calculated for each species per site. Any taxa that occurred at less than 5% of the prairie sites were excluded from the analysis. In all, 164 plant taxa were included in the analysis.

To determine if ant tending differed amongst the three prairie types, we analyzed the percentage of aphid colonies tended by ants using a one-way analysis of variance

(ANOVA). Only Aphis spp. were considered in the analysis due to the limited host range 65

of B. communis (Desneux et al. submitted). Our treatments consisted of the three prairie types: remnant, isolated reconstruction, and integrated reconstruction. Percent ant tended was used as the response variable. Each data point was taken from Aphis spp. when they were encountered during sampling. Data were analyzed using PROC GLM (SAS 2004).

Aphis monardae was analyzed individually utilizing the methods above as laboratory studies have shown that ant tending of this species reduces its use by B. communis

(Wyckhuys et al. 2007).

Results

Aphid abundance and diversity. Over the two years of our study, we found a total of

49 aphid species in 18 genera (Table 2). Of the 49 species, 13 belong to the genus Aphis.

Out of the 49 species found, 46 species were found while conducting monthly sampling and 26 species were found during weekly/bi-weekly sampling. Nearly one half (24) of the aphid species were only found during monthly sampling. Three species (Brachycaudus cardui, Nasonovia williamsi, and Uroleucon pieloui) were found only during weekly/bi- weekly and not found during monthly sampling. Thirty-eight species native to North

America were found along with eight exotic species. An additional six species (Table 3) of aphids were found from collections not made from transects. These aphids were collected from host plants which we had not previously collected aphids. These colonies tended to be larger and readily observed outside of transects or the prairies.

We were unable to assign a species name to three morpho-species and are identified with a “?” for status in Table 2. Host plants were identified for each I was able 66

to identify the host plant for each. The reason for the unknown status is that species determinations are lacking. This status exists even in the presence of identified host species for two species and the host genus for the other species. These three morpho- species collections could represent aberrant or new host records or possibly undescribed species. Further identification of these aphids was not complete in time for the preparation of this manuscript.

Seasonal abundance of aphids in prairies was greater in 2007 with >160,000 aphids surveyed compared to 2006 with >69,000 aphids observed. This increase in abundance was measured despite a reduction in the sampling effort (weekly sites were sampled on a bi-weekly schedule in 2007). When the reduction in sampling is taken into account, we found an average of 585 and 1,250 aphids per prairie visit in 2006 and 2007, respectively. Abundance of aphids for both monthly and weekly/bi-weekly sampling was generally greater in June, July, and August compared to May and September in both 2006

(Fig. 2A) and 2007 (Fig. 2B). A total of 18 and 21 aphid species were found during weekly/bi-weekly in 2006 (Fig. 3A) and 2007 (Fig. 4A), respectively. During monthly sampling 26 and 39 aphid species were observed in 2006 (Fig. 3B) and 2007 (Fig. 4B), respectively.

The most abundant aphid in our weekly/bi-weekly samples in 2006 was Aphis saniculae (31.96%) followed closely by Microparsus variabilis (31.79%)(Fig. 3A). Other abundant aphids (>5% of individuals) found were Aphis vernoniae (16.95%) and Aphis debilicornis (7.35%)(Fig. 3A). In 2007, by far the most abundant aphid in weekly/bi- weekly sampling was M. variabilis (48.44%)(Fig. 4A). Other aphids that were abundant 67

in 2007 (>5% of individuals) included A. debilicornis (11.62%), Uroleucon helianthicola

(10.67%), and Aphis nerii (9.42%). Our most abundant aphid species found during monthly sampling in 2006 was M. variabilis (27.74%)(Fig. 3B). Two other aphid species found in high abundance (>15% of individuals) were Aphis monardae (21.97%) and A. saniculae (18.55%). In 2007, by far the most abundant aphid species found while conducting monthly sampling was Carolinaia rhois (31.34%)(Fig. 4B). Other aphid species that were also considered abundant were M. variabilis (18.12%), A. debilicornis

(14.84%), U. helianthicola (9.87%), and A. monardae (6.54%)(Fig. 4B).

A number of the aphid genera found included species that were heavily tended by ants (>70% of aphid colonies). Genera that contained species with colonies heavily tended by ants are Aphis, Chaitophorous, Hysteronerva, Maculolachnus, Nearctaphis, and Thripsaphis (Table 4). Of the 13 species of Aphis found, ants heavily tended 9 species. Amongst the more abundant aphids (those comprised of >10 colonies observed) the most heavily tended species was A. vernoniae at 97.68%. Both the ant species and the aphid species they tended are reported in Table 5.

The percentage of parasitism observed in the field varied by aphid species (Table

54). The highest percentage of parasitism we observed for a species was

Pleotrichophorous psuedopatonkus at 60% (Table 5). Among the Aphis species, A. monardae had the highest percentage of parasitism at 3.94% (Table 5). Other Aphis species for which we observed parasitized aphids in the field were A. asclespiadis, A. debilicornis, A. pulchella, A. saniculae, and A. vernoniae with 2.89%, 0.19%, 0.36%,

0.02%, and 0.01% of aphids parasitized (Table 4), respectively. 68

Aphid Community Analysis. The aphid community was analyzed to determine the effect of prairie type. Hierarchical clustering analysis was used to divide the prairies sampled into clusters (e.g. groups). Cluster analysis produced three clusters (Fig. 5).

Cluster I was the largest cluster and contained a total of 11 sites with 7 remnants, 2 isolated reconstructed, and 2 integrated reconstructed prairie sites. Cluster II was comprised of 10 sites with 4, 5, and 1 remnant, isolated reconstructed, and integrated reconstructed prairie sites, respectively. Cluster III was made up of 5 sites with 3 and 2 isolated reconstructed and integrated reconstructed prairie sites, respectively, and was the only cluster to not include one of each of our three prairie types. Means for the aphid species in each cluster can be found in Table 6.

Our three clusters of prairie sites did not fall into the three types (remnant, isolated reconstruction, and integrated reconstruction)(Fig. 5). Rather, the clusters were influenced by particular aphid species. Cluster II was dominated by A. monardae and has a mean of 1,210.3 compared to 34.55 and 538.6 for clusters I and III, respectively for that species (Table 6). Portion A of cluster II contains four remnant prairies with high populations of A. monardae (Fig. 5). Similarly, cluster III was heavily influenced by

Microparsus variabilis with a mean of 6,404.2 compared to 117.1 and 0 for clusters I and

II, respectively. Cluster III contained three reconstructions and two integrated prairies, suggesting that high abundance of M. variabilis may indicate non-remnant prairie.

Cluster I lacked any dominant aphid species and those species with high means were 69

found in great abundance at one or only a few sites (i.e. Carolinaia rhois), relative to the rest of sites in the cluster.

To visually compare the differences in the aphid composition between the three prairie types we used NMDS. Data from 2006 and 2007 were combined and reported

(Fig. 6). The hulls represent the three prairie types. The overlapping hulls indicate that the aphid communities observed in reconstructed and integrated prairies are quite similar.

In contrast, the hulls representing remnant and integrated prairies did not overlap. The aphid community found in remnant prairies is different than that found in integrated and reconstructed prairies.

The plant community was also visually compared for differences in composition between the three prairie types using NMDS. Data from 2006 and 2007 were combined and reported (Fig. 7). The overlapping hulls indicate that the plant communities from the three types do overlap, at least partially. Integrated reconstructed prairie plant communities do not differ much from that of isolated reconstructed prairie plant communities. The hull representing remnant prairies did not overlap much with the hulls representing the other two prairie types. The remnant prairie plant community composition does differ from the other prairie types.

Results from analysis of ant tending were similar in 2006 and 2007. Therefore, the combined data are reported here. The percentage of Aphis colonies tended by ants did not vary across the three prairie types (df = 2, 245, F = 2.21, P = 0.1117). Also, ant tending of the most abundant Aphis, A. monardae, did not vary by prairie type either (df

= 2, 100, F = 2.54, P = 0.0836). 70

Discussion

We found 49 species of aphids in central Iowa prairies (Table 2). Of these, 13 were members of the genus Aphis including A. monardae that occurred in the highest abundance of the Aphis species. Aphis monardae was found to be a suitable host for B. communis (Wyckhuys and Hiempel 2007). Within a laboratory microcosm ant-tending of

A. monardae residing within flower heads of its host plant limited parasitism by B. communis (Wyckhuys et al. 2007). We observed that 84.82% of A. monardae colonies were tended by ants in the field (Table 5). This supports the conclusions of Wyckhuys et al. (2007) that ant tending of A. monardae along with a physical refuge will lower the risk of non-target impacts experienced from B. communis. Another eight species of Aphis were found to be heavily tended by ants (>70%; Table 5). Given the findings of

Wyckhuys et al. (2007), the high ant-tending rate of these species may lower exposure to and the risk of non-target impacts from B. communis.

