Lynch Wsu 0251E 10705.Pdf (796.4Kb)

EXPLORING CONSUMPTION BY TWO GENERALIST PREDATORS IN POTATOES

USING MOLECULAR GUT CONTENT ANALYSIS AND BEHAVIORAL STUDIES

CHRISTINE ANN LYNCH

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Entomology

MAY 2013

To the Faculty of Washington State University

The member of the Committee appointed to examine the dissertation of CHRISTINE ANN LYNCH find it satisfactory and recommend that it be accepted.

William E. Snyder, Ph.D., Chair

Carol M. Anelli, Ph.D.

Allan S. Felsot, Ph.D.

David R. Horton, Ph.D.

ACKNOWLEDGEMENT

I am grateful to everyone who assisted me during this research project. First, I

acknowledge Dr. William Snyder for challenging me with this project and answering my many

questions. The assistance and guidance of my committee members, Dr. Carol Anelli, Dr. Dave

Horton, and Dr. Allan Felsot, are greatly appreciated. For expanding my knowledge of

entomology, I want to thank Dr. William Turner, Dr. John Brown, Dr. Marc Klowden, and Dr.

Sanford Eigenbrode. For support with statistical analysis, I recognize Dr. Dave Crowder and

Elliott Moon. This project would not have been possible without the financial support from

USDA AFRI.

For help with field and greenhouse experiments, I want to thank Ben Hieb, Glen Chase,

Jake Gable, Elliott Moon, Courtney Malcom, Abbie Estep, Alicia Nutter, Max Savery, Tina

Miller, and Tom Walter. The assistance of Bailey Cox, Kerri Wheeler and Abbie Estep sorting

arthropods from insect community samples is greatly appreciated.

I would like to thank Dr. Gretchen Snyder for teaching me sampling field methods,

laboratory techniques, and greenhouse protocol. I would also like to thank the following lab

colleagues: Amanda Meadows, Carmen Castillo Carrillo, Elliott Moon, Karol Krey, Dr. Emily

Jones, Dr. Randa Jabbour, Dr. Joyce Parker, Dr. Daisy Zhen, Dr. Dave Crowder, Dr. Tadashi

Takizawa, and Dr. Tobin Northfield for their help with research experiments, support and/or advice.

I am greatly appreciative to Dr. Mark Pavek and his work group composed of Zach

Holden, Rudy Garza Jr., and Josh Rodriquez for their time, equipment, and assistance planting potato fields at WSU Othello Irrigated Agriculture Research and Extension Center (IAREC) all

iii

three field seasons. I would also like to thank John Steinbock and Mark Weber at IAREC for preparing my fields for planting, watering, and controlling weeds.

I also greatly appreciate Terry Nash from Adjuvant Brands Helena Products Group and Brian

Corbin from FMC for providing me with Dyne-Amic Surfactant (Helena) and Beleaf Flonicamid

(FMC) for my two season field experiment.

For inspiring me to study entomology, I thank Dr. Roblin Giblin-Davis and Dr. Rudolph

Scheffrahn. I would like to Dr. Paris Lambdin, Dr. Jerome Grant, Dr. Kevin Moulton, and Dr.

Reid Gerhardt at the University of Tennessee for preparing me well for this degree and future

endeavors.

I am very grateful to my parents for their encouragement and sacrifices made in order to

provide me with an excellent education. Finally, I thank Justin Toombs for his advice, support,

and willingness to listen.

EXPLORING CONSUMPTION BY TWO GENERALIST PREDATORS IN POTATOES

USING MOLECULAR GUT CONTENT ANALYSIS AND BEHAVIORAL STUDIES

Abstract

By Christine Ann Lynch, Ph.D. Washington State University May 2013

Chair: William E. Snyder

Generalist insect predators may search out prey species that are nutritious even when they are uncommon in the environment, such that rates of predation will not necessarily track prey abundance. This study examined patterns of predation and behavior for two species of insect predators, Nabis alternatus Parshley and Geocoris bullatus Stål, in laboratory, greenhouse and field experiments. The laboratory experiment detected the presence of DNA from Leptinotarsa decemlineata (Say) (Colorado potato beetle) and Myzus persicae (Sulzer) (green peach aphid) in both predator species at 0, 1, 2, 4, 6, 8, 16, and 24 hours after feeding, and Nabis alternatus was also tested using the same method for consumption of G. bullatus nymphs. Prey DNA was present up to 24 hours after consumption, and the digestion rate differed by predator species and prey item. An open field experiment manipulated densities of green peach aphid and Colorado potato beetle using pest specific insecticides to determine if changes in prey community structure were reflecting by changing predator feeding patterns. Both predator species consumed Colorado potato beetles and green peach aphids in this study, and N. alternatus also consumed G. bullatus.

in the greenhouse manipulated green peach aphid densities (10, 25, or 50 aphids) and predator community structure (8 G. bullatus, 8 N. alternatus, or 4 of each species) to determine if aphid consumption changed with pest density or predator species composition. Consumption increased as a higher number of green peach aphids were available to of the predators. Foraging activity was highest when single predator species were present, and it was reduced when both predator species were present. Altogether, this series of studies suggests that these generalist predators have remarkably rigid feeding patterns, and that predation of one predator species by another can be reduced though predator-avoidance behavior.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iii-iv

ABSTRACT ...... v-vi

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x-xiv

DEDICATION ...... xv

INTRODUCTION: Spud Web: Species Interactions and Biodiversity in Potatoes ...... 1

1. Abstract ...... 1-2

2. Introduction ...... 2-4

3. Plant biodiversity ...... 4-8

4. Herbivore biodiversity ...... 8-11

5. Natural enemy biodiversity ...... 11-16

6. Getting even with pests: Natural balance and biocontrol ...... 16-18

7. Summary and future directions ...... 18-21

8. References cited ...... 22-34

CHAPTER 1: Detecting Leptinotarsa decemlineata (Colorado potato beetle) in two generalist

predators, Geocoris bullatus and Nabis alternatus, and intraguild predation of Geocoris bullatus by Nabis alternatus over time ...... 39

1. Abstract ...... 39-40

2. Introduction ...... 40-42

3. Materials and methods ...... 42-46

4. Results ...... 46-47

vii

5. Discussion ...... 47-50

6. References cited ...... 51-57

CHAPTER 2: Manipulation of Myzus persicae, green peach aphid, and Leptinotarsa decemlineata, Colorado potato beetle, to determine predator feeding patterns in potatoes ...... 63

1. Abstract ...... 63-64

2. Introduction ...... 64-67

3. Materials and methods ...... 67-71

4. Results ...... 71-79

5. Discussion ...... 79-84

6. References cited ...... 86-89

CHAPTER 3: Density of green peach aphid, Myzus persicae, alters behavior of the predators

Geocoris bullatus and Nabis alternatus ...... 106

1. Abstract ...... 106-107

2. Introduction ...... 107-109

3. Materials and methods ...... 110-113

4. Results ...... 113-117

5. Discussion ...... 118-121

6. References cited ...... 123-126

CONCLUSIONS...... 135-137

ATTRIBUTIONS ...... 138

viii

LIST OF TABLES

1. Table 1: Primers used to detect the two different prey species in predators guts for the

feeding trials...... 62

3. Table 1: Primers used to detect the three different prey species in predators guts for the

field experiment...... 102

2. Table 2: Total number of insect and spider species collected from insect community

sampling for all twenty plots for 2010 and 2011 ...... 103-105

LIST OF FIGURES

INTRODUCTION: Figure legend ...... 35 Figure 1; Lucas et al. (1998) found that lacewing larvae fed not only on potato aphids, but also

on larvae of predatory Aphidoletes midges. “Intraguild” predation of this type has the

potential to disrupt aphid control, as lacewing larvae consume midge larvae that

otherwise might have eaten many aphids. Arrows denote energy flow and so point from

prey to predator; black arrows indicate predation of the herbivore by predators, while the

red arrow indicates predation of one predator by another ...... 36

Figure 2; In potato foliage, Nabis alternatus bugs and Hippodamia convergens beetles exert

complementary impacts on Myzus persicae aphids (Straub and Snyder 2008). The lady

beetles forage primarily at leaf edges, whereas the predatory bugs also forage at leaf

centers. Because of these differences in space use, aphids face heavy predation pressure

everywhere on the plant only when both predator species occur together ...... 37

Figure 3; In Washington potato crops, predators and pathogens exert complementary impacts on

Colorado potato beetles (Ramirez and Snyder 2009). Potato beetle eggs and larvae in the

foliage are eaten by a diverse group of predatory Hippodamia convergens (Hc) and

Pterostichus melanarius (Pmel) beetles, and Nabis alternatus (Nabis) bugs. Once the

beetles enter the soil to pupate they are infected by entomopathogenic Steinernema

carpocapsae (Scarp) and Heterorhabditis marelatus (Hmar) nematodes, and Beauveria

bassiana (Bbass) fungi. Because of the spatiotemporal separation of predators and

pathogens, potato beetles face attack throughout their life cycle only when both classes of

natural enemy are present ...... 38

CHAPTER 1: Figure legend ...... 58

Figure 1; Detection of Leptinotarsa decemlineata larvae DNA in Geocoris bullatus from time 0

to 24 hours later (r2 = 0.8512) ...... 59

Figure 2; Detection of Leptinotarsa decemlineata larvae DNA in Nabis alternatus from time 0 to

24 hours later (r2 = 0.9632) ...... 60

Figure 3; Detection of Geocoris bullatus nymph DNA in Nabis alternatus from time 0 to 24

hours later (r2 = 0.9897) ...... 61

CHAPTER 2: Figure legend ...... 90-91

Figure 1; Maps of field plot experimental design arrangement for 4 treatments (A = aphids

present, ACPB = aphids and Colorado potato beetle, CPB = CPB present, and O = neither

species) in (a) 2010 and (b) 2011. Plots were randomized each year using a random

number generator ...... 92

Figure 2; Average number of three different types of natural enemies. Predatory insects (a, b),

arachnids (c, d), and parasitoids (e, f) found during Dvac suction sampling in the four

treatments (A = aphids present, ACPB = aphids and Colorado potato beetle, CPB = CPB

present, and O = neither species) in 2010 (a, c, e) and 2011 (b, d, f) ...... 93

Figure 3; Average number of herbivores other than aphids and Colorado potato beetles (a, b),

aphids (c, d), and Colorado potato beetles (e, f) found during Dvac suction sampling in

the four treatments (A = aphids present, ACPB = aphids and Colorado potato beetle, CPB

= CPB present, and O = neither species) in 2010 (a, c, e) and 2011 (b, d, f)...... 94

Figure 4; Average number of the predators: Geocoris bullatus (a, b) and Nabis alternatus (c, d)

found during Dvac suction sampling in the four treatments (A = aphids present, ACPB =

aphids and Colorado potato beetle, CPB = CPB present, and O = neither species) in 2010

(a, c) and 2011 (b, d) ...... 95

Figure 5; Average number of detritivores (a, b), pollinators (c, d), and miscellaneous insects (e, f)

found during Dvac suction sampling in the four treatments (A = aphids present, ACPB =

aphids and Colorado potato beetle, CPB = CPB present, and O = neither species) in 2010

(a, c, e) and 2011 (b, d, f) ...... 96

Figure 6; Average number of aphids found during visual counts in the four treatments (A =

aphids present, ACPB = aphids and Colorado potato beetle, CPB = CPB present, and O =

neither species) for (a) 2010 and (b) 2011 ...... 97

Figure 7; Average number of Leptinotarsa decemlineata (Colorado potato beetle or CPB) found

during visual counts in the four treatments (A = aphids present, ACPB = aphids and

Colorado potato beetle, CPB = CPB present, and O = neither species) for (a) 2010 and (b)

2011...... 98

Figure 8; Proportion of Geocoris bullatus positive for consumption of (a, c) aphids and (b, d)

Colorado potato beetles in the four treatments (A = aphids present, ACPB = aphids and

Colorado potato beetle, CPB = CPB present, and O = neither species) in (a, b) 2010 and

(c, d) 2011 ...... 99

Figure 9; Proportion of Nabis alternatus positive for consumption of (a, c) aphids and (b, d)

Colorado potato beetles in the four treatments (A = aphids present, ACPB = aphids and

Colorado potato beetle, CPB = CPB present, and O = neither species) in (a, b) 2010 and

(c, d) 2011 ...... 100

Figure 10; Proportion of Nabis alternatus positive for consumption of Geocoris bullatus in the

four treatments (A = aphids present, ACPB = aphids and Colorado potato beetle, CPB =

CPB present, and O = neither species) in (a) 2010 and (b) 2011 ...... 101

CHAPTER 3: Figure legend ...... 127-128

xii

Figure 1; Number of aphids eaten by monocultures of Geocoris bullatus (G) and Nabis

alternatus (N), or a mix of G. bullatus and N. alternatus (GN), when foraging among 10,

25, or 50 green peach aphids per potato plant. The experiment was conducted within four

blocks: (a) block 1, (b) block 2, (c) block 3, and (d) block 4 ...... 129

Figure 2; Per capita number of times that predators were observed over 8 hours when Geocoris

bullatus foraged among only conspecifics (G) or when this species foraged alongside

Nabis alternatus (G with N) (panel a,), or when N. alternatus foraged among only

conspecifics (N) or alongside G. bullatus (N with G) (panels b). Predators were presented

with potato plants initially housing either 10, 25 or 50 green peach aphids ...... 130

Figure 3; Per capita number of times that predators were observed on top of the potato plant

when Geocoris bullatus foraged among only conspecifics (G) or when this species

foraged alongside Nabis alternatus (G with N) (panels a, c, e and g), or when N.

alternatus foraged among only conspecifics (N) or alongside G. bullatus (N with G)

(panels b, d, f and h). Predators were presented with potato plants initially housing either

10, 25 or 50 green peach aphids, and the experiment was conducted within four blocks:

block 1 (a, b), block 2 (c, d), block 3 (e, f) and block 4 (g, h) ...... 131

Figure 4; Per capita number of times that predators were observed on the middle of the potato

plant when Geocoris bullatus foraged among only conspecifics (G) or when this species

foraged alongside Nabis alternatus (G with N) (panels a, c, e and g), or when N.

alternatus foraged among only conspecifics (N) or alongside G. bullatus (N with G)

(panels b, d, f and h). Predators were presented with potato plants initially housing either

10, 25 or 50 green peach aphids, and the experiment was conducted within four blocks:

block 1 (a, b), block 2 (c, d), block 3 (e, f) and block 4 (g, h) ...... 132

xiii

Figure 5; Per capita number of times that predators were observed mating when Geocoris

bullatus foraged among only conspecifics (G) or when this species foraged alongside

Nabis alternatus (G with N) (panels a, c, e and g), or when N. alternatus foraged among

only conspecifics (N) or alongside G. bullatus (N with G) (panels b, d, f and h). Predators

were presented with potato plants initially housing either 10, 25 or 50 green peach aphids,

and the experiment was conducted within four blocks: block 1 (a, b), block 2 (c, d), block

3 (e, f) and block 4 (g, h)...... 133

Figure 6; Per capita number of times that Nabis alternatus were observed consuming Geocoris

bullatus when G. bullatus was present with N. alternatus (G with N) (panels a and b), or

when N. alternatus consumed other N. alternatus among only conspecifics (N) (panels b

and c). Predators were presented with potato plants initially housing either 10, 25 or 50

green peach aphids, and the experiment was conducted within four blocks: block 2 (a),

block 3 (b), and block 4 (c). There was no intraguild predation for block 1, so it is not

shown in this figure ...... 134

xiv

Dedication

This dissertation is dedicated to my parents and husband for all of their support and

encouragement throughout my two graduate degrees in entomology.

INTRODUCTION: Spud Web: Species Interactions and Biodiversity in Potatoes

Christine A. Lynch, David W. Crowder, Randa Jabbour and William E. Snyder

Department of Entomology, Washington State University, Pullman, WA 99164

ABSTRACT

Agroecologists often see greater biodiversity as the key to reducing pest problems on farms.

Others have suggested, however, that increasing species number only increases the risk of negative interactions among species, such as predation of one predator by another, which could disrupt biological control. Such multi-species interactions have long been a topic of interest among entomologists working in potato crops, and here we review the basic ecological knowledge that has come from work in this important model cropping system. We examine the effects of increasing biodiversity on potato-insect species interactions at multiple trophic levels: among plants, herbivores, and natural enemies. Increasing plant diversity at both local and landscape scales can help build predator populations and increase suppression of potato pests.

However, planting flowering plants near potato crops sometimes provides supplemental food for pests, making pest problems worse. Effects of herbivore diversity are equally complex. Feeding by early-season herbivores sometimes makes plants resistant to late-arriving herbivores, harming those pests most active later in the growing season. On the other hand, multiple herbivore species

can “distract” predators from feeding on particular target pests, providing less-preferred

herbivore species with protection from predation. Predator diversity likewise exerts varying

effects. In most cases predator species appear to complement one another by foraging in different

locations in the crop or by attacking different pest stages, thus improving biological control. In

some cases, however, predator species feed on one another, disrupting biological control. In

summary, although increasing biodiversity within potato crops sometimes worsens pest

problems, it most often makes pest outbreaks less likely. Thus, work in potatoes largely supports

the view of early agroecologists that increasing biodiversity restores natural balance among

plants, herbivores, and natural enemies.

Key words: predation; competition; intraguild predation; facilitation; complementarity

Introduction

Much early work in biological control focused on interactions between particular natural enemy species and their pestiferous prey (DeBach and Rosen 1991). This approach likely reflected the many successes of classical biological control, where natural enemies of an introduced pest were released into the invasive range and dramatically reduced pest densities. Classical biological control agents were sought that had a high degree of prey specificity and a reproductive rate as high as the target pest (DeBach and Rosen 1991). Such highly-specialized natural enemies are tightly linked to particular herbivores, and have the clear ability to exert density-dependent regulation of pests (Hawkins et al. 1997, 1999). However, recent years have seen growing interest in the community ecology of biological control where interactions among more than two

species are considered (e.g., Vandermeer 1995, Matson et al. 1997). First, classical biological

introductions have become more difficult and expensive because of growing societal concerns

about potential unintended, negative impacts of biocontrol agents on native species (Howarth

1991). Furthermore, this approach is only possible when pests are non-native exotics. Instead,

there is increasing interest in the conservation of native natural enemies, including generalist

predators, which often interact with many other species in addition to any single target pest

(Landis et al. 2000, Symondson et al. 2002). Second, consumer concerns about possible negative

human- and environmental-health effects of pesticides have increased adoption of organic

agriculture and other pesticide-reduction schemes (Crowder et al. 2010). With a reduction in

broad-spectrum pesticide applications by growers, the abundance and diversity of natural

enemies generally increases (Bengtsson et al. 2005, Hole et al. 2005, Straub et al. 2008). This

inevitably increases the complexity of agricultural food webs (e.g., Tylianakis et al. 2007).

The relationship between biodiversity and biocontrol has long been a topic of interest to basic

and applied ecologists. Early agroecologists thought that the relative ecological simplicity of

agricultural monocultures rendered them more prone to herbivore outbreaks than natural systems

(Pimentel 1961, Root 1973, van Emden & Williams 1974). If so, then the restoration of more

natural levels of biodiversity would improve natural pest control (Straub et al. 2008). This

viewpoint is consistent with the emerging sub-discipline of “biodiversity-ecosystem function” research, which suggests that ecosystem health is maximized only within species-rich communities (Ives et al. 2005, Hooper et al. 2006). Other viewpoints also exist. Simplest is the

“green-world hypothesis” of Hairston et al. (1960), who proposed that the world’s ecosystems break down into three simple trophic levels: plants, herbivores, and predators. Predators generally regulate herbivore densities, so that plants grow and proliferate largely unscathed.

Biodiversity is never explicitly discussed in the 1960 paper, but it can be inferred that predators

(and other natural enemies) form a cohesive third trophic level that acts consistently regardless of

the number of species present (Hairston and Hairston 1993, 1997). In contrast, the “trophic-level

omnivory hypothesis” suggests that complex feeding relationships among species blur the

formation of distinct trophic levels; without trophic levels, cascading predator effects on

herbivores and then plants cannot occur (Strong 1992, Polis and Strong 1996). For example,

ecological communities typically include many species of generalist predators, which often feed

on plants, detritivores and other predators in addition to herbivores (Polis 1991). Data from

particular studies in agricultural crops variously supports each of these hypotheses (Straub et al.

2008).

Potato crops often contain diverse communities of herbivores and their natural enemies

(Walsh and Riley 1868, Hough-Goldstein et al. 1993, Hilbeck and Kennedy 1996, Hilbeck et al.

1997, Koss et al. 2005), providing an ideal model system to study the ecological issues discussed

above. Traditionally, potatoes have been heavily treated with insecticides, due to the high value

of the crop and the severe damage caused by some insect and other pests (Hare 1990). However,

recent years have seen growing pressure to reduce insecticide use on this food crop, leading to a

growing organic sector and the adoption of pesticide-reduction schemes in conventionally- managed fields (Koss et al. 2005, Werling and Gratton 2010). Here, we explore species interactions among herbivores and natural enemies occurring in potato crops, and how these interactions are influenced by increasing biodiversity. We separately consider how biodiversity effects might operate at the plant, herbivore and predator trophic levels.

Plant Biodiversity

Root (1973) presented two hypotheses for how increasing plant diversity in agricultural fields

might decrease pest problems. First, specialist herbivores might have a more difficult time

finding their host plant against a background of other plant species. Second, diverse plantings

might support a more diverse community of non-pest prey and other foods, increasing the

abundance and diversity of natural enemies. Indeed, diversified plantings often house fewer pests

than do agricultural monocultures (Russell 1989, Andow 1991). However, differentiating

between the two above-described hypotheses as the root cause of this diversity effect has been

problematic (Bommarco and Banks 2003). Increasingly, agroecologists have examined crop

diversification plans as a means to conserve natural enemies (Landis et al. 2000). Plant

diversification can be accomplished either at local scales, with resource-providing plants

installed at field edges or within the crop (Landis et al. 2000), or at the scale of landscapes,

where regions including more crop species and more non-crop habitats might provide diverse

resources for highly-mobile natural enemies (Tscharntke et al. 2005).

In-Field Plant Diversity. Perhaps the most common approach to diversifying the in-field

habitat of potato crops has been the application of straw mulch. Straw mulch can benefit

predators by establishing more-benign environmental conditions relative to bare ground fields,

by providing protection from the predators’ own natural enemies (including intraguild predators),

and/or by providing food for detritivores that act as supplemental, non-pest prey (Settle et al.

1996, Langellotto and Denno 2004, Halaj and Wise 2002). Brust (1994) reported that potato plantings receiving straw mulch housed larger populations of generalist predators, and also lower densities of Colorado potato beetles. With fewer potato beetles, potato plants suffered less feeding damage. He also found no difference in potato beetle movement between mulched and

un-mulched plants, suggesting that the benefits of mulch were not due to potato beetles avoiding the colonization of mulched potatoes (Brust 1994). Several other studies confirmed that potato beetle densities decrease in straw-mulched plots (Zehnder and Hough-Goldstein 1990, Stoner

1993, Johnson et al. 2004). Often, the application of straw mulch has been associated with higher potato yields (Zehnder and Hough-Goldstein 1990, Stoner 1993), although it is not always clear whether this effect is due to reduced potato beetle densities, better moisture retention in the soil, or both (Stoner et al. 1996). Nonetheless, increased structural complexity is not universally beneficial. Szendrei and Weber (2009) found that potato crops planted into rye stubble attracted higher densities of predatory C. maculata lady beetles but lower densities of the predatory carabid beetle Lebia grandis. Mulching decreased overall beetle suppression by predators because the carabid was the most voracious predator of potato beetles (Szendrei and Weber

2009). Indeed, molecular gut-content analysis revealed that predators were more likely to have fed on potato beetles in un-mulched than in mulched plots (Szendrei et al. 2010).

Predators of aphid and potato beetle in potatoes, such as the lady beetle Coleomegilla maculata, are often highly mobile, suggesting they may be affected by the mixture of potato and other plant habitats within and around farms. For example, corn crops seem to be most attractive to predatory lady beetles, such that lady beetle densities in potato fields planted near corn can be too low to exert significant impacts on potato pests (Groden et al. 1990, Nault and Kennedy

2000). However, if the attractiveness of potato crops could be increased by, for example, planting flowering plants at field edges or between crop rows, potatoes might be more attractive to lady beetles and other natural enemies (e.g., Patt et al. 1997). This could increase biological control impacts in potatoes. For example, Groden et al. (1990) suggested timed cutting of adjacent alfalfa crops as a strategy to encourage C. maculata to move from alfalfa to potatoes as

potato-pest densities grow. Idoine and Ferro (1990) indicated that adult females of the Colorado

potato beetle egg parasitoid Edovum puttleri benefited from feeding on aphid honeydew,

suggesting that providing a sugar source for the wasps could benefit the control of potato beetles.

Such approaches to predator conservation are fraught with risk, however. For example, Baggen

and Gurr (1998) found that planting flowering plants near potatoes provided food for adults of

the parasitoid Copidosoma koehleri, an egg parasitoid of the pestiferous potato moth

Phthorimaea operculella. These “floral resources” extended parasitoid longevity and increased

fecundity, both of which would benefit potato moth biocontrol. However, in field trials,

providing flowers actually increased pest densities in potatoes and resulting crop damage; this

occurred because potato moths also benefitted from the use of floral resources (Baggen and Gurr

1998). Subsequent work showed that flower species could be selected which benefitted only the

parasitoid and not the moth (Baggen et al. 1999), such that the unintended benefits to the pest

could be eliminated and the parasitoid selectively promoted (Baggen et al. 1999).

Landscape-Scale Plant Diversity. Plant diversity can also be beneficial at scales larger than

single potato fields. Werling and Gratton (2010) examined how landscape structure and diversity impacted predation of green peach aphid (Myzus persicae) and Colorado potato beetle

(Leptinotarsa decimlineata) pests, and found that control of the two pests was impacted at different spatial scales. Green peach aphid predation was highest in field margins adjacent to potato fields that were imbedded within landscapes containing diverse non-crop habitats within

1.5 km; however, this effect of landscape diversity did not alter predation of green peach aphids within the potato fields themselves (Werling and Gratton 2010). In contrast, predation of

Colorado potato beetle eggs was unaffected by landscape diversity at larger scales, but instead increased as the ratio of field margin to crop increased. Thus, potato beetle predation was

greatest in smaller potato fields where relatively diverse edge habitats were always close by

(Werling and Gratton 2010). Ground beetle predation of weed seeds was greater in field margins compared to adjacent potato crops (Gaines and Gratton 2010), again suggesting that greater plant diversity in field margins increased predation relative to what was seen in the plant-species-poor potato crop. In conclusion, the presence and size of adjacent non-crop habitat can have important implications for predation of herbivores in potato fields, but there is limited evidence for the role of landscape complexity at larger scales.

Herbivore Biodiversity

While pest management decisions in potatoes may often focus on just one or two pests perceived to be of the greatest economic importance, these crops typically house a diverse community of many herbivore species (Lynch et al. 2006). Multiple species of herbivores have the potential to indirectly impact one another’s densities, with these interactions generally transmitted indirectly by the host plant (Karban and Baldwin 1997, Lill and Marquis 2001,

Ohgushi 2005, Rodriguez-Saona and Thaler 2005). These multi-herbivore effects can be either harmful or beneficial to particular key potato pests. Likewise, the presence of multiple herbivore species can either heighten or weaken the impact of natural enemies on a particular species of pest (e.g., Eubanks and Denno 2000). Below, we review research literature examining how herbivore diversity impacts interactions among herbivore species, and among predators and their herbivore prey, on potatoes.

Herbivore-Herbivore Interactions Mediated by Plants. Plant-mediated indirect interactions among herbivores generally fall into three categories: (1) those due to resource

depletion, where one herbivore species consumes plant resources that are then unavailable to a

second herbivore species, (2) those due to induced plant defenses, where feeding by one

herbivore species triggers the plant to deploy defensive strategies that harm a second, often later-

arriving, herbivore species, and (3) those due to alterations in plant chemistry following feeding

by one herbivore species that render a plant more susceptible and/or attractive to a second

herbivore species. Interactions of all types have been described in potato crops, or are likely to

occur.

The Colorado potato beetle is often on the losing end of plant-mediated competition with other herbivore species (e.g., Tomlin and Sears 1992a,b; Wise 2002, Lynch et al. 2006, Kaplan et al. 2007). This might be because the Colorado potato beetle generally does its greatest damage relatively late in the growing season, such that their feeding is influenced by changes in plant chemistry/quality that have been induced by earlier-arriving herbivores. For example, early- season feeding by both flea beetles (Wise and Weinberg 2002) and potato leafhoppers (Lynch et al. 2007) causes female potato beetles to avoid ovipositing on affected potato plants, and slows development of any larvae that do hatch. Slower larval development not only makes it more difficult for Colorado potato beetles to complete multiple generations each year, but also heightens the risk of beetle larvae falling victim to predators; predation risk is heightened because the beetles are forced to spend more time in vulnerable smaller stages (Kaplan et al.

2007).

Although the precise mechanism through which earlier feeding by flea beetles or leafhoppers harms potato beetles has not been demonstrated, potato plants defend themselves through some combination of altered nutritional quality, allelochemical defenses, and morphological alterations

(Tomlin and Sears 1992 a,b; Hlywka et al. 1994, Bolter and Jongsma 1995, Pelletier et al. 1999).

It is likely that one or more of these defenses are involved in induced resistance to Colorado

potato beetle. The indirect effect of leafhoppers on potato beetles depends on the density of the

leafhoppers, unlike chemical defenses that can be triggered to high levels by relatively little

herbivore feeding (Heil and Kost 2006), and leafhoppers are known to alter the amino acid

profile of potato foliage (Tomlin and Sears 1992a). This implies that leafhoppers harm potato

beetles, at least in part, by reducing the nutritional value of potato plants (Lynch et al. 2006).