Louda et al. (2003) have suggested that biological host range alone is simply not sufficient to warrant release of an organism for biological control, but that species with ecological similarities to the target organism should be taken into consideration (i.e. species that overlap spatially or temporally). A total of 40 species aphids found during the course of our survey occurred in July and/or August when A. glycines populations are increasing and reaching their peak (Table 2). Not all of these species would be at risk from direct impacts from B. communis utilizing them as alternate hosts given the host specificity for the genus Aphis (Wyckhuys and Hiempel 2007, Desneux et al. submitted). 71

The percent of parasitized aphids observed in the field varied depending on the aphid species. The greatest percentage of parasitism observed for an Aphis was 3.94% (A. monardae). This was not the greatest percentage of parasitism observed in the field as

Pleotrichophorous psuedopatonkus had the greatest percentage of individuals

(60.0%)(Table 5). In the case of the Aphis species, the rate of parasitism observed may be misleading, as ants could have preyed upon or removed aphid mummies (Hill and Hoy

2003, Persad and Hoy, 2004, Wyckhuys et al. 2007).

One aphid species, Hyalomyzus monardae, does share a host plant with A. monardae, Monarda fistulosa L. (Lamiaceae)(Table 2). Both species of aphids overlap in phenology (Fig. 8). We observed a total of 5 colonies of the two species have been found occurring together on individual M. fistulosa. With the ability of B. communis to utilize

A. monardae as a host, it is unclear what the risk would be to H. monardae. It is not known if B. communis can utilize H. monardae as a host. However, the occurrence of these two aphids side by side may result in exposure of this native aphid to B. communis to H. monardae. We rarely (3.26% of colonies) observed ants tending H. monardae

(Table 5). We did observe that H. monardae occurred on leaves that were extremely curled. The aphids were found feeding on the inside of the leaf curls which may be afford a physical refuge from B. communis as described for A. monardae (Wyckhuys et al.

2007).

The hierarchical cluster analysis did not provide us with a clear delineation between remnant, isolated reconstruction, and integrated reconstruction prairies.

However, the high abundance of two aphid species, A. monardae and M. variabilis, does 72

give a rough indication for the three prairie types. Aphis monardae was present at over two thirds (19/26) of our sites (Table 2), but the highest abundance of this species was found at remnant sites (Table 4, Fig. 5). Aphis monardae was found at all three prairie types, although our results indicate that remnant prairies are more favorable for greater abundance of A. monardae. Abundance of M. fistulosa was similar among the three site types. Similarly, M. variabilis was found in high abundance at isolated reconstructions and integrated reconstruction prairie sites (Table 4, Fig. 5). This species was found almost exclusively at isolated and integrated reconstruction prairies and only once at a remnant prairie (Moeckly). The host plant of M. variabilis, Desmodium spp., also occurs in higher abundance in reconstructed prairies compared to remnant prairies.

Aphid and plant communities differed in their composition between remnants and isolated and integrated reconstruction prairies (Fig. 6). Any future assessment of aphid communities in prairies will need to account for this difference. This will be especially true for future studies involving the impact or presence of B. communis in the aphid community in prairies.

Acknowledgements

We would like to thank B. Lammers, C. Hildebrand, D. Howell, J. Goerndt, J.

Pearson, J. Judson, K. Hansen, K. Cantu, K. Van Zante, M. Cosgrove, M. Meetz, L.

Meetz, M. Stegmann, S. Peterson, S. Lekwa, S. Rolfes, L. Lown, S. Moeckly, and M.

Moeckly for allowing us to conduct our research on the land they own or manage; G.

VanNostrand, E. VanNostrand, M. Payne, R. Dostalik, and D. Windsor for field support; 73

Dr. D. Voegtlin and D. Lagos for assistance with and identification of aphids; C. Tyrrell and W. Watson for conducting plant surveys. The North Central Soybean Research

Program provided funding for this work.

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Table 1. Prairies sampled for aphids in 2006 and 2007 including type, size in hectares (ha), and latitude and longitude

Site name Type1 Size County Lat, Long

A.C. & Lela Morris8 Remnant 8.1 ha Jasper N 41º46.26', W 92º57.69'

Boone Railroad9 Remnant ~3 ha Boone N 42º03.06', W 93º48.52'

Doolittle Prairie SP2, 8 Remnant 4.9 ha Story N 42º08.96', W 93º35.36'

Drobney8 Remnant 14.3 ha Jasper N 41º33.65', W 93º00.82'

Judson8, Remnant 6.1 ha Guthrie N 41º45.89', W 94º34.16' 79

Liska-Stanek Prairie SP2, 8 Remnant 8.1 ha Webster N 42º24.82', W 94º13.46'

Marietta Sand Prairie SP2, 8 Remnant 6.9 ha Marshall N 42º05.83', W 93º02.34'

Moeckly8, 15 Remnant 16.2 ha Polk N 41º46.94', W 93º39.90'

1 All integrated prairies were located at Chichaqua Bottoms Green Belt in Polk County, except Turtlehead fen in Jasper county, Iowa and directly adjacent to remnant areas

2 State Preserve

8 Sampled monthly

9 Sampled weekly/bi-weekly

Table 1. continued

Site name Type1 Size County Lat, Long

Puccoon CBG3, 8 Remnant 1.5 ha Jasper N 41º44.13', W 93º21.89'

Reichelt Unit SSF4, 8 Remnant 16.2 ha Jasper N 41º42.19', W 92º51.84'

Sheeder Prairie SP2, 8 Remnant 10.1 ha Guthrie N 41º41.36', W 94º35.29'

“Airport” CBG3, 8, Integrated 23.7 ha Polk N 41º44.13', W 93º21.89'

Engeldinger Marsh CBG3, 8 Integrated 2.0 ha Polk N 41º46.60', W 93º21.24' 80

Sandhill East CBG3, 8 Integrated 8.9 ha Polk N 41º46.36', W 93º23.32'

Sandhill West CBG3, 8 Integrated 16.7 ha Polk N 41º46.08', W 93º22.99'

Turtlehead Fen CBG3, 8 Integrated 8.7 ha Jasper N 41º47.11', W 93º30.67'

Big Creek WMA5, 8 Reconstructed 6.9 ha Polk N 41º48.22', W 93º43.77'

Brigg’s Woods Park8, Reconstructed 6.9 ha Hamilton N 42º26.17', W 93º47.90'

Colo Bogs WMA5, 8 Reconstructed 32.4 ha Story N 42º00.68', W 93º16.18'

3 Chichaqua Bottoms Greenbelt

4 Stephen’s State Forrest

5 Wildlife Management Area 81

Table 1. continued

Site name Type1 Size County Lat, Long

Grant Ridge9 Reconstruction 8.4 ha Story N 41º58.18', W 93º28.31'

Grimes Farm CA6, 8 Reconstruction 6.1 ha Marshall N 42º01.35', W 92º58.15'

McFarland Park8, Reconstruction 24.3 ha Story N 42º05.75', W 93º33.99'

Meetz8, Reconstruction 8.1 ha Story N 42º03.54', W 93º32.55'

Prairie Flower Campground8, Reconstruction 3.8 ha Polk N 41º44.99', W 93º41.41' 81

Richard’s Marsh NRA7, 8, Reconstruction 30.4 ha Story N 42º19.62', W 42º19.62'

Stargrass9 Reconstruction 2.5 ha Story N 41º59.68', W 93º33.24'

6 Conservation Area

7 Natural Resource Area 82

Table 2. Aphid species and their host plants collected from transect sampling in 2006 and 2007.

Aphid species Host plant(s) Locations1 Status2 Occurrence3

Acuticauda solidaginifoliae Solidago gigantea* 1 Native September

Solidago canadensis 1

Aphis asclepiadis Apocynum sibiricum 1 Native June-August

Asclepias syriaca 2

Aphis cornifoliae Cornus sp. 3 Native July 82

Aphis debilicornis Helianthus grosseserratus 3 Native May-September

Helianthus sp. 1

Solidago canadensis* 1

Aphis decepta Pastinaca sativa 1 Native May, July, September

Aphis fabae complex Cirsium sp. 1 Exotic August

1 Number of locations on which a particular species was found on a host plant

2 Classified as native or exotic to North America

3 Months in which specimens were collected during transect sampling

* New host plant record according to Blackman and Eastop (2006) 83

Table 2. continued

Aphid species Host plant(s) Locations1 Status2 Occurrence3

Aphis farinosa Salix sp. 1 Native June-July

Aphis monardae Monarda fistulosa 19 Native May-September

Aphis nerii Asclepias incarnata 1 Exotic July-August

Asclepias syriaca 1

Aphis oestlundi Oenothera sp. 1 Native August 83

Aphis pulchella Euphorbia corollata* 3 Native June-July

Aphis rumicis Rumex obtusifolius 1 Exotic June

Aphis saniculae Cicuta maculata* 1 Native May-August

Pastinaca sativa* 1

Zizia aurea 8

Aphis vernoniae Helenium autumnale 2 Native June-August

Vernonia baldwinii 1

Vernonia fasciculata 1

Brachycaudus cardui Onosmodium molle* 1 Exotic June 84

Table 2. continued

Aphid species Host plant(s) Locations1 Status2 Occurrence3

Brachycaudus tragopogonis Tragopogonan sp. 1 Exotic July

Capitophorus elaeagni Cirsium arvensis 1 Exotic June-September

Cirsium discolor 3

Cirsium sp. 1

Carolinaia rhois Elymus canadensis 10 Native July-August 84

Rhus glabra* 1

Chaitophorus nigrae Salix sp. 2 Native June-July

Chaitophorus populicola Populus sp. 1 Native July

Hyadaphis foeniculi Daucus carota 3 Exotic August

Hyalomyzus monardae Monarda fistulosa 11 Native May-August

Hysteroneura setariae Prunus americana 5 Native June-August

Prunus serotina 2

Macrosiphoniella ludovicianae Artemisia ludoviciana 3 Native May-August

Macrosiphoniella tapuskae Achillea millefolium 1 Native June, August 85

Table 2. continued

Aphid species Host plant(s) Locations1 Status2 Occurrence3

Macrosiphoniella tapuskae Artemisia ludoviciana* 1

Maculolachnus submacula Rosa arkansana* 2 Native July

Microparsus variabilis Desmodium spp. 6 Native June-September

Myzus cerasi Prunus serotina 1 Exotic June 85

Nasonovia sp. Polygonum amphibium* 1 ? June, July, August

Nasonovia williamsi Potentilla arguta 1 Native June

Nearctaphis bakeri Trifolium pratense 3 Native June-August

Pleotrichophorus psuedopatonkus Achillea millefolium* 1 Native May

Rhodobium porosum Rosa arkansana* 6 Native June-July

Thripsaphis ballii Carex sp. 1 Native August

Thripsaphis daviaulti Carex sp. 1 Native June

Uroleucon sp.** Ratibida pinnata* 8 Native May-July

** Undescribed species 86

Table 2. continued

Aphid species Host plant(s) Locations1 Status2 Occurrence3

Uroleucon (Lambersius) sp. Solidago sp. 1 ? June

Uroleucon ambrosiae Silphium integrifolium* 3 Native June-August

Silphium perfoliatum* 4

Uroleucon anomalae Aster novae-angliae 5 Native June-August 86

Uroleucon atripes Brickellia eupatorioides* 4 Native August

Uroleucon erigeronensis Conyza canadensis 1 Native July

Uroleucon helianthicola Helianthus grosseserratus* 14 Native May-August

Helianthus tuberosus 2

Uroleucon luteolum Solidago canadensis* 8 Native June-August

Solidago gigantea 2

Solidago regida* 1

Uroleucon macgilliwrayae Solidago sp. 1 Native June

Uroleucon nigrotuberculatum Solidago canadensis 10 Native June-August

Uroleucon olivei Solidago canadensis* 1 Native July 87

Table 2. continued

Aphid species Host plant(s) Locations1 Status2 Occurrence3

Uroleucon pieloui Solidago canadensis 1 Native July

Uroleucon sonchellum Lactuca canadensis 2 Native August

Uroleucon sp. Ambrosia trifida 1 ? June 87

Table 3. Aphid species and their host plants not collected from transect sampling in 2006 and 2007.

Aphid species Host plant(s) Locations1 Status2

Anoecia cornicola Cornus sp. 1 Native

Aphis craccivora Rumex obtusifolius* 1 Exotic

Aphis fabae complex Rumex obtusifolius 1 Exotic

Aphis impatientis Impatiens sp. 1 Native

Macrosiphom impatientis Impatiens sp. 1 Native

Uroleucon eupatoricolens Ambrosia artemisifolia* 1 Native

Uroleucon rurale Ambrosia trifida* 1 Native

1 Number of locations on which a particular species was found on a host plant

2 Classified as native or exotic to North America

Table 4. Aphid abundance, percent parasitism rate, and percent tended by ants in the field for 2006 and 2007

Aphid species Colonies1 aphid/colony2 Parasitism3 Ant Tended4

Acuticauda solidaginifoliae 42 31.8 0.00% 0.00%

Aphis asclepiadis 18 39.9 2.89 33.33

Aphis cornifoliae 6 107.5 0.00 33.33

Aphis debilicornis 299 92.1 0.19 93.99

Aphis decepta 4 37.0 0.00 100.00

Aphis fabae complex 1 15 0.00 100.00

Aphis farinosa 13 69.2 0.00 92.46

Aphis monardae 531 29.9 3.94 84.82

Aphis nerii 15 280.8 0.00 13.33

Aphis oestlundi 1 80 0.00 100.00

Aphis pulchella 14 19.8 0.36 78.57

Aphis rumicis 2 90.5 0.00 50.00

Aphis saniculae 257 81.9 0.02 79.72

Aphis vernoniae 349 29.1 0.01 97.68

Brachycaudus cardui 1 1 0.00 0.00

Brachycaudus tragopogonis 1 37 0.00 0.00

Capitophorus elaeagni 158 34.3 0.27 6.44

Carolinaia rhois 918 49.0 0.00 0.11

Chaitophorus nigrae 3 199.7 0.00 100.00 90

Table 4. continued

Aphid species Colonies1 aphid/colony2 Parasitism3 Ant Tended4

Chaitophorus populicola 10 128.7 0.00 100.00

Hyadaphis foeniculi 9 12.0 0.00 0.00

Hyalomyzus monardae 375 8.3 4.02 3.26

Hysteronerva setariae 39 31.5 0.16 87.21

Macrosiphoniella ludovicianae 230 3.5 0.00 0.00

Macrosiphoniella tapuskae 50 3.5 0.57 0.00

Maculolachnus submacula 9 3.0 0.00 77.44

Microparsus variabilis 1158 51.3 0.02 10.59

Myzus cerasi 7 88.6 0.00 43.00

Nasonovia sp. 10 12.5 0.00 0.00

Nasonovia williamsi 7 3.6 4.00 0.00

Nearctaphis bakeri 23 42.3 0.00 86.96

Pleotrichophorus psuedopatonkus 8 1.3 60.00 0.00

Rhodobium porosum 137 9.1 0.00 17.96

Thripsaphis ballii 2 13.5 0.00 100.00

Thripsaphis daviaulti 3 10.3 0.00 100.00

Uroleucon sp. (Ratibida) 210 25.6 0.02 0.00

Uroleucon (Lambersius) sp. 1 18.0 0.00 0.00

Uroleucon ambrosiae 39 35.9 0.07 13.08

Uroleucon anomalae 28 10.1 0.00 0.00 91

Table 4. continued

Aphid species Colonies1 aphid/colony2 Parasitism3 Ant Tended4

Uroleucon atripes 16 4.9 0.00 0.00

Uroleucon erigeronensis 2 137.5 0.00 0.00

Uroleucon helianthicola 714 21.9 0.02 0.70

Uroleucon luteolum 419 2.4 0.00 0.00

Uroleucon macgilliwragae 14 2.6 0.00 0.00

Uroleucon nigrotuberculatum 64 16.5 3.02 1.56

Uroleucon olivei 2 4.5 0.00 0.00

Uroleucon pieloui 2 20.0 0.00 0.00

Uroleucon sonchellum 4 64.8 0.00 0.00

Uroleucon sp. (Ambrosia) 1 6.0 0.00 0.00

1 Total number of aphid colonies found during transect sampling in 2006 and 2007

2 Estimated mean size of aphid colonies found during transect sampling in 2006 and 2007

3 Estimated parasitism rates of aphids found during transect sampling in 2006 and 2007

4 Estimated percentage of aphid colonies tended by ants found during transect sampling in 2006 and 2007

Table 5. Aphid species and the ant species found tending them in central Iowa prairies in

2006 and 2007

Aphid species Ant species

Aphis asclepiadis Formica montana

Aphis cornifoliae Formica incerta

Aphis craccivora Formica montana

Aphis debilicornis Formica montana

Formica subsericea

Aphis decepta Formica montana

Aphis farinosa Formica montana

Aphis monardae Camponotus americanus

Crematogaster cerasi

Dolichoderus taschenbergi

Formica argentea

Formica dakotensis

Formica dolosa

Formica exsectoides

Formica incerta

Formica montana

Formica obscuripes

Formica pallidefulva

Formica subsericea 93

Table 5. continued

Aphid species Ant species

Aphis monardae Lasius alienus

Lasius neoniger

Myrmica sp.