It is worth noting that previous feeding by other herbivore species does not invariably deter

subsequent potato beetle attack. For example, in one study potato plants that had cabbage looper

regurgitant applied to wounds in the foliage, simulating caterpillar attack, attracted more potato

beetle adults than did un-damaged plants (Landolt et al. 1999). Similarly, potato plants attacked

by beet armyworm larvae were more attractive to colonizing potato beetles (Bolter et al. 1997).

In the Bolter et al. (1997) study, previous feeding by Colorado potato beetles also heightened

attractiveness of damaged plants to later-arriving potato beetles, suggesting that plant damage by any chewing herbivore rendered potato plants more attractive.

Herbivore-Herbivore Interactions Mediated by Shared Predators. Herbivore species can also indirectly impact one another by changing the behavior of shared predators. For example,

“apparent competition” occurs when the presence of one herbivore species draws in larger numbers of natural enemies than might otherwise be found. When these enemies switch to feeding also on a second herbivore species, the second herbivore species is harmed (Holt 1977,

Harmon and Andow 2004). Thus, one herbivore species harms another by increasing the second species’ risk of predation. Apparent competition among potato herbivores has not been directly demonstrated, but there is good circumstantial evidence to suggest it might occur. For example, green peach aphid and other aphid pests of potato can attract large numbers of aphid-associated

lady beetles and other generalist predators (Koss et al. 2005), which likely opportunistically feed on potato beetle eggs and other vulnerable herbivore species (e.g., Chang and Snyder 2004).

Similarly, potato plants damaged by Colorado potato beetles are more attractive to the predatory pentatomid Perillus bioculatus than are un-damaged plants (Weissbecker et al. 1999, 2000); presumably, once these generalist predators are drawn to a crop they would feed also on prey other than potato beetles.

However, the presence of one herbivore species will not necessarily lead to higher predator attack rates on a second, co-occurring herbivore species. In fact, the presence of a preferred prey might draw predator attacks away from a second, unpalatable or less-preferred herbivore species

(Harmon and Andow 2004). For example, in laboratory arenas Coleomegilla maculata lady beetles eat fewer Colorado potato beetle eggs when aphids are available as alternative prey

(Groden et al. 1990, Hazzard and Ferro 1991, Mallampalli et al. 2005). Similarly, in field cages where predators cannot aggregate at aphid infestations, the presence of green peach aphids protects Colorado potato beetles from attack by a diverse guild of spider and predatory bug generalist predators (Koss and Snyder 2005). Disruption of potato beetle predation in the presence of aphids apparently occurs because aphids are moreattractive prey for most predator species. Consistent with this interpretation, the presence of Colorado potato beetles as prey has no impact on these same predators’ likelihood of attacking aphids (Koss et al. 2004).

Natural Enemy Biodiversity

Potato crops often house a remarkably high diversity of predator, pathogen and parasitoid natural enemies (Walsh and Riley 1868, Hough-Goldstein et al. 1993, Alyokhin and Sewell

2004, Straub and Snyder 2006, Ramirez and Snyder 2009). For example, in North America,

Colorado potato beetles are attacked by a diverse guild of generalist egg and larval predators, egg and larval parasitoids, and entompathogenic nematodes and fungi (Lopez et al. 1993, Hilbeck and Kennedy 1996, Berry et al. 1997, Koss et al. 2005, Crowder et al. 2010). Indeed, both observational studies and predator surveys using molecular gut content analysis have found a vast array of predators feeding on Colorado potato beetles under entirely natural, open-field conditions (Chang and Snyder 2004, Greenstone et al. 2010, Szendrei et al. 2010).

As we have seen for plants and herbivores, increasing biodiversity among natural enemies can have either positive or negative consequences for particular potato pests. On the one hand, as more enemy species are added to a community this increases the risk that one enemy will feed on another. Such “intraguild” predation has the potential to greatly disrupt biological control (Polis et al. 1989, Rosenheim et al. 1995, Ives et al. 2005; Fig. 1). On the other hand, combining natural enemies that fill different ecological niches can lead to complementary impacts on a pest species, where different enemy species eliminate spatial or temporal refuges from predation that the pest might otherwise enjoy (Wilby and Thomas 2002, Casula et al. 2006, Straub et al. 2008; Fig. 2-3).

Furthermore, natural enemies sometimes facilitate one another’s prey capture. For example, a predator may chase a prey species from one habitat to another second predator species located somewhere else in the environment (e.g., Losey and Denno 1998). In these cases, pest control is strongest where several natural enemy species co-occur. We next discuss examples of negative, and then positive, predator-predator interactions that have been found to impact biological control in potatoes.

Negative Predator-Predator Interactions. Natural enemies often feed upon one another, and this has the potential to greatly limit biological control (Rosenheim et al. 1995). Some

evidence for this comes from potato crops. For example, Mallampali et al. (2002) showed that

the spined soldier bug (Podisus maculiventris) fed heavily on larvae of the twelve-spotted lady

beetle (Coleomegilla maculata), which disrupted predation of Colorado potato beetle eggs by

that lady beetle. Similarly, in laboratory feeding trials the predatory bug Anthocoris nemorum

was as likely to feed on green peach aphids parasitized by the wasp Aphidius colemani as on

unparasitized aphids (Meyling et al. 2004), and Beauveria bassiana fungi pathogenic to green

peach aphids and Colorado potato beetles also attacked C. maculata lady beetles (Todorova et al.

2000). If these interactions also occur in the field, they could disrupt biological control.

One of the more detailed series of studies on intraguild predation among aphid predators

examined interactions among predatory midges (Aphidoletes aphidomyza), lacewings

(Chrysoperla rufilabris), and lady beetles (C. maculate) attacking potato aphids (Macrosiphum

euphorbiae) (Fig. 1). The midges are highly susceptible to intraguild predation by the lacewing

and lady beetle; lacewing and lady beetle larvae also attack one another (Lucas et al. 1998).

Apparently in response to its high risk of falling victim to intraguild predation, the midge has

developed several strategies to reduce this danger. First, midge larvae will “hide” at the center of

aphid aggregations, so that aphids at the colony periphery will absorb most predator attacks

(Lucas and Brodeur 2001). Second, midge eggs are deposited on plants with high trichome

density, which makes them relatively immune from predation by C. maculata predators (Lucas

and Brodeur 1999).

The parasitoid wasp Aphidius nigripes attacks green peach aphids (Myzus persicae) in potato

crops in North America, where it faces attack by hyperparasitoids (Brodeur and McNeil 1992).

As the parasitoid larva nears the end of its development, it somehow alters the host aphid’s

behavior, causing the doomed aphid to walk to the top of the potato plant before being killed by

the parasitoid. The wasp then pupates beneath its former host’s exoskeleton. Brodeur and

McNeil (1992) found that parasitoid pupae located at the tips of plants were unlikely to fall victim to hyperparasitoids. In alfalfa crops, this placement of parasitoid pupae was found to make the wasps less accessible for ground-foraging, predatory carabid beetles that occasionally climb onto plants to forage (Snyder and Ives 2001). Altogether, this suggests that the selective pressures exerted by intraguild predators have led the parasitoid to develop its ability to change host behavior, in order to be delivered to a pupation site largely out of reach of the parasitoid’s own natural enemies (Brodeur and McNeil 1992). Similarly, the lady beetle C. maculata leaves aphid-infested potato plants to pupate elsewhere, apparently in order to avoid intraguild predation by the many predators that aphids attract (Lucas et al. 2000).

Positive Predator-Predator Interactions. There is growing evidence that species that occupy different niches consume more resources than any single species can consume (Hooper et al. 2006, Cardinale et al. 2006). The same may hold true when herbivorous agricultural pests are the “resource”, and predator, parasitoid and pathogen natural enemies are the “consumers”

(Straub et al. 2008). After all, natural enemy species differ from one another in where they hunt in the environment, the time of day or year that they are active, and in the particular hunting style they use, such that pests are likely to face a broad-based attack only when several natural enemy species co-occur (Wilby and Thomas 2002, Snyder 2009). In this sense, natural enemy species are likely to complement one another, and some of the best evidence of this comes from work in potato crops.

Working with potato plants enclosed in large field cages, Straub and Snyder (2006, 2008) compared the effects of single natural enemy species on green peach aphid prey to those of diverse mixes of 3 or 4 enemy species (Fig. 2). The biocontrol agents considered in this work

were a diverse group composed of Nabis and Geocoris true bugs and parasitoid wasps. All

experiments manipulated predators following a substitutive design, in which total predator

density (or, in some experiments, predator biomass) was held constant across species richness

levels. Such designs isolate the impacts of changes in species number by eliminating differences

among treatments in species abundance (Loreau and Hector 2001). Initial experiments showed

that the different predator species strongly differed in how effective they were at killing aphids,

but that diverse mixes of predator species killed only slightly more aphids than did single enemy

species (Straub and Snyder 2006). This suggested that predator species did not strongly

complement one another. However, subsequent work painted a more complex picture. When

predator diversity was manipulated simultaneously on both collard (Brassica oleracea) and potato plants colonized by the green peach aphids – in that experiment, plants of the two species were present in different cages – diverse predator communities killed more aphids than any single enemy species on plants of both species (Straub and Snyder 2008). Predators complemented one another quite strongly on collard plants, where diverse predator communities killed 200 more aphids than single species, but quite weakly on potatoes, where diverse predator communities killed just 6 more aphids than single predator species (Straub and Snyder 2008).

Behavioral observations indicated that lady beetles foraged largely at the edges of leaves, while predatory bugs and parasitoids foraged at leaf centers (Fig. 2). Because collard leaves are larger, these space-use niche differences, and thus predator-predator complementarity, were greater on collards than potatoes (Straub and Snyder 2008).

Natural enemy facilitation occurs when the presence of one natural enemy species indirectly heightens prey capture by a second predator species. In one well-known example of this phenomenon, lady beetles foraging in alfalfa foliage cause aphids to drop to the ground; once on

the ground, aphids are readily eaten by ground beetles on the soil surface (Losey and Denno

1998). Something analogous has been reported for Colorado potato beetle prey, where predators facilitate prey infection by pathogens (Ramirez and Snyder 2009; Fig. 3). During their development, the beetles move from the foliage where they feed to the soil where they pupate, and as they do so they transition between two quite distinct communities of natural enemies (Fig.

3). Aboveground, the beetles are attacked by lady beetles, ground beetles, and Nabis true bugs.

Belowground, they face infection by entomopathogenic nematodes (Heterorhabditis spp. and

Steinernema spp.), and Beauveria bassiana fungi. Ramirez and Snyder (2009) manipulated species number among these predator and pathogen natural enemies, and then measured potato beetle survival from egg to adult in large field cages. Enemy species number was again manipulated following a substitutive design, so that natural enemy densities did not differ as species richness was changed. They found that potato beetle mortality increased as predator/pathogen biodiversity increased. This happened because predators and pathogens were increasingly likely to co-occur at higher species-richness levels, and predator-pathogen pairings were particularly lethal to potato beetles. The authors suggest that energetically costly antipredator defenses of the potato beetle larvae, deployed to escape from predators early in potato beetle development, weakened the beetles’ later ability to fight off pathogen infection (Ramirez and Snyder 2009, Ramirez et al. 2010). Thus, exposure to predators indirectly weakened beetle immune function. A similar type of facilitation appears to occur even within entomopathogen communities, as exposure to entomopathogenic fungi increases potato beetle susceptibility to entomopathogenic nematodes (Jabbour et al. 2011).

Getting Even With Pests: Natural Balance and Biocontrol

The term “biodiversity” is often used as if it were synonymous with the number of

species present, known as “species richness”. However, ecologists have long suspected that

ecosystem function might also improve when species’ relative abundances are evenly matched,

known as greater “evenness” (Hillebrand et al. 2008). Indeed, most common biodiversity indices

include some measure of evenness, in addition to richness, in their calculation. It is thought that

communities with just a few very common species, typical for example of highly-disturbed areas

dominated by weedy and/or invasive species, are less healthy than communities where many

species are similarly-common. Unfortunately, ecologists’ intuition has, until recently, been backed by relatively little empirical evidence that more-even communities are in fact more stable and productive than their uneven counterparts (Hillebrand et al. 2008).

Interestingly, some of the best evidence for the importance of greater evenness comes from potato crops. Crowder et al. (2010) examined how the transition from conventional farming practices, dominated by intense insecticide use, to organic farming with its greater reliance on natural processes, influenced richness and evenness among natural enemies of insects in

Washington potato fields. As in the Ramirez and Snyder (2009) study discussed previously, natural enemies of insects in Washington potatoes are dominated by insect generalist predators and nematode and fungus pathogens. In a regional survey of predators and pathogens spanning a broad geographic area and several years, Crowder et al. (2010) found, surprisingly, no increase in predator/pathogen species richness in fields using organic practices. However, natural enemy evenness was significantly higher in organic than conventional potato fields. This meant that just one or two natural enemies were abundant in conventional fields, but many enemies were similarly common in organic fields. A meta-analysis of predator surveys for crops worldwide,

not just potatoes, showed that greater evenness among natural enemy communities was generally

greater in organic than conventional fields across these many crops and world regions. When the

authors constructed natural enemy communities that ranged from very even to very uneven, they

found that control of potato beetle pests was significantly stronger when predators and/or

pathogens were evenly abundant. Indeed, greater enemy evenness translated into significantly

larger potato plants, and thus presumably higher potato yields (although yields were not

measured). Thus, balance among natural enemies may be as important for strong pest

suppression as having a large number of natural enemy species (Crowder et al. 2010).

Summary & Future Directions

Agroecologists often suggest that greater biodiversity is the key to reducing pest problems.

Work in potato crops, however, paints a more nuanced picture. Increasing plant structural

diversity in the crop by mulching often appears to augment natural enemy populations and

increase enemy impacts on pests (Brust 1994). Likewise, more diverse landscapes can foster

greater impacts of natural enemies (Werling and Gratton 2010), as can the addition of flowering

plants that provide resources to natural enemies (Baggen et al. 1999). However, poorly-chosen flowering plants can feed potato pests in addition to their predators and parasitoids, worsening pest problems (Baggen and Gurr 1998).

Increasing herbivore diversity has similarly complex effects. Herbivores such as flea beetles and leafhoppers that attack potato plants early in the growing season trigger induced resistance in potato plants that renders plants less attractive and/or nutritious to later-occurring herbivores like

Colorado potato beetles (e.g., Lynch et al. 2007). As a result, early-season herbivory can dampen

later herbivory by other species. However, the presence of highly-attractive prey like aphids

might draw predator attacks away from less-desirable prey like potato beetles, such that aphids

indirectly protect potato beetles (e.g., Koss and Snyder 2005). Thus, increasing herbivore

diversity can either harm or benefit particular pest species.