Paratrechina parvula

Prenolepsis impairs

Temnothorax ambiguous

Aphis nerii Crematogaster cerasi

Aphis oestlundi Crematogaster cerasi

Aphis pulchella Formica dolosa

Formica subsericea

Myrmica sp.

Aphis rumicis Lasius alienus

Aphis saniculae Formica montana

Formica pallidefulva

Formica subsericea

Myrmica sp.

Aphis vernoniae Dolichoderus taschenbergi

Formica montana

Myrmica fracticornis

Crematogaster cerasi 94

Table 5. continued

Aphid species Ant species

Chaitophorus nigrae Formica montana

Chaitophorus populicola Formica subsericea

Hyalomyzus monardae Lasius alienus

Hysteroneura setariae Crematogaster cerasi

Formica dakotensis

Formica exsectoides

Formica incerta

Formica subsericea

Lasius alienus

Myrmica sp.

Prenolepsis imparis

Maculolachnus submacula Formica obscuripes

Microparsus variabilis Formica montana

Formica subsericea

Lasius alienus

Lasius neoniger

Myrmica sp.

Paratrechina parvula

Myzus cerasi Formica subsericea

Nearctaphis bakeri Formica montana 95

Table 5. continued

Aphid species Ant species

Nearctaphis bakeri Lasius alienus

Thripsaphis balli Dolichoderus taschenbergi

Uroleucon ambosiae Formica subsericea

Uroleucon sonchellum Formica incerta

Table 6. Cluster means for aphid species analyzed with hierarchical clustering.

Cluster means

Taxa 1 2 3

Acuticauda solidaginifoliae 0 121.5 24.6

Aphis asclepiadis 11.2 6.1 0

Aphis cornifoliae 0 64.5 0

Aphis debilicornis 2,025.18 25.1 0

Aphis monardae 34.55 1,210.3 216.6

Aphis pulchella 24.18 1.1 0

Aphis saniculae 792.5 13.8 15.2

Aphis vernoniae 111.9 8.0 0

Capitophorus elaeagni 109.6 3.5 328.0

Carolinaia rhois 3,835.6 246.7 41.0

Caitophorus nigrae 51.9 2.8 0

Hyadaphis foeniculi 0.6 4.0 12.2

Hyalomyzus monardae 7.0 114.7 216.6

Hysteronerva setariae 11.9 106.5 6.2

Macrosiphoniella ludovicianae 71.73 0.8 0

Macrosiphoniella tapuskae 16.0 0 0

Microparsus variabilis 117.1 0 6,404.2

Nearctaphis bakeri 11.5 2.8 82.8

Rhodobium porosum 112.2 1.5 0 97

Table 6. continued

Cluster means

Taxa 1 2 3

Uroleucon sp. (Ratibida) 40.5 61.0 473.8

Uroleucon ambrosiae 32.3 103.7 1.6

Uroleucon anomolae 17.7 2.2 9.8

Uroleucon atripes 4.73 2.6 0

Uroleucon helianthicola 1,118.6 75.1 253.8

Uroleucon luteolum 1.9 3.7 189.8

Uroleucon nigrotuberculatum 11.4 15.2 144.6

Uroleucon sonchellum 0 12.00 27.80

Figure 1. Map of the location of prairies sampled in central Iowa in 2006 and 2007 for aphids.

Figure 2. Mean seasonal abundance of aphids + SEM by month and week of sampling season at the 26 monthly and 3 weekly/bi-weekly sampled prairies in (A) 2006 and (B)

2007.

Figure 3. Proportion of the total number of aphids surveyed on a monthly sampling period belonging to a particular species in (A) 2006 and (B) 2007.

Figure 4. Proportion of the total number of aphids surveyed on a weekly/bi-weekly sampling period belonging to a particular species in (A) 2006 and (B) 2007.

Figure 5. Dendrogram from hierarchical cluster analysis of aphid species collected from the 26 prairie sites classified as remnant (Rm), isolated reconstruction (Rc), or integrated reconstruction (I).

Figure 6. Representation of the aphid community by NMDS across the three prairie types from combined years (2006 and 2007). Non-overlapping hulls indicate different species compositions.

Figure 7. Representation of the plant community by NMDS across the three prairie types from combined years (2006 and 2007). Non-overlapping hulls indicate different species compositions.

Figure 8. Season abundance of Aphis monardae and Hyalomyzus monardae at weekly/bi- weekly sampled sites in 2006. 100

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Aphis monardae

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111

Chapter 4

Are prairies a source of Aphis glycines (Hemiptera: Aphididae) predators?

A paper to be submitted to Journal of Insect Conservation

Wayne J. Ohnesorg, Jessica M. Orlofske, and Matthew E. O’Neal

Abstract

Prairies once covered the majority of the landscape in Iowa. Prairie restoration and reconstruction has been suggested for the improvement ecosystem services.

Specifically, this native, perennial habitat may serve as reservoirs and refugia for natural enemies for biological control of annual crop pests. We investigated central Iowa prairies for aphidophagous and generalist predators that can contribute to A. glycines management. During 2006 and 2007 prairies were sampled for predators using sweep nets. Prairies were selected from three types; 11 native prairies that had not been seeded into (remnants), 10 reconstructed prairies formerly agricultural fields that had been seeded and isolated from remnants (isolated reconstructions), and 5 integrated reconstructed prairies adjacent to remnant prairies (integrated). Sampling was conducted weekly/bi-weekly on 3 sites, while the remaining sites were sampled once a month. We found arthropods from 7 orders with 34 families that are or could be considered potential predators of A. glycines, of which, ~5% were coccinellids and Araneid spiders being the most abundant predator collected (>1,500). The diversity of the coccinellid community was dominated by native species (Brachiacantha decempustulata, Brachiacantha ursina,

Colleomegilla maculatta, Cycloneda munda, Diomus sp., Hippodamia convergens, 112

Hippodamia parenthesis, Hyperaspis undulata, and Microweisea sp.). However, the exotic Coccinella septempunctata was the single most abundant coccinellid species found. Only one other species of exotic coccinellid was found, Harmonia axyridis, but at a very low abundance. This is in sharp contrast to Iowa soybean fields were H. axyridis dominates. We suggest that prairies may not serve as a source of H. axyridis, but may serve as a source of other aphid predators such as Syrphidae. Furthermore, our data suggests that the state of prairie restoration does not affect the natural enemy community.

Introduction

Aphis glycines Matsumura (Hemiptera: Aphididae) has become a pest of soybean

(Glycine max) in North America. First discovered in Wisconsin in 2000 (Wedburg 2000,

Alleman et al. 2002), it has since spread across the Midwest and several Canadian provinces and by 2003 could be found across Iowa (O’Neal 2006). In 2003, more than

2007). Endemic predators can regulate populations of A. glycines (Fox et al. 2004,

Rutledge et al. 2004, Fox et al. 2005, Rutledge and O’Neil 2005, Costamagna and Landis

2006, Desneux et al. 2006, Mignault et al. 2006, Schmidt et al. 2007). However, A. glycines still produces outbreaks despite the mortality produced by predators.

Conservation has been proposed as an approach to biological control wherein the environment or management practices are modified to enhance natural enemies (i.e., 113

aphid predators) and their deleterious effects on pests (Hajek 2004). Habitat management is one method of conservation biological control that has been successful (Landis et al.

2000) in reducing the impact of some agricultural insect pests. Non-crop area provided for habitat management can enhance natural enemies by providing alternate hosts

(DeBach and Rosen 1991, Menalled et al. 1999), shelter (Gurr et al. 1998), and non-host food (Baggen et al. 1999, Wilkinson and Landis 2005). Cereal crops bordered with

Phacelia tanacetifolia (Hydrophyllaceae) had higher rates of syrphid-derived aphid predation, attributed to adult flies utilizing this floral resource (Sengonça and Frings

1988, Hickman and Wratten 1996). Recent evidence suggests that natural enemies that require a floral resource may benefit more from native, perennial plants than other commonly used exotic plant species (Fiedler and Landis 2007a,b). Of the 43 native species examined by Fiedler and Landis (2007a), 35 occur in prairies. Prairies are grassland communities consisting of primarily grasses and forbs. This community was once a major component of the landscape of the Midwest, prior to European settlement.

In Iowa, prairie now covers less than 0.1% of the area it once occupied (Smith 1998).

With the interest to improve conservation practices to improve ecosystem services

(Secchi et al. 2008), there may be potential for reconstructed prairies to serve as a source of natural enemies for the biological control of A. glycines.

Recent interest in prairie restoration has resulted in reconstructed prairies being planted into old agricultural fields. In a study by Shepherd and Debinski (2005), butterflies (Lepidoptera) responded differently to three qualitative types of prairies: remnants, isolated reconstructions, and integrated reconstructions. Remnant prairies 114

supported a greater abundance and diversity of butterflies. Integrated reconstructions were an intermediate between remnant and isolated reconstructed prairies in both abundance and diversity of butterflies.

Prairies could possibly conserve predators of A. glycines, such as Coccinellidae

(Coleoptera), Chrysopidae (Neuroptera), Hemerobiidae (Neuroptera), Syrphidae

(Diptera), Nabidae (Hemiptera), and Opiliones and specifically Harmonia axyridis Pallas

(Coleoptera: Coccinellidae), and Orius insidiosus (Say) (Hemiptera: Anthocoridae),

(Rutledge et al. 2004, Allard and Yeargan 2005). Of particular interest are H. axyridis and O. insidiosus, both considered to be key predators of A. glycines in soybean fields

(Rutledge et al. 2004, Rutledge and O’Neil 2005).

We hypothesize that prairies in central Iowa could act as a source of predators for biological control of A. glycines. Our objective was to describe the diversity and abundance of the foliar active aphidophagous arthropod community found in prairies in central Iowa. A second objective was to determine if the natural enemy community will differ between the 3 prairie types: remnant, isolated reconstruction, and integrated reconstruction.

Methods and Materials

isolated reconstructions, and 5 integrated reconstructions. Sites that were classified as remnants were areas where the original seed bank has remained and has not received additional seed from other sources. Isolated reconstructed prairies were agricultural fields that had been converted into prairie plantings with a minimum age of 7 years. Integrated reconstructed prairies were sites where reconstructed prairies were located directly adjacent to remnant prairies and other restored areas with a minimum age of 3 years. All integrated reconstruction sites were located at Chichaqua Bottoms Greenbelt in Polk

County, Iowa with the exception of Turtlehead Fen in Jasper County. Chichaqua Bottoms

Greenbelt is a large-scale remnant/reconstructed natural area encompassing more than

2,600 hectares.

Natural Enemy Sampling. Natural enemy sampling was conducted at 26 prairie sites once a month starting in May and continuing through August in 2006 and 2007. A subset of eight sites was also sampled in September of both years. Of the 26 sites, three were sampled on a weekly basis from 24 May, 2006 through the end of July in 2006 and on a biweekly basis in August and September in 2006. In 2007, those same three sites were sampled on a biweekly basis beginning 1 May and sampling continued through

September. Sampling was conducted along three 25-m transects at each site at each sampling period. Transects were randomly selected at each site on each sampling date.

Prior to sampling a prairie, the prairie was divided roughly into quarters. Random numbers were obtained from a random number table to determine which quarter of each prairie to sample. From the center of the quarter, another random number was obtained to 116

determine in which of four directions the transect would be laid out: right, left, forward, or back. Samples were taken at least 10-m from the closest edge of each site to avoid edge effects. Samples were taken using a 38.1-cm diameter sweep net. Each sample consisted of twenty pendulum sweeps along each transect. Samples were kept cool during transportation back to the laboratory and were subsequently stored at -20º C. Once samples were frozen, they were sorted and transferred into 70% ethyl alcohol.

Aphid Monitoring. Aphid populations in our prairies were monitored for availability of prey for natural enemies using a transect method modified from New (1998). Three random 2-m by 25-m transects were sampled for aphids at each sampling time. The above ground portion of all plants was inspected for aphids. Aphids were identified to morpho-species based on color and host plant. The total number of colonies found on each transect was recorded along with size of the first 10 colonies for each morpho- species. The mean colony size for each morpho-species was calculated and then multiplied by the number of colonies found and summed among all aphid morpho-species to attain total aphid abundance on each transect. Total sampled aphid abundance at a prairie was calculated to be the mean of the total aphid abundance of the three transects.

Natural Enemy Identification. The taxonomic level, for aphidophagous and generalist predatory natural enemies identification varied. All natural enemies were identified to order and nearly all to family. Only Opiliones were identified to order only. Two families, Cantharidae and Nabidae, were identified to the generic level. Generic level is 117

necessary to distinguish between predatory and non-predatory Cantharidae. The

Anthocoridae, Pentatomidae, and Coccinellidae were identified to species.

Statistical Analysis. The data were prepared as follows. All sites that were sampled monthly were included in the analysis. Data from weekly/bi-weekly sampling was taken from the sampling period closest to the middle of the month when possible. Only sampling months containing complete data sets were analyzed. Data from September were not included as only a sub-sample of the sites was sampled. In 2006, only natural enemies sampled in June and July were included in the analysis. All months were included from 2007, except September. The total abundance of each taxon was tallied for each site at the family level, except for Opiliones, which were not identified to family.

All months were included from 2007, except September. Any species that occurred at less than 5% of the prairie sites was excluded from the following analyses. Utilizing this criteria taxa which occurred at a single were not included and reduced the total number of taxa analyzed.

To determine the association between prairie type and the abundance and diversity of the natural enemy community, natural enemies were analyzed utilizing a hierarchical cluster analysis. This analysis places sites into groups based upon the abundance and composition of the natural enemy community sampled. If prairie type does have an association with natural enemy abundance and diversity, we would expect three groups or clusters with a specific prairie type dominating each cluster. We utilized a

Bray-Curtis distance metric for analysis with Ward’s linkage method. Bray-Curtis is 118

robust measure of distance for ecological data (Faith et al. 1984). Data were analyzed using R statistical software (version 2.5.1, © The R Foundation for Statistical

Computing).

An ordination analysis, non-metric multidimensional scaling (NMDS) was also used to compare the natural enemy community across the three prairie types. NMDS summarizes relationships between all pairs of species data in order to represent it in multiple dimensions as distances, so the closer two points are, the more similar the communities (i.e., same species composition; Kenkel and Orlóci 1986). We utilized the

Bray-Curtis distance measure for analysis. Data were analyzed using R statistical software (version 2.5.1, © The R Foundation for Statistical Computing). Drawing convex hulls for each prairie type visualized differences in species composition.

Results

Natural Enemy Sampling. Aphidophagous arthropods and generalist predators that may also feed on aphids collected in 2006 and 2007 are summarized in Table 2. In all, two classes of arthropods were collected comprising 7 orders, and 34 families. Further identification of selected taxa included 15 genera and 13 species. In both 2006 and 2007, the most abundant families of foliage-dwelling predators were from the class Araneae.

Specifically, Salticidae was the single most abundant family of predatory arthropods in

2006, while Araneidae was the most abundant in 2007. The most abundant predatory insect was adult Syrphidae (Diptera) in both 2006 and 2007. While adult syrphids are not predatory, their larvae are important aphid predators. A number of known natural 119

enemies of A. glycines found in soybean fields were also found in prairies. This included

H. axyridis and O. insidiosus, key predators of A. glycines (Rutledge et al. 2004, Rutledge and O’Neil 2005). Other aphid predators found included other Coccinellidae,

Chrysopidae, Hemerobiidae, Syrphidae, Opiliones, and Nabidae.

More specifically we collected 12 species in 9 genera of Coccinellidae. Total seasonal abundance of Coccinellidae was similar in both 2006 (Fig. 2A) and 2007 (Fig.

2B). The diversity of the coccinellid community was dominated by native species

(Brachiacantha decempustulata, Brachiacantha ursina, Colleomegilla maculatta,

Cycloneda munda, Diomus sp., Hippodamia convergens, Hippodamia parenthesis,

Hyperaspis undulata, and Microweisea sp.). However, the exotic Coccinella septempunctata was the single most abundant coccinellid species found. Only one other species of exotic coccinellid was found, Harmonia axyridis, but at a very low abundance.

We found a total of 8 individuals of H. axyridis combined in both years of sampling.