The impacts of predator diversity are equally multifaceted. In a few cases, adding predators

that mostly eat other predators disrupts overall biological control (e.g., Mallampalli et al. 2002;

see also Fig. 1). More often, however, predators, parasitoids and pathogens complement one another, attacking pests in different habitats and/or during different life stages. As a result,

biological control is most effective when several enemy species are present (Straub and Snyder

2008, Ramirez and Snyder 2009; Fig. 2-3). In summary, while greater diversity sometimes

makes pest problems worse, it appears that increasing biodiversity and species evenness within

potato crops is more likely to make pest problems less frequent.

While entomologists have made great progress in delineating interactions among potato

arthropods, several topics, in our opinion, are still worthy of further exploration:

1. The Role of Behavioral Interactions among Species. Species interact not only by killing one another (or by outcompeting one another for food), but also by changing one another’s behavior

(Lima and Dill 1990). For example, predators that chase herbivores from preferred feeding sites can protect plants from damage even when the pest is not killed (Schmitz et al. 1997, 2004,

Werner and Peacor 2003, Preisser et al. 2005). Such behavior-mediated or “nontrophic” interactions have been shown to be important in other cropping systems, but relatively little attention has been paid to them in potatoes. Similarly, interference among predator species occurs not only when predators actually eat one another, but also when a predator flees a

particular habitat to avoid being eaten by another predator (Moran and Hurd 1994); whether a predator is truly killed or simply retreats, herbivore suppression can be equally disrupted

(Schmitz 2008, Steffan and Snyder 2010). The obvious anti-predator behaviors of potato-pests such as the Colorado potato beetle (e.g., Ramirez et al. 2010), and existing good evidence for natural enemies in potato behaviorally avoiding one another (e.g., Lucas and Brodeur 2001), together suggest that nontrophic interactions might be an important way that species interact in potatoes.

2. Biodiversity Aspects Other Than Species Richness. Ecologists interested in biodiversity’s effects most often manipulate the number of species present, or species richness (Hooper et al.

2006, Cardinale et al. 2006). However, biodiversity includes a second component, species’ relative abundances or evenness (Hillebrand et al. 2008). Communities with more-evenly- abundant species are often thought to be more stable than those with highly skewed relative abundances, although this assumption has rarely been tested. Recently, Crowder et al. (2010) found that communities of predator and pathogen natural enemies were more evenly-balanced in organic than conventionally-managed potato fields, and this greater enemy evenness translated into significantly improved biocontrol of Colorado potato beetles. More work is needed, however, to see if these benefits of greater evenness are widespread in potato and other crops.

Furthermore, the impacts of in-field and landscape factors on promoting or reducing evenness warrants further consideration.

3. The Mechanisms of Induced Plant Defenses in Potatoes. Robert Denno and his colleagues have clearly shown that early-season feeding by leafhopper herbivores renders potato plants less-

susceptible to Colorado potato beetles later in the growing season (Lynch et al. 2006, Kaplan et

al. 2007). Potatoes are known to have many possible defenses against these and other herbivores,

but it is not always clear precisely which defenses are activated against which herbivore species, and whether chewing and sucking herbivores are battled in the same ways. Increasing knowledge about the molecular bases of defenses within closely related plant species, such as tomato, should facilitate our ability to learn about the specific operations of anti-herbivore defenses in potato

(Mueller et al. 2005). Such information would increase our understanding of tradeoffs for the plant in defending against one herbivore species versus another (e.g., Kaplan et al. 2009).

Acknowledgments

The authors were supported during the preparation of this book chapter by the National

Research Initiative of the USDA National Institute of Food and Agriculture (NIFA), grant

#2007-02244.

References Cited

Alyokhin, A., and G. Sewell. 2004. Changes in lady beetle community following the

establishment of three alien species. Biol. Invasions 6: 463-471.

Andow, D. A. 1991. Vegetational diversity and arthropod population response. Annu. Rev.

Entomol. 36: 561-586.

Baggen, L. R., and G. M. Gurr. 1998. The influence of food on Copidosoma koehleri

(Hymenoptera: Encyrtidae), and the use of flowering plants to enhance biological control of

potato moth, Phthorimaea operculella (Lepidoptera: Gelechiidae). Biol. Control 11: 9-17.

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.

Entomol. Exp. Appl. 91: 155-161.

Bengtsson, J., J. Ahnström, and A. Weibull. 2005. The effects of organic agriculture on

biodiversity and abundance: a meta-analysis. J. Appl. Ecol. 42: 261-269.

Berry, R. E., J. Liu, and G. Reed. 1997. Comparison of endemic and exotic entomopathogenic

nematode species for control of Colorado potato beetle (Coleoptera: Chrysomelidae). J.

Econ. Entomol. 90: 1528-1533.

Bolter, C. J. and M. A. Jongsma. 1995 Colorado potato beetles (Leptinotarsa decemlineata)

adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. J. Insect Physiol.

41: 1071-1078.

Bolter, C. J., J. M. Dicke, J. J. Van Loop, J. H. Visser, and M. A. Posthumus. 1997. Attraction of

Colorado potato beetle to herbivore-damaged plants during herbivory and after its

termination. J. Chem. Ecol. 23: 1003-1023.

Bommarco, R., and J. E. Banks. 2003. Scale as a modifier in vegetation diversity experiments:

effects on herbivores and predators. Oikos 102: 440-448.

Brodeur, J., and J. M. McNeil. 1992. Host behavior-modification by the endoparasitoid Aphidius

nigripes – a strategy to reduce hyperparasitism. Ecol. Entomol. 17: 97-104.

Brust, G. E. 1994. Natural enemies in straw-mulch reduce Colorado potato beetle populations

and damage in potato. Biol. Control 4: 163-169.

Cardinale, B. J., D. S. Srivastava, J. E. Duffy, J. P. Wright, A. L. Downing, M. Sankaran, and C.

Jouseau. 2006. Effects of biodiversity on the functioning of trophic groups and ecosystems.

Nature 443: 989-992.

Casula, P., A. Wilby, and M. B. Thomas. 2006. Understanding biodiversity effects on prey in

multi-enemy systems. Ecol. Lett. 9: 995-1004.

Chang, G. C., and W. E. Snyder. 2004. The relationship between predator density, community

composition, and field predation on Colorado potato beetle eggs. Biol. Control 31: 453-461.

Crowder, D. W., T. D. Northfield, M. R. Strand, and W. E. Snyder. 2010. Organic agriculture

promotes evenness and natural pest control. Nature 466: 109–112.

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

Press, New York, New York, USA.

Eubanks, M. D., and R. F. Denno. 2000. Host plants mediate ominivore-herbivore interaction

and influence prey suppression. Ecology 81: 936-947.

Gaines, H. R. and C. Gratton. 2010. Seed predation increases with ground beetle diversity in a

Wisconsin (USA) potato agroecosystem. Agric. Ecosyst. Environ. 137: 329-336.

Greenstone, M. H., Z. Szendrei, M. E. Payton, D. L. Rowley, T. C. Coudron, and D. C. Weber.

2010. Choosing natural enemies for conservation biological control: use of the prey

detectability half-life to rank key predators of Colorado potato beetle. Entomol. Exp. Appl.

136: 97-107.

Groden, E., F. A. Drummond, R. A. Casagrande, and D. L. Haynes. 1990. Coleomegilla

maculata (Coleoptera: Coccinellidae): its predation upon the Colorado potato beetle

(Coleoptera: Chrysomelidae) and its incidence in potatoes and surrounding crops. J. Econ.

Entomol. 83: 1306–1315.

Halaj, J., and D. H. Wise. 2002. Impact of a detrital subsidy on trophic cascades in a terrestrial

grazing food web. Ecology 83: 3141-3151.

Hairston, N. G. Jr., and N. G. Hairson, Sr. 1993. Cause-effect relationships in energy flow,

trophic structure, and interspecies interactions. Am. Nat. 142: 379-411.

Hairston, N. G. Jr., and N. G. Hairson, Sr. 1997. Does food-web complexity eliminate trophic

level dynamics? Am. Nat. 149: 1001-1007.

Hairston, N. G., F. E. Smith, and L. B. Slobotkin. 1960. Community structure, population control

and competition. Am. Nat. 94: 421-425.

Hare, D. 1990. Ecology and management of the Colorado potato beetle. Annu. Rev. Entomol.

35: 81-100.

Harmon, J. P., and D. A. Andow. 2004. Indirect effects between shared prey: Predictions for

biological control. Biocontrol 49: 605-626.

Hawkins, B. A., H. V. Cornell, and M. E. Hochberg. 1997. Predators, parasitoids, and pathogens

as mortality agents in phytophagous insect populations. Ecology 78: 2145–2152.

Hawkins, B. A., N. J. Mills, M. A. Jervis, and P. W. Price. 1999. Is the biological control of

insects a natural phenomenon? Oikos 86: 493-506.

Hazzard, R. V. and D. N. Ferro. 1991. Feeding response of adult Coleomegilla maculata

(Coleoptera: Coccinellidae) to eggs of Colorado potato beetle (Coleoptera: Chrysomelidae)

and green peach aphids (Homoptera: Aphididae). Environ. Entomol. 20: 644–651.

Heil, M. and C. Kost. 2006. Priming of indirect defenses. Ecol. Letters. 9: 813–817.

Hilbeck, A. and G. G. Kennedy. 1996. Predators feeding on the Colorado potato beetle in

insecticide-free plots and insecticide-treated commercial potato fields in eastern North

Carolina. Biol. Control 6: 272–282.

Hilbeck, A., C. Eckel, and G. G. Kennedy. 1997. Predation on Colorado potato beetle eggs by

generalist predators in research and commercial potato plantings. Biol. Control 8: 191–196.

Hillebrand, H., D. M. Bennett, and M. W. Cadotte. 2008. Consequences of dominance: a review

of evenness effects on local and regional ecosystem processes. Ecology 89: 1510–1520.

Hlywka, J. J., G. R. Stephenson, M. K. Sears, and R. Y. Yada. 1994. Effects of insect damage on

glycoalkaloid content in potatoes (Solanum tuberosum). J. Agri. Food Chem. 42: 2545-2550.

Hole, D. G., A. J. Perkins, J. D. Wilson, I. H. Alexander, F. Grice, and A. D. Evans. 2005. Does

organic farming benefit biodiversity? Biol. Conserv. 122: 113-139.

Holt, R. D. 1977. Predation, apparent competition, and the structure of prey communities. Theor.

Pop. Biol. 12: 197-229.

Hooper, D. U., F. S. Chapin, J. J. Ewel, A. Hector, P. Inchausti, S. Lavorel, J. H. Lawton, D. M.

Lodge, M. Loreau, S. Naeem, B. Schmid, H. Setala, A. J. Symstad, J. Vandermeer, and D. A.

Wardle. 2005. Effects of biodiversity on ecosystem functioning: A consensus of current

knowledge. Ecol. Monogr. 75: 3-35.

Hough-Goldstein, J.A., Heimpel, G.E., Bechmann, H.E., and C. E. Mason. 1993. Arthropod

natural enemies of the Colorado potato beetle. Crop Prot. 12: 324–334.

Howarth, F. G. 1991. Environmental impacts of classical biological control. Annu. Rev.

Entomol. 36: 485-509.

Idoine, K., and D. N. Ferro. 1990. Persistence of Edovum putleri (Hymenoptera: Eulophidae) on

potato plants and parasitism of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae):

Effects of resource availability and weather. Environ. Entomol. 19: 1732-1737.

Ives, A. R., B. J. Cardinale, and W. E. Snyder. 2005. A synthesis of subdisciplines: predator-prey

interactions, and biodiversity and ecosystem functioning. Ecol. Lett. 8: 102-116.

Jabbour, R., D. W. Crowder, E. A. Aultman, and W. E. Snyder. 2011. Entomopathogen

biodiversity increases host mortality. Biol. Control 59: 277-283.

Johnson, J. M., J. A. Hough-Goldstein, and M. J. Vangessel. 2004. Effects of straw mulch on

pest insects, predators, and weeds in watermelons and potatoes. Environ. Entomol. 33: 1632-

1643.

Kaplan, I., G. P. Dively and R. F. Denno. 2009. The costs of anti-herbivore traits in agricultural

crop plants: a case study involving leafhoppers and trichomes. Ecol. Appl. 19: 864-872.

Kaplan, I., M. E. Lynch, G. P. Dively, and R. F. Denno. 2007. Leafhopper-induced plant

resistance enhances predation risk in a phytophagous beetle. Oecologia 152: 665-675.

Karban, R., and I. T. Baldwin. 1997. Induced responses to herbivory. University of Chicago

Press. Chicago, Ill.

Koss, A. M., and W. E. Snyder. 2005. Alternative prey disrupt biocontrol by a guild of generalist

predators. Biol. Control 32: 243–251.

Koss, A. M., G. C. Chang, and W. E. Snyder. 2004. Predation of green peach aphids by

generalist predators in the presence of alternative, Colorado potato beetle egg prey. Biol.

Control 31: 237-244.

Koss, A. M., A. S. Jensen, A. Schreiber, K. S. Pike, and W. E. Snyder. 2005. A comparison of

predator and pest communities in Washington potato fields treated with broad-spectrum,

selective or organic insecticides. Environ. Entomol. 34: 87-95.

Landis, D. A., S. D. Wratten, and G. M. Gurr. 2000. Habitat management to conserve natural

enemies of arthropod pests in agriculture. Annu. Rev. Entomol. 45: 175-201.

Landolt, P. J., J. H. Tumlinson, and D. H. Alborn. 1999. Attraction of Colorado potato beetle

(Coleoptera: Chrysomelidae) to damaged and chemically induced potato plants. Environ.

Entomol. 28: 973-978.

Langellotto, G. A., and R. F. Denno. 2004. Responses of invertebrate natural enemies to

complex-structured habitats: a meta-analytical synthesis. Oecologia 139: 1-10.

Levin, D. A. 1976. The chemical defenses of plants to pathogens and herbivores. Annu. Rev.

Ecol. Syst. 7: 121-159.

Lill, J. T., and R. J. Marquis. 2001. The effects of leaf quality on herbivore performance and

attack from natural enemies. Oecologia 126: 418–428.

Lima, S.L., and L. M. Dill. 1990. Behavioral decisions made under the risk of predation: a

review and prospectus. Can. J. Zool. 68: 619–640.

Lopez, E. R., D. N. Ferro, and R. G. Van Driesche. 1993. Direct measurement of host and

parasitoid recruitment for assessment of total losses due to parasitism in the Colorado potato

beetle Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) and Myiopharus

doryphorae (Riley) (Diptera: Tachinidae). Biol. Control 3: 85-92.

Loreau, M., and A. Hector. 2001. Partitioning selection and complementarity in biodiversity

experiments. Nature 412: 72–76.

Losey, J. E., and R. F. Denno. 1998. Positive predator–predator interactions: enhanced predation

rates and synergistic suppression of aphid populations. Ecology 79: 2143–2152.

Lucas, E., and J. Brodeur. 1999. Oviposition site selection by the predatory midge Aphidoletes

aphidimyza (Diptera: Cecidomyiidae). Environ. Entomol. 28: 622-627.

Lucas, E., and J. Brodeur. 2001. A fox in sheep’s clothing: Furtive predators benefit from the

communal defense of their prey. Ecology 82: 3246-3250.

Lucas, E., D. Coderre, and J. Brodeur. 1998. Intraguild predation among aphid predators:

Characterization and influence of extraguild prey density. Ecology 79: 1084-1092.

Lucas, E., D. Coderre, and J. Brodeur. 2000. Selection of molting sites by Coleomegilla

maculata (Coleoptera: Coccinellidae): Avoidance of intraguild predation. Environ. Entomol.

29: 454-459.

Lynch, M. E., I. Kaplan, G. P. Dively, and R. F. Denno. 2006. Host-plant-mediated competition

via induced resistance: interactions between pest herbivores on potatoes. Ecol. Appl. 16:

855–864.

Mallampalli, N., I. Castellanos, and P. Barbosa. 2002. Evidence for intraguild predation by

Podisus maculiventris on a ladybeetle, Coleomegilla maculata: Implications for biological

control of Colorado potato beetle, Leptinotarsa decemlineata. Biocontrol 47: 387-398.

Mallampalli, N., F. Gould, and P. Barbosa. 2005. Predation of Colorado potato beetle eggs by a

polyphagous lady beetle in the presence of alternative prey: potential impact on resistance

evolution. Entomol. Exp. Appl. 114: 47-54.

Matson, P. A., W. J. Parton, A. G. Power, and M. J. Swift. 1997. Agricultural intensification and

ecosystem properties. Science 277: 504–509.

Meyling, N. V., A. Enkegaard, and H. Brodsgaard. 2004. Intraguild predation by Anthocoris

nemorum (Heteroptera: Anthocoridae) on the aphid parasitoid Aphidius colemani. Biocontrol

Sci. Tech. 14: 627-630.

Moran, M. D., and L. E. Hurd. 1994. Short-term responses to elevated predator densities:

noncompetitive intraguild interactions and behaviour. Oecologia 98: 269-273.

Mueller, L. A., T. H. Solow, N. Taylor, B. Skwarecki, R. Buels, J. Binns, C. Lin, M. H. Wright,

R. Ahrens, Y. Wang, E. V. Herbst, E. R. Keyder, N. Menda, D. Zamir and S. D. Tanksley.

2005. The SOL genomics network: a comparative resource for Solanaceae biology and

beyond. Plant Physiol. 138: 1310-11317.

Nault, B. A., and G. C. Kennedy. 2000. Seasonal changes in habitat preference by Coleomegilla

maculata: Implications for Colorado potato beetle management in potato. Biol. Control 17:

164-173.

Ohgushi, T. 2005. Indirect interaction webs: herbivore-induced effects through trait change in

plants. Annu. Rev. Ecol. Evol. Syst. 36: 81-105.

Patt, J. M., J. H. Lashomb, and G.C. Hamilton. 1997. Impact of strip-insectary interplanting

with flowers on conservation biological control of the Colorado potato beetle. Advances

Hort. Science 11: 175-181.

Pelletier, Y., G. Grondin, and P. Maltais. 1999. Mechanism of resistance to the Colorado potato

beetle in wild Solanum species. J. Econ. Entomol. 92: 708-713.

Pimentel, D. 1961. Species diversity and insect population outbreaks. Ann. Entomol. Soc. Am.

54: 76-86.

Polis, G. A. 1991. Complex trophic interactions in deserts: an empirical critique of food-web

theory. Am. Nat. 138: 123-155.

Polis, G. A., and D. R. Strong. 1996. Food web complexity and community dynamics. Am. Nat.

147: 813-846.

Polis, G. A., C. A. Myers, and R. D. Holt. 1989. The ecology and evolution of intraguild

predation: potential competitors that eat each other. Annu. Rev. Ecol. Syst. 20: 297-330.

Preisser, E.L., D. I. Bolnick, and M. F. Benard. 2005. Scared to death? The effects of

intimidation and consumption on predator–prey interactions. Ecology 86: 501–509.

Ramirez, R.A. and W. E. Snyder. 2009. Scared sick? Predator-pathogen facilitation enhances the

exploitation of a shared resource. Ecology 90: 2832–2839.

Ramirez, R. A., D. W. Crowder, G. B. Snyder, M. R. Strand, and W. E. Snyder. 2010.

Antipredator behavior of Colorado potato beetle larvae differs by instar and attacking

predator. Biol. Control 53: 230–237.

Rodriguez-Saona, C. and J. S. Thaler. 2005. The jasmonate pathway alters herbivore feeding

behaviour: consequences for plant defenses. Entomol. Exp. Appl. 115: 125–134.

Root, R. B. 1973. Organization of a plant-arthropod association in simple and diverse habitats:

the fauna of collards. Ecol. Monographs 43: 95-124.

Rosenheim, J. A., H. K. Kaya, L. E. Ehler, J. J. Marois, and B. A. Jaffee. 1995. Intraguild

predation among biological-control agents: theory and practice. Biol. Control 5: 303-335.

Russell, E. P. 1989. Enemies hypothesis: a review of the effect of vegetational diversity on

predatory insects and parasitoids. Environ. Entomol. 18: 590-599.

Schmitz, O. J. 2008. Predators avoiding predation. Proc. Nat. Acad. Sci. USA 39:14749-14750.

Schmitz, O. J., A. P. Beckerman, and K. M. O’Brien. 1997. Behaviorally mediated trophic

cascades: effects of predation risk on food web interactions. Ecology 78: 1388–1399.

Schmitz, O.J., V. Krivan, and O. Ovadia. 2004. Trophic cascades: the primacy of trait-mediated

interactions. Ecology Letters: 7: 153–163.

Settle, W.H., H. Ariawan, E. T. Astuti, W. Cahyana, A. L. Hakim, D. Hindayana, A. S. Lestari,

and S. Pajarningsih. 1996. Managing tropical rice pests through conservation of generalist

natural enemies and alternative prey. Ecology 77: 1975–1988.

Snyder, W. E. 2009. Coccinellids in diverse communities: which niche fits? Biological Control

51: 323–335.

Snyder, W. E., and A. R. Ives. 2001. Generalist predators disrupt biological control by a

specialist parasitoid. Ecology 82: 705–716.

Steffan, S. A., and W. E. Snyder. 2010. Cascading diversity effects transmitted exclusively by

behavioral interactions. Ecology 91: 2242-2252.

Stoner, K. A. 1993. Effects of straw and leaf mulches and trickle irrigation on the abundance of

Colorado potato beetles (Coleoptera: Chrysomelidae) on potato in Connecticut. J. Entomol.

Sci. 28: 393-403.

Stoner, K. A., F. J. Ferrandino, M. P. N. Gent, W. H. Elmer, and J. A. Lamonida. 1996. Effects

of straw mulch, spent mushroom compost, and fumigation on the density of Colorado potato

beetle (Coleoptera: Chyrsomelidae) in potatoes. J. Econ. Entomol. 89: 1267–1280.

Straub, C. S. and W. E. Snyder. 2006. Species identity dominates the relationship between

predator biodiversity and herbivore suppression. Ecology 87: 277–282.

Straub, C. S. and W. E. Snyder. 2008. Increasing enemy biodiversity strengthens herbivore

suppression on two plant species. Ecology 89: 1605-1615.

Straub, C. S., D. L. Finke, and W. E. Snyder. 2008. Are the conservation of natural enemy

biodiversity and biological control compatible goals? Biological Control 45: 225-237.

Strong, D. R. 1992. Are trophic cascades all wet? Differentiation and donor-control in speciose

systems. Ecology 73: 747-754.

Symondson, W. O. C., K. D. Sunderland, and M. H. Greenstone. 2002. Can generalist predators

be effective biocontrol agents? Annu. Rev. Entomol. 47: 561–594.

Szendrei, S., and D. C. Weber. 2009. Response of predators to habitat manipulation in potato

fields. Biol. Control 50: 123-128.

Szendrei, S., M. H. Greenstone, M. E. Payton, and D. C. Weber. 2010. Molecular gut-content

analysis of predator assemblage reveals the effect of habitat manipulation on biological

control in the field. Basic Appl. Ecol. 11: 153-161.

Todorova, S. I., D. Coderre, and J. C. Cote. 2000. Pathogenicity of Beauveria bassiana isolates

toward Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), Myzus persicae

(Homoptera: Aphididae) and their predator Coleomegilla maculata lengi (Coleoptera:

Coccinellidae). Phytoprotection 81: 15-22.

Tomlin, E. S. and M. K. Sears. 1992a. Indirect competition between the Colorado potato beetle

(Coleoptera: Chrysomelidae) and the potato leafhopper (Homoptera: Cicadellidae) on potato:

laboratory study. Environ. Entomol. 21: 787–792.

Tomlin, E. S. and M. K. Sears. 1992b. Effects of Colorado potato beetle and potato leafhopper

on amino acid profile of potato foliage. J. Chem. Ecol. 18: 481-488.

Tscharntke, T., A. M. Klein, A. Kruess, I. Steffan-Dewenter, and C. Thies. 2005. Landscape

perspectives on agricultural intensification and biodiversity-ecosystem service management.

Ecol. Lett. 8: 857-874.

Tylianakis, J. M., T. Tscharntke, and O. T. Lewis. 2007. Habitat modification alters the structure

of tropical host–parasitoid food webs. Nature 445: 202–205.

Vandermeer, J. 1995. The ecological basis of alternative agriculture. Annu. Rev. Ecol. Syst. 26:

201-224.

van Emden, H. F., and G. F. Williams. 1974. Insect stability and diversity in agro-ecosystems.

Annu.Rev. Entomol. 19: 455-475. van Loon, J. J. A., E. W. de Vos, and M. Dicke. 2000. Orientation behaviour of the predatory

hemipteran Perillus bioculatus to plant and prey odours. Entomol. Exp. Appl. 96: 51–58.

Walsh, B. D., and C. V. Riley. 1868. Potato bugs. Am. Entomol. 1: 21–49.

Weissbecker, B., J. J. A. Van Loon, and M. Dicke. 1999. Electroantennogram responses of a

predator, Perillus bioculatus, and its prey, Leptinotarsa decemlineata, to plant volatiles. J.

Chem. Ecol. 25: 2313–2325.

Weissbecker, B., J. J. A. Van Loon, M. A. Posthumus, H. J. Bouwmeester, and M. Dicke. 2000.

Identification of volatile potato sesquiterpenoids and their olfactory detection by the two-

spotted stinkbug Perillus bioculatus. J. Chem. Ecol. 26: 1433–1445.

Werling, B. P., and C. Gratton. 2010. Local and landscape structure differentially impact

predation of two potato pests. Ecol. Appl. 20: 1114-1125.

Werner, E. E. and S. D. Peacor. 2003. A review of trait-mediated indirect interactions in

ecological communities. Ecology 84: 1083–1100.

Wilby, A., and M. B. Thomas. 2002. Natural enemy diversity and pest control: patterns of pest

emergence with agricultural intensification. Ecol. Lett. 5: 353-360.

Wise, M. J. and A. M. Weinberg. 2002. Prior flea beetle herbivory affects oviposition

preferences and larval performance of a potato beetle on their shared host plant. Ecol.

Entomol. 27: 115-122.

Zehnder, G. W., and J. Hough-Goldstein. 1990. Colorado potato beetle (Coloptera:

Chrysomelidae) population development and effects on yield of potatoes with and without

straw mulch. J. Econ. Entomol. 83: 1982–1987.

This chapter is published:

Lynch, C. A, D. W. Crowder, R. Jabbour, and W. E. Snyder. 2013. Spud web: Species interactions and biodiversity in potatoes, pp. 271-290. In P. Giordanengo and A. Alyokhin (eds.), Insect Pests of Potatoes: Global perspectives on biology and management. Academic Press (Elsevier) Oxford, UK. , Waltham, MA.

Figures

Fig. 1. Lucas et al. (1998) found that lacewing larvae fed not only on potato aphids, but

also on larvae of predatory Aphidoletes midges. “Intraguild” predation of this type has the potential to disrupt aphid control, as lacewing larvae consume midge larvae that otherwise might have eaten many aphids. Arrows denote energy flow and so point from prey to predator; black arrows indicate predation of the herbivore by predators, while the red arrow indicates predation of one predator by another.

Fig. 2. In potato foliage, Nabis alternatus bugs and Hippodamia convergens beetles exert complementary impacts on Myzus persicae aphids (Straub and Snyder 2008). The lady beetles forage primarily at leaf edges, whereas the predatory bugs also forage at leaf centers. Because of these differences in space use, aphids face heavy predation pressure everywhere on the plant only when both predator species occur together.

Fig. 3. In Washington potato crops, predators and pathogens exert complementary impacts on Colorado potato beetles (Ramirez and Snyder 2009). Potato beetle eggs and larvae in the foliage are eaten by a diverse group of predatory Hippodamia convergens (Hc) and

Pterostichus melanarius (Pmel) beetles, and Nabis alternatus (Nabis) bugs. Once the beetles

enter the soil to pupate they are infected by entomopathogenic Steinernema carpocapsae (Scarp)

and Heterorhabditis marelatus (Hmar) nematodes, and Beauveria bassiana (Bbass) fungi.

Because of the spatiotemporal separation of predators and pathogens, potato beetles face attack throughout their life cycle only when both classes of natural enemy are present.

Fig. 1

Fig. 2

Fig. 3

CHAPTER 1: Detecting Leptinotarsa decemlineata (Colorado potato beetle) in two generalist predators, Geocoris bullatus and Nabis alternatus, and intraguild predation of Geocoris bullatus

in Nabis alternatus over time

Christine A. Lynch, Gretchen B. Snyder, Eric Chapman, James Harwood and William E. Snyder

Department of Entomology, Washington State University, Pullman, WA 99164, USA

ABSTRACT

Molecular gut-content analysis is an established and increasingly important approach for tracking arthropod predation in the field. It is difficult to know what hemipteran generalist predators consume because there are no identifiable prey pieces in their gut. In order to relate the proportion of predator individuals found to contain pest DNA to the number of pests eaten over a given time period, it is necessary to determine how long pest DNA can be detected after predation occurred. This study examined digestion rates for two species of insect predators, adult

Nabis alternatus and Geocoris bullatus, using Colorado potato beetle larvae, and G. bullatus nymphs as prey items. After feeding was observed on a prey item, predators were then killed 0,

1, 2, 4, 6, 8, 16, and 24 hours later by being placed in 95% ethanol and freezing immediately. For each predator-prey combination, 10 females and 10 males were used at each time point for a total of 80 females and 80 males. Prey-species-specific PCR primers were used to test for prey DNA in predators’ guts. Detection of Colorado potato beetle and G. bullatus nymphs occurred for both

G. bullatus and N. alternatus over time, and a small percentage were positive for prey items up to 24 hours later. This study helps confirm results of molecular gut-content analysis that is used for studying predator-prey interactions among these species in potato fields; successful detection of prey DNA indicates that predation was most likely to have occurred within the previous 24 hours.