Proportionally, the native Coccinellidae community was more abundant than the exotic

Coccinellidae community in 2006 (Fig. 3A) and 2007 (Fig. 3B). The only month in which natives did not occupy the greatest proportion of the community was June in both years (Figs. 3A,B). In June, C. septumpunctata was the single most abundant coccinellid.

In addition to known A. glycines predators, we found other predatory taxa that feed on aphids or are generalist predators that may potentially use A. glycines as a food source. In this category we include Dolichopodidae (Diptera), two genera of Cantharidae

(Coleoptera: Podabrus, Rhagonycha), and Reduviidae (Hemiptera).

120

Natural Enemy Community Analysis. The natural enemy community was analyzed to determine the effect of prairie type. Hierarchical clustering analysis was used to divide the prairies sampled into clusters (e.g. groups). The cluster analysis did not produce a hierarchy that corresponded to prairie type and yielded four clusters. Our analysis of the natural enemy community produced four clusters (Fig. 4), only one of which was dominated by specific type of prairie. Cluster I contained only reconstructed prairies (2 isolated and 1 integrated reconstructions). The three remaining clusters contained a mix of the three categories of prairies surveyed. Seven sites comprised the cluster II with 3,

2, and 2 remnant, isolated reconstruction and integrated reconstruction prairie sites, respectively. Cluster III was the largest cluster, and it was made up of 12 sites with 6, 5, and 1 remnant, isolated reconstructed and integrated reconstructed prairie sites, respectively. Cluster IV was comprised of two remnants, one isolated reconstruction, and one integrated reconstruction. The clusters are characterized by the natural enemy community found in them rather than by prairie type. Means for the natural enemy taxa in each cluster can be found in Table 3. Specific taxa were often more abundant in one cluster than in others. Cluster I contained the lowest means for may of the natural enemy taxa (e.g., Anthocoridae and Syrphidae), but had the highest mean for Lycosidae

(Araneae) (2.67). As for cluster II, mid-level means are typical yet it had the highest mean for Syrphidae (21.71). Cluster III was made up of mostly mid-level to high means.

Some of the highest means were for Braconidae, Eulophidae (Hymenoptera),

Dolichopodidae (Diptera), and Oxyopidae (Araneae) with means of 19.17, 10.42, 11.17, and 3.58, respectively. Cluster IV contained the highest means for Araneidae (Araneae), 121

Salticidae (Araneae), Thomisdae (Araneae), Coccinellidae, and Nabidae (Hemiptera) with means of 61.25, 48.75, 27.25, 12.5, and 7, respectively.

An additional visual comparison of the natural enemy community composition amongst the three prairie types was produced using NMDS (Fig. 6). Data from 2006 and

2007 was combined and reported. The hulls represent the three prairie types and are overlapping, suggesting that the natural enemy communities observed in three prairie types are very similar.

Aphid Monitoring. Aphid abundance was monitored throughout the duration of our study. Over the two years of our study, we found a total of 49 aphid species in 18 genera

(Table 4) while conducting transect sampling. Aphid abundance was greater in 2007 than in 2006. In 2006, weekly/bi-weekly sampling revealed that aphidophagous and generalist predator natural enemy abundance appeared to track aphid abundance (Fig. 6A). As natural enemy abundance increased in August, a decrease in aphid abundance was also observed (Fig. 6A). Monthly sampling in 2006 produced a similar trend to the weekly/bi- weekly sampling in that natural enemies increased as the season progressed (Fig. 7A).

Decreases in aphid abundance were observed with the increased natural enemy abundance (Fig. 7A). The decrease in aphid abundance may have been due to other causes, including both biotic and abiotic factors.

In 2007, bi-weekly sampling yielded a similar trend to 2006 with aphidophagous and generalist predator natural enemy abundance seeming to track aphid abundance (Fig.

6B). However, at the end of August, a drop in aphid abundance did not show a 122

corresponding increase in natural enemies. Monthly sampling for natural enemies and corresponding aphid abundance in 2007 generated a trend of increasing abundance as the season progressed with abundance beginning to decrease in September (Fig. 7B). Aphid abundance also increased as the season progressed and decreased in September (Fig. 7B).

Discussion

Prairies in central Iowa may serve as sources of predatory and aphidophagous arthropods for conservation biological control of A. glycines and possibly other pests. The results of our study show that a number of A. glycines predators can be found in central

Iowa prairies. These results support our hypothesis that prairies can serve as a source of predatory natural enemies that could be important for biological control of A. glycines

(Table 2). Of the A. glycines natural enemies found, one is a key natural enemy of A. glycines, O. insidiosus (Rutledge et al. 2004, Rutledge and O’Neil 2005).

In a study conducted by Evans (2003) in alfalfa fields in Utah, it was demonstrated that C. septempunctata, our most abundant coccinellid in both years, had displaced native coccinellids. Upon further inspection, the mechanism was found to be reductions in the amount of prey available for forage (Evans 2003). Wheeler and

Hoebeke (1995) have suggested that C. septempunctata may have caused the disappearance of the once common native coccinellid Coccinella novemnotata. While our study lacks baseline data prior to the introduction of C. septempunctata, it appears that C. septempunctata is currently the dominant individual coccinellid species found in central 123

Iowa prairies. This suggests that C. septempunctata has displaced native coccinellids as the dominant species in prairies in central Iowa.

In contrast, the abundance of H. axyridis was low relative to C. septempunctata and native coccinellids. Native coccinellids, as a group dominated in our prairie sites.

Prairies do not appear to be desirable habitat for H. axyridis. In soybean, Schmidt et al.

(2008) found that H. axyridis was the dominant coccinellid followed by C. septempunctata and then native coccinellids. This difference in community composition suggests that prairies would not act as a source of H. axyridis for biological control of A. glycines.

The use of H. axyridis for biological control and its subsequent invasions has been marked with non-target impacts to other coccinellids and crops (i.e., grapes), which have been reviewed by Koch (2003), Koch et al. (2006), and Pervez and Omkar (2006). The invasion of H. axyridis into different habitats has generated concern for other arthropods and potential for non-target impacts (Boettner et al. 2000). From our study, it seems that

H. axyridis may not have considerable non-target impact on aphids or other Coccinellidae in prairies.

A number of other predators were found in central Iowa prairies. Some of these predators are potential and recorded aphid predators with not all of them found by

Schmidt et al. 2008. Dolichopodidae adults are predaceous on smaller insects, which could include aphids. While their use of aphids as prey is not known, spiders (Araneae) composed over half of the predatory arthropods sampled in both 2006 and 2007. This large population of predatory arthropods may not have direct implications for biological 124

control of A. glycines, but they may be beneficial for biological control of other pest species. We also found two genera, each with two morpho-species, of predatory soldier beetles (Podabrus and Rhagonycha) that had not been found in soybean fields in previous studies (Beschinski and Pedigo 1981, Schmidt et al. 2008). Other predatory soldier beetles (Cantharis spp.) have been found feeding on aphids in agricultural fields, namely sugar beets (Beta vulgaris)(Landis and Van Der Werf 1997). Adults in the genus

Rhagonycha are generalist predators, while Podabrus adults are predatory on soft-bodied insects, including aphids (Ramsdale 2001).

The three types prairie (remnant, isolated reconstruction and integrated reconstruction) does not appear to play a role in the natural enemy community at the scope of this study. Results from the hierarchical cluster analysis and NMDS suggest that the generalist and aphidophagous natural enemy community we examined responds to the three prairie types in the same manner. Other factors may be involved in determining the suitability of prairies as a source of natural enemies. Recent work with selected native plants has shown that some are more attractive than others to natural enemies (Fiedler and Landis 2007a,b). This would suggest that the plant community found within individual prairies might have more influence on natural enemy abundance than our three prairie types.

Prairies could act as a source for the key A. glycines predators of syrphids and O. insidiosus. Syrphids were the most abundant predatory insect found, and were part of a shift observed in the soybean natural enemy community that occurred after the arrival of

A. glycines (Schmidt et al. 2008). In the comparison made by Schmidt et al. (2008), it 125

was observed that the exotic Coccinellidae had become more abundant than native

Coccinellidae. Also, syrphids had not been previously recorded from the soybean natural enemy community (Bechinski and Pedigo 1981, Schmidt et al. 2008). A key A. glycines predator O. insidiosus was not present in high numbers relative to other aphid predators, but was found at a much higher abundance (17x) than that of H. axyridis. In this context, prairies may serve as a reservoir for both syrphids and O. insidiosus for the biological control of A. glycines.