Keywords: Generalist predators; Damsel bug; Big-eyed bug; Colorado potato beetle, DNA, molecular gut content analysis

1. Introduction

The available prey species, prey handling times, and changing field microhabitats could change the species of prey eaten and the predator’s ability to capture prey items over time, since generalist predators feed on multiple prey species (Symondson et al. 2002). Therefore, predators’ densities are not tied to those of any particular prey (Harmon and Andow 2004).

Generalists can impact many different pest species because they switch feeding to different pest species as each pest becomes abundant (Symondson et al. 2002). For example, many predators also receive nutrients from plant tissues that include pollen, seeds, floral nectars, extrafloral nectars and honeydew (Lundgren 2009). Insect pests are often attacked by a variety of natural enemies, and some natural enemies will be more effective at pest suppression than other species

(Loreau et al 2001, Straub and Snyder 2006). Determining which natural enemies to conserve is a difficult question to answer, when there are multiple promising candidates in an agroecosystem with many species (Cardinale et al 2003, Greenstone and Sunderland 1999,

Greenstone et al 2010). The detection and quantification of predation patterns is multifaceted

and is important to the expansion and improvement of conservation biological control

(Symondson et al. 2002). Direct observation of predation events, controlled manipulation of predator and prey densities to determine predation mechanisms, and the detection of prey markers in predators are methods used to determine the occurrence, frequency and impact of predators on prey populations (Weber and Lundgren 2009).

Molecular gut-content analysis is an established and increasingly important approach for tracking arthropod predation in the field (Hagler et al. 1992, Hagler & Naranjo 1994,

Symondson 2002, Harwood et al. 2007, Juen & Traugott 2007, Kuusk et al. 2008, Lundgren et al. 2009). Predation is a difficult interspecific interaction to study in the field (Sunderland 1988,

Greenstone and Morgan 1989), and gut analysis is considered the least disruptive approach for ecosystem processes (Greenstone et al. 2010). This is because gut-content analysis can be conducted using predators freshly collected from the field, where they have been allowed to forage entirely naturally (Greenstone et al. 2010). Monoclonal antibody based assays

(Greenstone 1996, Harwood et al. 2004) and polymerase chain reaction (PCR) amplification of prey deoxyribonucleic acid (DNA) sequences (Symondson 2002, Sheppard & Harwood 2005,

Gariepy et al. 2007) are the two most common molecular gut-content analysis methods

(Greenstone et al. 2010). Remains detected in the gut of a particular predator may not show predation on a live prey item, but it may actually be scavenging on a dead animal or reflects secondary predation of one predator eating another that consumed a prey item (Harwood et al.

2001, Calder et al. 2005, Foltan et al. 2005, Juen & Traugott 2005, Sheppard et al. 2005).

However, studies have repeatedly shown these biases to be extremely minor with nearly all

“positive” tests reflecting predation of live prey (Harwood et al. 2001). Predator species with low digestion rates could house detectable prey DNA much longer than predators that digest

quickly, with slow-digesters giving the impression of being much more important predators of a

given prey even though the feeding histories were identical to those of fast digesters

(Greenstone et al. 2010). For this reason, it is important to compare among predator species the

length of time that prey DNA can be detected.

We are interested in determining digestion curves for the two main omnivorous generalist

predator species in potatoes, which are the big-eyed bug Geocoris bullatus Stål and the damsel

bug Nabis alternatus Parshley (Koss et al. 2004; Tamaki and Weeks 1972a, b). Both of these

predators consume the major pests species in Washington, which are Myzus persicae (Sulzer),

the green peach aphid, and Leptinotarsa decemlineata (Say), the Colorado potato beetle (Biever and Chauvin 1992, Mowry 2001). Intraguild predation may also be important in this predator community: in simple laboratory arenas, Nabis alternatus (damsel bug) and Geocoris bullatus

(big-eyed bug) will feed on each other depending on which predator is in the larger

developmental stage (Raymond 2000). We investigated the detection limits of target prey DNA

using G. bullatus and N. alternatus predators with L. decemlineata (Colorado potato beetles or

CPB) and .G bullatus nymphs (for N. alternatus only) as prey items. This study will help us

know possibly how long field collected predators would test positive for a prey item.

2. Materials and methods

2.1. Field collection of generalist predators

Predators, Nabis alternatus and Geocoris bullatus, were collected live using a D-vac suction sampler (B&S Model 24 Ventura, CA). An alfalfa field at the WSU Research Farm near

Othello, WA was used to collect predators until the potato field had grown tall enough to support

an insect community. Mesh D-vac bags were brought back in coolers with ice packs from the

field and were transferred to a cool incubator (10 °C) back at the laboratory. Predators were later

sorted individually into 60 mm x 15 mm sterile polystyrene Petri dishes (Fisher Scientific) with

damp cotton dental wicks and were fed Acyrthosiphon pisum (Harris), pea aphids, if they were

not going to be used immediately for an experiment. Predators used in experiments were held

without aphids for 48 hours before use.

2.2. Rearing potato plants, green peach aphids, and Colorado potato beetles

Potato plants for the greenhouse microcosm experiments were grown in the greenhouse

at ambient day length and 22–25 °C. Whole certified organic Ranger russet potato seed (Basin

Gold Cooperative, Warden, WA) were planted in decagonal planting containers (McConkey and

Co; Sumner, WA) filled with Sunshine Professional growing mix potting soil (Sungro

Horticulture; Seba Beach, AB Canada). The containers were put in the greenhouse and were

watered. Containers were watered once a week, and potato plants were broken off of the main

tuber around 10.16 cm tall and were transplanted into separate 15.24 cm square pots with WIL-

GRO professional products pro balance slow release granular fertilizer (16-16-16-7S, Wilbur-

Ellis Co.; Halsey, OR) sprinkled on top of the Sunshine Professional growing mix potting soil

(Sungro Horticulture; Seba Beach, AB Canada). Plants were watered as needed (three times a

week) before use in insect colonies, when they were about 15.24 cm tall or taller.

A pea aphid, Acyrthosiphon pisum (Harris), colony was originally started from pea aphids

collected on alfalfa at the WSU Research Farm near Othello, WA, in 2011 was used to feed predators being held until use in feeding trials; pea aphids were reared on pea plants, Pisum

sativum, in 60 x 60 x 60 cm fine mesh BugDorms (Megaview Science; Taiwan, China). This colony was maintained under the same conditions and in the same greenhouse as the potato plants but in a separate room as the potato plants. The same square pots, soil, and fertilizer as the potato plants were used to start pea plants from seeds (4 seeds in each pot). Potato and pea plants in the aphid colonies were watered 3 times a week.

Adult Colorado potato beetles (CPB) collected in eastern Washington potato fields produced the eggs and larvae used in our feeding trials. Beetles in the colony were fed live potato plants and were housed in the same greenhouse but different room as the aphid colony in 60 x 60 x 60 cm fine mesh BugDorms (Megaview Science; Taiwan, China). The greenhouse was maintained at 22–25°C with ambient day length. Eggs were harvested daily and placed in a refrigerator (5°C) to retard egg development.

2.3. Feeding trials

Adult N. alternatus and G. bullatus were the life stage of the predators used, and G.

bullatus nymphs and Colorado potato beetle first instar larvae were the life stages of the prey used. After field collection and cool storage, N. alternatus and G. bullatus were sorted in the lab into clean individual petri dishes with moistened dental wicks, and they were fed pea aphids for about seven days to eliminate current prey DNA in their guts. After predators were fed pea aphids, they were moved to a clean petri dish, and a new damp cotton dental wick was added after each predator was sexed and dish labeled with sex. Then the predators were starved for 48 hours before starting the feeding trials. Next, each predator was fed one individual of focal prey (CPB or G. bullatus). Once a predator initiated feeding, the petri dish was marked

with a dot, and the predator was allowed to feed for at least 5-10 minutes. If a predator just probed a prey item or fed for less time, it was not used in the experiment.

After the predator fed on the focal prey item for 5 to 10 minutes, then the prey item was removed and was replaced with one pea aphid. Predators that did not feed on the pea aphid

“chaser” were also not used in the experiment. The time was marked on the dish after a predator fed on both prey items, and a moistened dental wick was added to the petri dish. Predators were either killed right away for time zero or one of the other seven time points (0, 1, 2, 4, 6, 8, 16, and 24 hours), and each predator was killed by being placed into an Eppendorf 1.5 ml microcentrifuge tube containing 95% ethanol and freezing them immediately. For each predator-prey combination, 10 females and 10 males were used at each time point for a total of

80 females and 80 males. There were two predators and three prey items. If not enough predators fed on the prey items to complete the entire experiment, then it was broken into blocks with the same number of males and females for each time point (e.g. 2 males and 2 females all at times 0, 1, 2, 4, 6, 8, 16, and 24 hours).

Each predator was crushed using a 0.5 ml mortar-and-pestle microcentrifuge tube with a buffer (eg, Harwood et al. 2009), and total DNA was extracted from each crushed specimen using QIAGEN DNeasy Tissue kits (QIAGEN Inc., Chatsworth, CA, USA). The manufacturer’s animal tissue protocol was used for all of the DNA extractions (Chapman et al.

2010). Specific primers for each prey item were used to detect CPB (Greenstone et al 2010), or

G. bullatus in each predator gut (Table 1). PCR (50 µL) consisted of 1U Takara buffer (Takara

Bio Inc., Shiga, Japan), 0.2 mM of each dNTP, 0.2 m M of each primer, 1U Takara Ex Taq and template DNA (1–5 µL of total DNA). PCR were carried out in Bio-Rad PTC-200 and C1000 thermal cyclers (Bio-Rad Laboratories, Hercules, CA, USA). The PCR cycling protocol for the

Colorado potato beetle primers were 94.5 °C for 3 minutes, followed by 40 cycles 94.5 °C at 45 seconds, 37 °C for 1 minute, 72° C for 2 minutes, and a final extension of 72 °C for 5 minutes

(Greenstone et al. 2007). The PCR cycling protocol for the G. bullatus primers were 94 °C for 1 minute followed by 50 cycles of 94 °C for 45 seconds, 61 °C for 45 seconds, 72 °C for 30 seconds. Reaction success was determined by electrophoresis of 10 µL o f PCR product in 3%

SeaKem agarose (Lonza, Rockland, ME, USA) stained with ethidium bromide (0.1 mg / µL).

The results of each predator were recorded after viewing the results of the gel.

2.4. Statistics

We examined the relationship between time and probability that prey DNA was detected using nonlinear logistic regression. We investigated the detection limits of target prey DNA using G. bullatus and N. alternatus predators with CPB as prey items for both species, and G. bullatus nymphs as a prey item for N. alternatus. The regressions for all five feeding trials were fitted in SigmaPlot (Systat Software, San Jose, CA). Durbin-Watson, Normality test, and

Constant variance test were also done for each curve, and all of data sets passed these tests .

3. Results

3.1. Feeding trials Colorado potato beetles

Both G. bullatus and N. alternatus were fed a single Colorado potato beetle and a chaser pea aphid for each predator tested. The chaser pea aphid was used as a control to make sure that the predator was consuming each prey item. Detection of prey DNA decreased over time for both

predators between 0 and 24 hours. The digestion curve decreased significantly over time for

Geocoris bullatus (Fig. 1) feeding on CPB (F3, 8 = 9.5328, P = 0.0165). There was 100 %

detection of CPB at time zero and this slowly decreased to 60 % detection around 8 hours after

feeding and about 50 % detection by 24 hours. The digestion curve decreased significantly over

time for N. alternatus (Fig. 2) feeding on CPB (F3, 8 = 43.6457, P = 0.0005). There was 100 % detection of CPB at time zero and this slowly decreased to 70 % detection around 8 hours after feeding and about 15 % detection by 24 hours.

3.2. Feeding trials Geocoris bullatus nymphs

The predators N. alternatus were fed a single G. bullatus nymph and a chaser pea aphid

for each predator tested. The chaser pea aphid was used as a control to make sure that the

predator was consuming each prey item. Detection of prey DNA decreased over time for N.

alternatus between 0 and 24 hours. The digestion curve decreased significantly over time for N.

alternatus (Fig.3) feeding on G. bullatus nymphs (F3, 8 = 159.6697, P < 0.0001). There was 95 %

detection of GPA at time zero and this gradually decreased to 50 % detection around 8 hours

after feeding and about 5 % detection by 24 hours.

4. Discussion

Molecular gut-content analysis has transformed the way that researchers determine the

role of predators in suppressing insect populations, and using the incidence of a pest in a

predator’s gut to rank predators importance is a logical first step in ranking them for

conservation biological control (Greenstone et al. 2010). Detection of CPB and G. bullatus nymphs occurred for both G. bullatus and N. alternatus over time, although the length of DNA detection in predator guts differed by prey item and predator species. By 24 hours, only a small

percentage of predators showed a low detection rate for prey items in both predator species and

most prey items. For Colorado potato beetle, G. bullatus (Fig. 1) and N. alternatus (Fig. 2)

percent positive for CPB was similar at 8 hours but dropped much lower for N. alternatus at 24

hours. The intraguild predation feeding trial with Nabis alternatus consuming G. bullatus

nymphs (Fig. 3) showed the lowest percent positive predators for the N. alternatus at 8 hours and

24 hours, so G. bullatus fluids were digested quickly by N. alternatus. This is similar to a study

with Orius insidiosus, a minute pirate bug, consuming soybean aphids, thrips and eggs/larvae of

Asian multi-colored ladybeetles where this predator showed a low detection of prey DNA for the

ladybeetle and thrips between 10 and 24 hours (Harwood et al. 2007). This species is also a

hemipteran predator, so it is not surprising the digestion of prey would be similar. Digestion in

Coleomegilla maculata, spotted lady beetle, was very rapid in starved predators being fed

Colorado potato beetle eggs and slower with predators that were fed potato aphids, and this predator’s digestive system aggressively degraded the prey (Weber and Lundgren 2009), which has been shown in other systems (eg; Zhang et al. 2007, Nejstgaard et al. 2008)

Variables that affect the detectability of samples positive for prey DNA include the number and size of prey eaten, stage of prey eaten, time since prey consumption, temperature from consumption to collection, and the consumption of alternate prey during feeding (Sopp &

Wratten 1986, Sopp et al. 1992, Hagler & Naranjo 1997, Weber and Lundgren 2009). For conventional Polymerase chain reaction, the target DNA decay rates are not available because the data is presence or absence. Logistic models can be used to characterize the decline in

detectability (Weber and Lundgren 2009), which includes the detectability half-life for PCR

(Chen et al. 2000, Greenstone et al. 2007). The detectability half-life is defined as the time after

which only half of the target meals can be detected in a cohort of predators as estimated by probit

analysis, and it is an index of the detectability interval (Chen et al. 2000). We used a logistic model because it fit our data, and either logit or probit models are acceptable to use for the data

(Dey and Astin 1993, Trexler and Travis 1993). Chaser prey (e.g., Hagler and Naranjo 1997,

Symondson et al. 1999, Greenstone et al. 2007, Harwood et al. 2007) and starvation (e.g.,

Hagler 1998, Chen et al. 2000, Harper et al. 2006) are commonly used in studies to determine the rate of target prey marker disappearance (Weber and Lundgren 2009). Non-target chaser prey is used to simulate normal feeding rates and eliminate the adverse effects of starvation on digestion rate and DNA detectability (Greenstone & Hunt 1993, Chen et al. 2000, Harwood et al. 2007).

There are limitations to using feeding trials and using PCR for molecular gut content analysis. Predators were not used for a feeding trial if they did not consume both the target prey item and the chaser prey. A least three times more predators were used for each feeding trial, which means that a large number of live predators must be collected to complete a feeding trial.

Smaller predators like G. bullatus often did not feed on both the target CPB larvae and chaser aphid prey, so more insects were used to have enough to run each trial. PCR only detects the presence or absence of the prey DNA, but it does not tell you how many prey items or prey stages have been consumed (Greenstone et al. 2010).

This study helps us know possibly how long field collected predators would test positive for a prey item. For both species of predators, prey DNA is low around 24 hours, so most predators collected from fields and tested positive for prey most likely ate the prey less than 24 hours before collection. It would also be possible to map networks of intraguild predators and

alternate prey by using multiple DNA primers for predators and prey present in more complex

systems (Agustý´ et al. 2003, Harwood et al. 2007, Saccaggi et al. 2008). Results of a large field

study would provide more insight into the mechanism of biological control, and it would help

managers enhance biological control (Greenstone et al. 2010).

Acknowledgments

We thank Ben Hieb, Glen Chase, and Jake Gable for collecting live predators from the alfalfa field. Amanda Meadows assisted Gretchen Snyder with running the feeding trials. This project

was supported by a grant from USDA AFRI.

References

Agusti, N., Shayler, P., Harwood, J. D., Vaughan, I. P., Sunderland, K. D. & W. O. C.

Symondson. 2003. Collembola as alternative prey sustaining spiders in arable

ecosystems: prey detection within predators using molecular markers. Molecular

Ecology. 12: 3467– 3475.

Biever, K.D. and R. L. Chauvin. 1992. Timing of infestation by the Colorado potato beetle

(Coleoptera: Chrysomelidae) on the suppressive effect on field released stinkbugs

(Hemiptera: Pentatomidae) in Washington. Environ. Entomol. 21: 1212–1219.

Calder, C. R., Harwood, J. D., and W. O. C. Symondson. 2005. Detection of scavenged material

in the guts of predators using monoclonal antibodies: a significant source of error in

measurement of predation? Bulletin Entomol. Res. 95(1): 57-62.

Cardinale, B. J., C. Harvey, K. Gross and A. R. Ives. 2003. Biodiversity and biocontrol:

emergent impacts of a multi-enemy assemblage on pest suppression and crop yield in an

agroecosystem. Ecology Letters. 6: 857–865.

Chapman, E. G., Romero, S. A., and J. D. Harwood. 2010. Maximizing collection and

minimizing risk: does vacuum suction sampling increase the likelihood for

misinterpretation of food web connections? Molecular Ecology Resources. 10 (6): 1023-

1033.

Chen, Y. K., Giles, K. L., Payton, M. E. and M. H. Greenstone. 2000. Identifying key cereal

aphid predators by molecular gut analysis. Molecular Ecology. 9: 1887–1898.

Dey, E. L. and A. W. Astin. 1993. Statistical alternatives for studying college student retention: a

comparative analysis of logit, probit, and linear regression. Research in Higher

Education. 34(5): 569-581.

Foltan, P., Sheppard, S., Konvicka, M., and W. O. C. Symondson. 2005. The significance of

facultative scavenging in generalist predator nutrition: detecting decayed prey in the guts

of predators using PCR. Molecular Ecol. 14(13): 4147-4158.

Gariepy, T. D., Kuhlman, U., Gillot, C. & M. Erlandson. 2007. Parasitoids, predators, and PCR:

the use of diagnostic molecular markers in biological control of arthropods. Journal of

Applied Entomology. 131: 225–240.

Greenstone, M. H. 1996. Serological analysis of arthropod predation: past, present and future.

The Ecology of Agricultural Pests – Biochemical Approaches. W. O. C. Symondson & J.

E. Liddell(ed.). Chapman and Hall, London, UK. pp. 265–300.

Greenstone, M. H. and J. H. Hunt. 1993. Determination of prey antigen half-life in Polistes

metricus using a monoclonal antibodybased immunodot assay. Entomologia

Experimentalis et Applicata. 68: 1–7.

Greenstone, M. H. and C. E. Morgan. 1989. Predation on Heliothis zea (Lepidoptera:

Noctuidae): an instar-specific ELISA for stomach analysis. Annals of the Entomological

Society of America. 82: 45–49.

Greenstone, M. H. and K. D. Sunderland. 1999. Why a symposium on spiders in agroecosystems

now? Journal of Arachnology. 27: 267–269.

Greenstone, M. H., Rowley.D. L., Weber, D. C., Payton, M. E., and D. J. Hawthorne. 2007.

Feeding mode and prey detectability half-lives in molecular gut-content analysis: an

example with two predators of Colorado potato beetle. Bulletin Entomological Research.

97: 201-209.

Greenstone, M. H., Szendrei, Z., Payton, M. E., Rowley.D. L., Coudron, T. C., and D.

C. Weber. 2010. Choosing natural enemies for conservation biological control: use of the

prey detectability half-life to rank key predators of Colorado potato beetle. Entomologia

Experimentalis et Aplicata. 136: 97-107.

Hagler, J. R. 1998. Variation in the efficacy of several predator gut content immunoassays.

Biological Control. 12: 25-32.

Hagler, J. R., Cohen, A. C., Bradley-Dunlop, D., and F. J. Enriquez. 1992. Field evaluation of

predation on Lygus hesperus (Hemiptera: Miridae) using a species- and stage-specific

monoclonal antibody. Environmental Entomology. 21: 896–900.

Hagler, J. R. and S. E. Naranjo. 1994. A qualitative survey of two coleopteran predators of

Bemisia tabaci (Homoptera: Alyerodidae) and Pectinophora gossypiella (Lepidoptera:

Gelechiidae) using a multiple prey gut content ELISA. Environmental Entomology. 23:

193–197.

Hagler, J. R. and S. E. Naranjo. 1997. Measuring the sensitivity of an indirect predator gut

content ELISA: detectability of prey remains in relation to predator species, temperature,

time, and meal size. Biological Control 9: 112–119.

Harmon, J. P. and D. A. Andow. 2004. Indirect effects between shared prey: predictions for

biological control. Biocontrol. 49: 605-626.

Harper, G. L, S. K. Sheppard, J. D. Harwood, D. S. Read, D. M. Glen, M. W. Bruford, and W. O.

C. Symondson. 2006. Evaluation of temperature gradient gel electrophoresis for the

analysis of prey DNA within the guts of invertebrate predators. Bulletin of Entomological

Research. 96: 295-304.

Harwood, J. D., Sunderland, K. D. and W. O. C. Symondson. 2004. Prey selection by linyphiid

spiders: molecular tracking of the effects of alternative prey on rates of aphid

consumption in the field. Molecular Ecology. 13: 3549–3560.

Harwood, J. D., Desneux, N., Yoo, H. J. S., Rowley, D. L., Greenstone, M. H., Obrycki, J., and

R. J. O’Neil. 2007. Tracking the role of alternative prey in soybean aphid predation by

Orius insidiosus: a molecular approach. Molecular Ecology. 16: 4390–4400.

Harwood, J. D., Sunderland, K. D., and W. O. C. Symondson. 2001. Living where the food is:

web location by linyphiid spiders in relation to prey availability in winter wheat. J.

Applied Ecol. 38(1): 88-99.

Juen, A. and M. Traugott. 2005. Detecting predation and scavenging by DNA gut-content

analysis: a case study using a soil insect predator–prey system. Oecologia. 142: 344–352.

Juen, A and M. Traugott. 2007. Revealing species-specific trophic links in soil food webs:

molecular identification of scarab predators. Molecular Ecology. 16: 1545–1557.

Koss, A.M., Chang, G.C., and W. E. Snyder. 2004. Predation of green peach aphids by generalist

predators in the presence of alternative, Colorado potato beetle egg prey. Biol. Control.

31: 237-244.

Kuusk, A. K., Cassel-Lundhagen, A., Kvarnheden, A., and B. Ekbom. 2008. Tracking aphid

predation by lycosid spiders in spring-sown cereals using PCR-based gut-content

analysis. Basic and Applied Ecology. 9: 718–725.

Loreau, M., S. Naeem, P. Inchausti, J. Bengtsson, J. P. Grime, A. Hector, D. U. Hooper, M. A.

Huston, D. Raffaelli, B. Schmid, D. Tilman, and D. A. Wardle. 2001. Biodiversity and

ecosystem functioning: current knowledge and future challenges. Science 294: 804–808.

Lundgren, J. G., Ellsbury, M. E., and D. A. Prischmann. 2009. Analysis of the predator

community of a subterranean herbivorous insect based on polymerase chain reaction.

Ecological Applications. 19: 2157–2166.

Mowry, T.M. 2001. Green peach aphid (Homoptera: Aphididae) action thresholds for controlling

the spread of potato leafroll virus in Idaho. J. Econ. Entomol. 94: 1332–1339.

Nejstgaard, J. C., M. E. Frischer, P. Simonelli, C. Troedsson, M. Brakel, F. Adiyaman, A. F.

Sazhin, and F. Artigas. 2008. Quantitative PCR to estimate copepod feeding. Marine

Biology. 153: 565-577.

Raymond, B. 2000. Cannibalism and intraguild predation involving three predatory Heteroptera,

Nabis roseipennis, Geocoris punctipes and Orius insidiosus. Dissertation. University of

Kentucky, Lexington, Kentucky, USA.

Saccaggi, D. L., Kruger, K. and G. Pietersen. 2008. A multiplex PCR assay for the simultaneous

identification of three mealybug species (Hemiptera: Pseudococcidae). Bulletin of

Entomological Research. 98: 27–33.

Sheppard, S.K., Bell, J., Sunderland, K. D., Fenlon, J., Skervin, D., and W. O. C. Symondson.

2005. Detection of secondary predation by PCR analysis of gut contents of invertebrate

generalist predators. Molecular Ecol. 14(14): 4461-4468.

Sheppard, S. K. and J. D. Harwood. 2005. Advances in molecular ecology: tracking trophic links

through predator-prey food webs. Functional Ecology. 19: 751–762.

SigmaPlot, Version 10. Systat Software, San Jose, CA.

Sopp, P. I. and S. D. Wratten. 1986. Rates of consumption of cereal aphids by some polyphagous

predators in the laboratory. Entomologia Experimentalis et Applicata. 41: 69–73.

Sopp, P. I., K. D. Sunderland, J. S. Fenlon, and S. D. Wratten. 1992. An improved quantitative

method for estimating invertebrate predation in the ﬁeld using an enzyme-linked

immunosorbent assay (ELISA). Journal of Applied Ecology. 79: 295–302.

Sunderland, K. D. 1988. Quantitative methods for detecting invertebrate predation in the field.

Annals of Applied Biology. 112: 201–224.

Straub, C. S. and W. E. Snyder. 2006. Species identity dominates the relationship between

predator biodiversity and herbivore suppression. Ecology 87: 277–282.

Symondson, W .O. C. 2002. Molecular identification of prey in predator diets. Molecular

Ecology. 11: 627–641.

Symondson, W. O. C, M. L. Erickson, and J. E. Liddell. 1999. Development of a monoclonal

antibody for the detection and quantification of predation on slugs within the Arion

hortensis agg (Mollusca: Pulmonata). Biological Control. 16: 274-282.

Symondson, W. O. C., K. D. Sunderland, and M. H. Greenstone. 2002. Can generalist predators

be effective biocontrol agents? Annu. Rev. Entomol. 47: 561–594.

Tamaki, G. and R. E. Weeks. 1972a. Efficiency of three predators, Geocoris bullatus, Nabis

americoferus, and Coccinella transversoguttata, used alone or in combination against

three insect prey species, Myzus persiace, Ceramica picata, and Mamestra configurata,

in a greenhouse study. Environ. Entomol. 1: 258–263.

Tamaki, G. and R. E. Weeks. 1972b. Biology and ecology of two predators, Geocoris pallens

Stål and G. bullatus (Say). Technical Bulletin 1446, US Department of Agriculture.

Trexler, J. C. and J. Travis. 1993. Nontraditional regression analyses. Ecology. 74: 1629-1637.

Weber, D. C. and J. G. Lundgren. 2009. Detection of predation using qPCR: effect of prey

quantity, elapsed time, chaser diet, and sample preservation on detectable quantity of

prey DNA. Journal of Insect Science. 9: 41.

Zhang, G. L, Z. C. Lu, F. H. Wan, and G. L. Lovei. 2007. Real-time PCR quantiﬁcation of

Bemisia tabaci (Homoptera: Aleyrodidae) B-biotype remains in predator guts. Molecular

Ecology Notes. 7: 947–954.

Figure legends

Fig. 1. Detection of Leptinotarsa decemlineata larvae DNA in Geocoris bullatus from time 0 to

24 hours later (r2 = 0.8512).

Fig. 2. Detection of Leptinotarsa decemlineata larvae DNA in Nabis alternatus from time 0 to

24 hours later (r2 = 0.9632).

Fig. 3. Detection of Geocoris bullatus nymph DNA in Nabis alternatus from time 0 to 24 hours later (r2 = 0.9897).

Fig. 1

Fig. 2

Fig. 3

Table 1. Primers used to detect the two different prey species in predators guts for the feeding trials.

Prey species tested Primer

Leptinotarsa Cpb5s-F: 5’- CCTTTTCTCTTGGGCAGTTAT-3’ decemlineata Cpb6A-R: 5’- TTATCCCAAATCCAGGTAGAAT-3’

Geocoris bullatus Geo-294-F: 5’- TATCAAGAAGTATAGTAGAAATAGGAGCT-3’

Geo-449-R: 5’- AAATAAAATTAATAGCTCCAAGAATAGAAC-3’

CHAPTER 2: Manipulation of Myzus persicae, green peach aphid, and Leptinotarsa

decemlineata, Colorado potato beetle, to determine predator feeding patterns in potatoes

Christine A. Lynch*, Eric Chapman, James Harwood, and William E. Snyder

Department of Entomology, Washington State University, Pullman, WA 99164, USA

ABSTRACT

Generalist predators, by definition, feed on many different prey species. This can make it difficult to predict how changes in overall prey abundance or diversity will alter these predators’ impacts on particular target pests. We independently manipulated densities of two herbivores common on potatoes, the green peach aphid (Myzus persicae) and the Colorado potato beetle

(Leptinotarsa decemlineata), using flonicamid to target aphids and Bacillus thuringiensis

Tenebrionis to target potato beetles. We then used molecular gut-content analysis to track the effects of these manipulations on patterns of predation by the polyphagous predators Geocoris bullatus and Nabis alternatus on the two pests; to measure intraguild predation we also examined predation of the smaller G. bullatus by the larger N. alternatus. Aphids and potato beetles were reduced by the insecticides that targeted them, and both G.bullatus and N. alternatus reached lower densities in plots where aphids were reduced. However, these changes in predator and prey community structure did not translate into significant effects on either predator’s likelihood of

having eaten aphids or potato beetles, or of N. alternatus eating G.bullatus. This suggests either

that the predators’ diets were relatively unaffected by prey availability, or that predators mixed

their diets by moving freely among our plots. Our study provides evidence that, through

movement or innate preferences, the diets of generalist predators may remain surprisingly rigid

in the face of a mosaic of prey availability.