The predatory cantharids Rhagonycha spp. and Podabrus spp. may be examples of un-utilized predator species available for biological control of A. glycines. However, these species may not be adapted to ephemeral crop habitats. The movement of predatory arthropods from prairies into surrounding agricultural areas needs to be measured and documented. Also, the role of spiders in aphid predation needs to be better understood.

Further work must be done in these two areas to gain a more complete understanding of the ecosystem services provided by prairies.

Acknowledgements

We would like to thank B. Lammers, C. Hildebrand, D. Howell, J. Goerndt, J.

Pearson, J. Judson, K. Hansen, K. Cantu, K. Van Zante, M. Cosgrove, M. Meetz, L.

Meetz, M. Stegmann, S. Peterson, S. Lekwa, S. Rolfes, L. Lown, S. Moeckly, and M.

Moeckly for allowing us to conduct our research on the land they own or manage; E.

VanNostrand, M. Payne, R. Dostalik, & D. Windsor for field support; and G. 126

VanNostrand for spider identifications and field support. The North Central Soybean

Research Program provided funding for this work.

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Table 1. Prairies sampled for aphids in 2006 and 2007 including type, size in hectares (ha), and latitude and longitude

Site name Type1 Size County Lat, Long

A.C. & Lela Morris8 Remnant 8.1 ha Jasper N 41º46.26', W 92º57.69'

Boone Railroad9 Remnant ~3 ha Boone N 42º03.06', W 93º48.52'

Doolittle Prairie SP2, 8 Remnant 4.9 ha Story N 42º08.96', W 93º35.36'

Drobney8 Remnant 14.3 ha Jasper N 41º33.65', W 93º00.82'

Judson8, Remnant 6.1 ha Guthrie N 41º45.89', W 94º34.16' 132 2, 8

Liska-Stanek Prairie SP Remnant 8.1 ha Webster N 42º24.82', W 94º13.46'

Marietta Sand Prairie SP2, 8 Remnant 6.9 ha Marshall N 42º05.83', W 93º02.34'

Moeckly8, 15 Remnant 16.2 ha Polk N 41º46.94', W 93º39.90'

1 All integrated prairies were located at Chichaqua Bottoms Green Belt in Polk County, except Turtlehead fen in Jasper county, Iowa and directly adjacent to remnant areas

2 State Preserve

8 Sampled monthly

9 Sampled weekly/bi-weekly

134

Table 1. continued

Site name Type1 Size County Lat, Long

Puccoon CBG3, 8 Remnant 1.5 ha Jasper N 41º44.13', W 93º21.89'

Reichelt Unit SSF4, 8 Remnant 16.2 ha Jasper N 41º42.19', W 92º51.84'

Sheeder Prairie SP2, 8 Remnant 10.1 ha Guthrie N 41º41.36', W 94º35.29'

“Airport” CBG3, 8, Integrated 23.7 ha Polk N 41º44.13', W 93º21.89'

Engeldinger Marsh CBG3, 8 Integrated 2.0 ha Polk N 41º46.60', W 93º21.24' 133 3, 8

Sandhill East CBG Integrated 8.9 ha Polk N 41º46.36', W 93º23.32'

Sandhill West CBG3, 8 Integrated 16.7 ha Polk N 41º46.08', W 93º22.99'

Turtlehead Fen CBG3, 8 Integrated 8.7 ha Jasper N 41º47.11', W 93º30.67'

Big Creek WMA5, 8 Reconstructed 6.9 ha Polk N 41º48.22', W 93º43.77'

Brigg’s Woods Park8, Reconstructed 6.9 ha Hamilton N 42º26.17', W 93º47.90'

Colo Bogs WMA5, 8 Reconstructed 32.4 ha Story N 42º00.68', W 93º16.18'

3 Chichaqua Bottoms Greenbelt

4 Stephen’s State Forrest

5 Wildlife Management Area 135

Table 1. continued

Site name Type1 Size County Lat, Long

Grant Ridge9 Reconstruction 8.4 ha Story N 41º58.18', W 93º28.31'

Grimes Farm CA6, 8 Reconstruction 6.1 ha Marshall N 42º01.35', W 92º58.15'

McFarland Park8, Reconstruction 24.3 ha Story N 42º05.75', W 93º33.99'

Meetz8, Reconstruction 8.1 ha Story N 42º03.54', W 93º32.55'

Prairie Flower Campground8, Reconstruction 3.8 ha Polk N 41º44.99', W 93º41.41' 134 7, 8,

Richard’s Marsh NRA Reconstruction 30.4 ha Story N 42º19.62', W 42º19.62'

Stargrass9 Reconstruction 2.5 ha Story N 41º59.68', W 93º33.24'

6 Conservation Area

7 Natural Resource Area 135

Table 2. Taxa of aphidophagous and generalist predatory arthropods and total number of individuals collected during 2006 and 2007 in central Iowa prairies

#Individuals

Order Family Species 2006 2007

Araneae Anyphaenidae 1 24

Araneidae 585 997

Clubionidae 2 17

Dyctinidae 1 27

Dysderidae 0 1

Linyphiidae 132 28

Lycosidae 11 17

Oxyopidae 143 34

Philodromidae 91 98

Salticidae 629 765

Tetragnathidae 45 62

Thomisidae 397 235

Undetermined 19 46

Opiliones 18 20

Coleoptera Cantharidae Podabrus spp. 11 15

Rhagonycha spp. 3 26

Carabidae 10 16

Cicindelidae 2 0 136

Table 2. continued

#Individuals

Order Family Species 2006 2007

Coleoptera Cleridae 24 23

Coccinellidae Brachiacantha decempustulata 2 2

Brachiacantha ursina 7 9

Brachiacantha sp. 0 1

Coccinella septumpunctata 37 41

Coleomegilla maculata 17 3

Cycloneda munda 2 9

Diomus sp. 6 0

Harmonia axyridis 2 6

Hippodamia convergens 10 8

Hippodamia parenthesis 32 23

Hyperaspis undulata 7 13

Microweisea sp. 1 0

Diptera Dolichopodidae 36 264

Syrphidae 167 513

Hemiptera Anthocoridae Orius insidiosus 92 44

Nabidae Nabicula spp. 2 3

Nabis spp. 42 103

Pentotomidae Podisus maculiventris 18 73 137

Table 2. continued

#Individuals

Order Family Species 2006 2007

Hemiptera Reduviidae 87 142

Hymenoptera1 Aphelinidae 11 4

Braconidae 357 281

Encyrtidae 22 9

Eulophidae 123 201

Neuroptera Chrysopidae 110 165

Hemerobiidae 2 3

Totals 3332 4400

1 Families of Hymenoptera included are those that have been recorded to parasitize

aphids

138

Table 3. Cluster means for natural enemy taxa analyzed with hierarchical clustering for differences in the natural enemy community caused by prairie type.

Cluster means

Taxa 1 2 3 4

Anthocoridae 0.00 2.29 2.17 0.25

Anyphaenidae 2.00 0 0.58 1.00

Aphelindae 0.33 0.14 0 0.25

Araneidae 28.33 13.29 22.08 61.25

Braconidae 9.00 15.71 19.17 15.00

Cantharidae 0.67 1.00 1.83 2.75

Carabidae 0 0.86 0.42 0.50

Chrysopidae 6.67 7.43 6.58 8.50

Cleridae 1.33 0.86 1.08 1.25

Clubionidae 0.33 0.14 0.50 0.25

Coccinellidae 5.00 5.14 6.17 12.50

Dictynidae 0.33 0.14 0.917 2.00

Dolichopodidae 6.33 10.00 11.17 7.50

Eulophidae 0.67 5.29 10.42 5.75

Encyrtidae 0.33 0.43 0.67 0.75

Hemerobidae 0 0 0.08 0.50

Linyphiidae 2.00 1.57 2.33 1.75

Lycosidae 2.67 1.00 0.17 0.50 139

Table 3. continued

Cluster means

Taxa 1 2 3 4

Nabidae 2.33 6.29 3.67 7.00

Opiliones 0 0.71 1.00 2.25

Oxyopidae 2.67 3.29 3.58 3.25

Pentatomidae 0.33 2.57 2.08 6.25

Philodromidae 6.67 3.00 2.33 2.75

Reduviidae 2.00 1.86 5.67 8.25

Salticidae 14.33 15.14 34.25 48.75

Syrphidae 2.33 21.71 14.92 10.25

Tetragnathidae 2.67 5.57 1.00 3.00

Thomisidae 6.67 13.43 11.00 27.25

140

Table 4. Aphid species sampled from transect sampling in 2006 and 2007 including number of locations found at and status.

Aphid species Locations1 Status2

Acuticauda solidaginifoliae 2 Native

Aphis asclepiadis 2 Native

Aphis cornifoliae 3 Native

Aphis debilicornis 4 Native

Aphis decepta 1 Native

Aphis fabae complex 1 Exotic

Aphis farinosa 1 Native

Aphis monardae 20 Native

Aphis nerii 2 Exotic

Aphis oestlundi 1 Native

Aphis pulchella 3 Native

Aphis rumicis 1 Exotic

Aphis saniculae 8 Native

Aphis vernoniae 4 Native

Brachycaudus cardui 1 Exotic

Brachycaudus tragopogonis 1 Exotic

Capitophorus elaeagni 1 Exotic

1 Number of locations on which a particular species was found on a host plant

2 Classified as native or exotic to North America 141

Table 4. continued

Aphid species Locations1 Status2

Carolinaia rhois 11 Native

Chaitophorus nigrae 2 Native

Chaitophorus populicola 1 Native

Hyadaphis foeniculi 3 Exotic

Hyalomyzus monardae 11 Native

Hysteroneura setariae 7 Native

Macrosiphoniella ludovicianae 3 Native

Macrosiphoniella tapuskae 2 Native

Maculolachnus submacula 2 Native

Microparsus variabilis 7 Native

Myzus cerasi 1 Exotic

Nasonovia sp. 1 ?

Nasonovia williamsi 1 Native

Nearctaphis bakeri 5 Native

Pleotrichophorus psuedopatonkus 1 Native

Rhodobium porosum 6 Native

Thripsaphis ballii 1 Native

Thripsaphis daviaulti 1 Native

Uroleucon sp. (Ratibida) 8 Native

Uroleucon (Lambersius) sp. 1 ? 142

Table 4. continued

Aphid species Locations1 Status2

Uroleucon ambrosiae 5 Native

Uroleucon anomalae 5 Native

Uroleucon atripes 4 Native

Uroleucon erigeronensis 1 Native

Uroleucon helianthicola 14 Native

Uroleucon luteolum 8 Native

Uroleucon macgilliwragae 1 Native

Uroleucon nigrotuberculatum 11 Native

Uroleucon olivei 1 Native

Uroleucon pieloui 1 Native

Uroleucon sonchellum 2 Native

Uroleucon sp. (Ambrosia) 1 ? 143

Figure 1. Map of the location of prairies sampled in central Iowa in 2006 and 2007 for aphidophagous and generalist predatory arthropod natural enemies.

Figure 2. Total seasonal abundance of the Coccinellidae collected in (A) 2006 and (B)

2007 combined for all collection dates from all prairie sites.

Figure 3. Proportion of the total seasonal Coccinellidae collected that were Harmonia axyridis, Coccinella septempunctata, or natives in 2006 (A) and (2007).

Figure 4. Dendrogram from hierarchical cluster analysis of natural enemies collected from the 26 prairie sites classified as remnant (Rm), isolated reconstruction (Rc), or integrated reconstruction (I).

Figure 5. Representation of the natural enemy community by NMDS across the three prairie types from combined years (2006 and 2007). Overlapping hulls indicate similar species compositions.

Figure 6. Seasonal abundance by month and week of study for aphidophagous and generalist predatory arthropod natural enemies (NE) along with aphid prey representing the three prairie sites that were sampled weekly/biweekly in (A) 2006 and (B) 2007.

144

Figure 7. Seasonal abundance of aphidophagous and generalist predatory arthropod natural enemies (NE) along with aphid prey representing the 26 prairies sampled monthly in (A) 2006 and (B) 2007.

145

Fig. 1

146

Fig. 2A

n re w u o n k mat n Im U

Native Exotic 147

Fig. 2B

n re w u o n k mat n Im U

Native Exotic

148 Fig. 3A

Native

149

Fig. 3B

Native

150

Fig. 4

IV III I II L

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151

Fig. 5

Isolated Reconstruction

Integrated Reconstruction

Remnant

152

Fig. 6A

153

Fig. 6B

154

Fig. 7A

155

Fig. 7B

156

Chapter 5

General Conclusions

Chapter 2:

• The seed treatments of thiomethoxam and imidacloprid are not effective in reducing

Aphis glycines populations and unable to protect soybean yield.

• The neonicotinoid seed treatments of thiomethoxam and imidacloprid did not affect

the abundance of natural enemies.

• The reduced-risk insecticide pymetrozine protected soybean from Aphis glycines

herbivory as did the broad-spectrum insecticide -cyhalothrin.

• Pymetrozine was an intermediate in the abundance of natural enemies between the

untreated control and -cyhalothrin.

• Consistent results are needed from the foliar application of imidacloprid to conclude

its impacts on Aphis glycines and natural enemies.

Chapter 3:

• We found 49 species of aphids in central Iowa prairies, which included 13 species in

the genus Aphis.

• The 9 Aphis species heavily tended by ants (>70 % of colonies) may have a lower risk

of attack by Binodoxys communis.

• Prairie type had no effect on the amount of ant tending experienced by Aphis species

and specifically Aphis monardae. 157

• High abundance of Aphis monardae was associated with remnant prairies while high

abundance of Microparsus variabilis was associated with reconstructed or integrated

prairies.

• The aphid community found in remnant prairies is different than that found in

reconstructed or integrated prairies.

• Non-Aphis species that share a host plant with an Aphis species may be at risk for non-

target impacts by Binodoxys communis.

Chapter 4:

• We found arthropods from 7 orders with 34 families that are or could be considered

potential predators of A. glycines, of which, including 9 species of coccinellids

accounting for ~5% of the total natural enemies collected.

• Prairies in central Iowa would not act as a source of Harmonia axyridis for biological

control of Aphis glycines.

• Prairies in central Iowa could serve as a source of Syrphidae and Orius insidiosus for

biological control of Aphis glycines.

• The type of prairie does not affect the aphidophagous and generalist predator natural

enemy community found in a given prairie.

What does all this mean?

We now know that neonicotinoid seed treatments are not effective in reducing

Aphis glycines populations and protecting soybean yield. Our data support that a well- timed foliar application of an insecticide is sufficient to protect soybean yield. Use of a 158

reduced-risk insecticide does reduce the impact on the abundance natural enemies while protecting yield. With Binodoxys communis having been released for biological control of

Aphis glycines, future assessments of its non-target impacts will likely be made. These assessments will need to take into account the differences in the aphid community we found between remnant prairies and reconstructed and integrated prairies. However, our data suggest that this community difference does not exist for the aphidophagous and generalist predatory natural enemy community. From the natural enemy data, prairies would not act as a source of Harmonia axyridis, a key predator of Aphis glycines.

However, our data indicate that prairies would serve as a source of Syrphidae for biological control of Aphis glycines. 159

Acknowledgements

First and foremost I would like to thank my family. My wife Gina has been a

great support and encouragement during the past three years spent working on this degree.

Roger and Gwen, my parents, for their encouragement and allowing me to pursue my

passion in entomology from a young age. I can’t forget to thank everyone (friend and

family) who has endured the budding entomologist who would and still does stop to

investigate any insect or arthropod that happens to cross his path.

I am grateful for Dr. Matthew Elliot O’Neal for taking me on as a graduate

student in his laboratory here at Iowa State University. Even through the times when we

don’t see eye-to-eye, he has challenged me to put my best foot forward and be able to

explain my research thoughtfully and thoroughly.

My committee has been a great reservoir of wisdom and advice. Dr. Coats’

knowledge of insect toxicology and Dr. Debinki’s experience with prairies have proved

to be excellent resources.

Also the Iowa State University Department of Entomology for housing me and

allowing me to use their expertise. I can’t forget to thank the graduate student secretary

Kelly Kyle for all of her assistance and for the “candy dish.” Just for kicks and in

response to her prodding, I have now mentioned Beth Minner.

The collaborators on these projects have also proven invaluable; Jessica Orlofske

for her partnership in designing and accomplishing the all work done in prairies; Claudio

Gratton, George Heimpel, Bob O’Niel, Thelma Heidel for the non-target aphid study;

Dave Voegtlin and Doris Lagos for aphid identifications. 160

Finally, to all of the student workers who spent countless hours counting aphids and sampling for natural enemies in soybean fields and prairies; Scott Kostohryz,

Amanda Nodelman, Pankaj Makhija, Greg VanNostrand, Michaela Payne, Ruth Dostalik,

Drake Windsor, Satish Hanmanthgasi, Andrea Miller, Robert Moore, Alex Kleiman,

Dustin Paulson, Daniel Stull, and Limari Huesrtas.