Keywords: Prey diversity; Arthropod generalist predators; Intraguild predation: Colorado potato

beetle; Green peach aphid;

1. Introduction

By definition, generalist predators feed on multiple prey species (Symondson et al. 2002).

Therefore, their densities are not tied to those of any particular prey (Harmon and Andow 2004).

Some authors have suggested that this lack of a tight dynamical linkage with particular pests

might limit the ability of generalists to tightly regulate pest densities (Snyder and Ives 2008).

Rather, the available prey species, prey handling times, and changing field microhabitats might

change the species of prey eaten (Symondson et al. 2002). In contrast, other authors have argued

that, at least in some circumstances, the polyphagy of generalists might actually improve their

biocontrol impact. For example, generalists may already be present in a field feeding on non-pest prey when the pests first invade; in this way, generalists can form a “first line of defense” (e.g.,

Settle et al. 1996). Likewise, by switching among feeding on different pests as each pest

becomes abundant, generalists can impact many different pest species active at different times of

the year (Symondson et al. 2002). Indeed, the use of non-pest prey can be exploited to improve

biological control. For example, many predators also feed on plant tissues that include pollen, seeds, floral nectars, extrafloral nectars and honeydew (Lundgren 2009). By managing agroecosystems to increase the availability of these resources, it may be possible to build predator densities, and thus impacts on pests, across a farm (Landis et al. 2000).

However, increasing availability of alternative prey does not reliably improve biological control. For example, Prasad and Snyder (2006) showed that alternative prey disrupted biological control by ground and rove beetles because the predators preferred to feed on aphids in Brassica fields rather than the cabbage root maggots that were the main pest. Omnivory is perhaps most disruptive to biological control when one predator species feeds on another (Polis et al. 1989, Rosenheim 1998). For example, generalist carabid ground beetles indirectly disrupted biological control of aphids by eating a specialist parasitoid of the aphids; predation of the parasitoid eventually led to higher aphid densities over time even though aphid predation by the beetles was initially intense (Snyder and Ives 2001). Likewise, Mallampalli and colleagues

(2002) showed that intraguild predation between the spined soldier bug, Podisus maculiventris

Say (Heteroptera: Pentatomidae), and the twelve-spotted ladybeetle, Coleomegilla maculata

Lengi (Coleoptera: Coccinellidae) reduced overall predation on Colorado potato beetle eggs.

Some studies have shown that the presence of extraguild prey can reduce intraguild predation, with the frequency of intraguild predation decreasing as extraguild prey density increases (e.g.;

Bailey and Polis 1987, Lucas et al. 1998).

Two pests affecting potatoes in Washington are Myzus persicae (Sulzer), the green peach aphid (GPA), and Leptinotarsa decemlineata (Say), the Colorado potato beetle (CPB) (Biever and Chauvin 1992, Mowry 2001). Aphids harm potatoes primarily by transmitting plant viruses

(Radcliffe 1982, Blackman and Estop 2000), while potato beetles harm potatoes by consuming

plant foliage (Boiteau 2001). The predator guild on plant foliage in Washington State potato

fields is dominated by omnivorous generalist hemipteran predators, primarily the big-eyed bug

Geocoris bullatus Stål and the damsel bug Nabis alternatus Parshley (Koss et al. 2004; Tamaki and Weeks 1972a, b). Previous feeding trials in simple laboratory arenas have shown that while the presence of aphids reduced these predators’ predation rate on potato beetles, when potato beetle eggs fill the role of “alternative prey” they do not reduce the predators’ predation rate on aphids (Koss et al. 2004, Koss and Snyder 2005). Likewise, a field cage study showed that potato beetle suppression by a complex of generalist predators was reduced when aphids were also present (e.g., Koss and Snyder 2005). Intraguild predation may also be important in this predator community: in simple laboratory arenas, Nabis alternatus (damsel bug) and Geocoris bullatus

(big-eyed bug) will feed on each other depending on which predator is in the larger developmental stage (Raymond 2000). In field-cage studies in Washington potato crops, results sometimes suggested the occurrence of intraguild predation between Nabis and Geocoris (e.g.,

Koss and Snyder 2005, Straub and Snyder 2006) whereas other studies found no evidence for predation among these predators (e.g., Straub and Snyder 2008).

Here, we report the results of two open-field experiments wherein we experimentally manipulated densities of green peach aphids (GPA) and Colorado potato beetles (CPB) to determine if these prey manipulations altered patterns of predation by Nabis alternatus and

Geocoris bullatus. Densities of these pests were reduced using specific insecticides, or left unaltered, to form a fully-factorial manipulation of both pest species (Figure 1). Predation of

CPB and GPA by both predator species and of G. bullatus by N. alternatus was measured by molecular gut content analysis using prey-specific PCR primers. The use of molecular gut

content analysis allowed us to track predation without the need to use field cages or to directly

observe cryptic predator behavior.

2. Materials and methods

2.1. Field site and planting methods

This open field experiment was conducted at the Washington State University Research

Farm in Othello, WA during 2010 and 2011. The field was first hilled, and then the cut potato

seed was planted using a potato planting machine that planted potatoes 0.03 meters apart in rows

that were the full length of the field. The potato seed was planted all in one day (April 20, 2010

and April 29, 2011), and the field contained furrows between each row without dammer dike.

The length of the field was 43.89 meters, and the width across each row was 109.97 meters. The

field was 1.19 acres (0.48 hectares) with twenty 5 by 10 meter plots that contained

approximately 160 potato plants and were marked with flags within the full rows of Ranger

russet potatoes. The rest of the field was broken down into 5 by 10 meter buffer plots around all

four edges of the field and between the rows of 20 plots. Each treatment plot was completely

surrounded by buffer plots of the same size (Fig. 1). The potato plants were spaced 0.03 meters

apart with 0.86 meters in between rows, and there were 52 rows of potatoes. A random number

generator was used to determine the order of each different treatment for all of the plots in the

field. The field was sampled 4 times (every two weeks starting mid June) for herbivores and

predators in 2010 and was sampled 3 times in 2011. The first sample of the predators in the field

was taken in June each year before any pesticide was sprayed.

Using selective insecticides that specifically targeted each pest, we conducted a fully-

factorial manipulation (i.e., Sih et al. 1998) of GPA and CPB within an open-field experiment

that was conducted in each of 2 years (2010 and 2011). Each year, there were five replicates of

each of the four pest-manipulation combinations, which were no spray for GPA or CPB, spray to

suppress CPB only, spray to suppress GPA only, and sprays to suppress both pest species (Figure

1). Novodor (Bacillus thuringiensis Tenebrionis) was applied to reduce densities of Colorado

potato beetles, while Beleaf (Flonicamid) was sprayed to reduce densities of green peach aphids.

The control plots were sprayed with water, and a separate Chapin® 15.14 liter (4 gallon)

backpack sprayer with piston pump (Model 61800 Batavia, NY) was used for each insecticide or

water application. All of the treatments in the field were sprayed three times throughout the

experiment on 1 July, 21 July, and 11 August in 2010; and on 11 July, 22 July, and 9 August in

2011 (in both year plots were first sprayed one week after the introduction of green peach aphids, as described below). Each plot was completely sprayed and an extra meter-wide area on each side of each plot was also sprayed for a total spray area of 7 meters by 12 meters. All 20 plots were sprayed by two different people on the same day.

CPB naturally established in our plots as overwintering adults emerged in the spring. For

GPA, in addition to natural colonization by overwintering alates, we also artificially infested those plots designated to house robust populations of GPA (Figure 1) by adding aphids from a long-term greenhouse colony of aphids not infected with any potato virus; this was done because

GPA densities can vary greatly from year-to-year (Koss and Snyder 2005). Aphids in our colony were originally collected on potatoes near Prosser, WA USA and reared on potato (organically- produced Russet and Yukon) plants in the greenhouse (16:8 light-dark cycle and temperatures of

22–25 °C). We first established field-hardy aphid colonies by transferring GPA from our

greenhouse colony to four 2 x 2 x 2-m field cages covered on all sides, except for the bottom,

with a 32 x 32 Lumite mesh screen (BioQuip, Gardena, California, USA). Each of these field

cages contained 6 Ranger russet potato plants from the greenhouse that were watered three times

a week. After allowing six weeks for aphids to adjust to field conditions and reproduce, we

attached leaflets containing 10 aphids each to 10 different plants using a stick-pin in each aphid+ plot. GPA were added to plots with aphids or control plots on 25 June, 6 July, and 28 July in

2010, and on 1 July, 14 July, and 1 August in 2011.

Potato plants for aphid field cages were started on whole certified organic Ranger russet

potato seed (Basin Gold Cooperative, Warden, WA)and were planted in decagonal planting

containers (McConkey and Co; Sumner, WA) filled with Sunshine Professional growing mix

potting soil (Sungro Horticulture; Seba Beach, AB Canada). The containers were put in the

greenhouse and were watered. Containers were watered once a week, and potato plants were

broken off of the main tuber around 10.16 cm tall and were transplanted into separate 15.24 cm

square pots with WIL-GRO professional products pro balance slow release granular fertilizer

(16-16-16-7S, Wilbur-Ellis Co.; Halsey, OR) sprinkled on top of the Sunshine Professional

growing mix potting soil (Sungro Horticulture; Seba Beach, AB Canada).

2.2. Sampling

The field was sampled for the arthropod community and for predators. A sample of the

predators in the field was taken one week after the pesticides were first sprayed (on 15 June in

2010 and on 30 June 2011), and then again periodically thereafter throughout the growing

season (on 15 July, 5 August and 16 August in 2010, and on 28 July and 18 August in 2011).

Predators and herbivores were sampled using a D-vac suction sampler (B&S Model 24 Ventura,

CA). Two sets of D-vac samples were collected with one set for identification (predators and herbivores) and one for molecular analysis for each sample date (only predators). The survey samples of herbivores and predators were stored at 0 °C. The predators from the second sample were processed using molecular gut content analysis to determine what they were feeding on in the field. The arthropod community from the first sample were sorted, identified and placed into labeled vials with 95% EtOH in the lab. Concurrent with these D-vac collections, but on different plants than those sampled using the D-vac, visual counts of GPA and CPB were recorded for 10 plants per plot. During these visual surveys, on each plant we spent 30 seconds each searching the ground around the base of each plant, the lower half of the foliage, and the upper half of the foliage. There were three sampling dates for each year (2010 and 2011).

For molecular gut content analyses, G. bullatus and N. alternatus predators were put on dry ice in the field and individually placed into a 1.5 mL microcentrifuge tube containing 100%

EtOH. All samples were stored at -12 °C until analysis. To prepare for DNA extraction, each predator was crushed using a 0.5 ml mortar-and-pestle microcentrifuge tubes with a buffer (e.g.,

Harwood et al. 2009). Total DNA was extracted from each crushed specimen using QIAGEN

DNeasy Tissue kits (QIAGEN Inc., Chatsworth, CA, USA). The manufacturer’s animal tissue protocol was used for all of the DNA extractions (Chapman et al. 2010). Specific primers for each prey item were used to detect aphids (Chapman et al. 2010), CPB (Greenstone et al 2010), or G. bullatus in each predator gut (Table 1). PCR (50 µL) consisted of 1U Takara buffer

(Takara Bio Inc., Shiga, Japan), 0.2 mM of each dNTP, 0.2 m M of each primer, 1U Takara Ex

Taq and template DNA (1–5 µL of total DNA). PCR were carried out in Bio-Rad PTC-200 and

C1000 thermal cyclers (Bio-Rad Laboratories, Hercules, CA, USA). The PCR cycling protocol

for the aphid primers were 94 °C for 1 minute followed by 50 cycles of 94 °C for 50 seconds, 40

°C for 45 seconds, 72 °C for 45 seconds and a ﬁnal extension of 72 °C for 5 min. The PCR cycling protocol for the CPB primers were 94.5 °C for 3 minutes, followed by 40 cycles 94.5 °C at 45 seconds, 37 °C for 1 minute, 72° C for 2 minutes, and a final extension of 72 °C for 5

minutes (Greenstone et al. 2007). The PCR cycling protocol for the G. bullatus primers were 94

°C for 1 minute followed by 50 cycles of 94 °C for 45 seconds, 61 °C for 45 seconds, 72 °C for

30 seconds. Reaction success was determined by electrophoresis of 10 µL o f PCR product in 3%

SeaKem agarose (Lonza, Rockland, ME, USA) stained with ethidium bromide (0.1 mg / µL).

The results for each predator were recorded after viewing the results of the gel.

2.3. Statistics

The experimental design was a 2 x 2 factorial (Sih, et al. 1998) with the two years as temporal blocks and five replicates of each treatment. We compared the seasonal trends in predator and prey densities along with the proportion of predators testing positive for having eaten each prey item using repeated measures MANOVA in JMP (JMP, Cary, North Carolina,

USA). Predator consumption data from molecular gut content analysis were arcsin square root- transformed before analysis to improve normality and homogeneity of variance. For the arthropod community samples, the different arthropod species were summed into functional groups that included herbivores (other than aphids or potato beetles), predators (other than G. bullatus or N. alternatus), detritivores, pollinators, arachnids, parasitoids, pollinators, and miscellaneous insects by plot.

3. Results

3.1. Arthropod community sampling

For the arthropod community sampling using a D-vac suction sampler, 10 different insect orders were found along with three other arthropod orders, i.e., Order Araneae, Opiliones, and

Collembola (Table 2). Eleven different families of pests or possible prey items were found including aphids and CPB, and more than twelve families of predators were also found in these plots including G. bullatus and N. alternatus. All of arthropods were divided into functional groups that included herbivores, detritivores, predators, pollinators, arachnids, parasitoids, and miscellaneous insects for those species with an unknown diet or not fitting into the other categories. First, the functional group data were summed for all of the sampling dates for each

year. Next, the functional data were broken down into treatments by sampling date for each year.

Arthropods that consume other arthropods (predators, parasitoids, arachnids) were displayed

together (Fig. 2) and herbivorous groups (herbivores, aphids, and Colorado potato beetles) were

displayed together (Fig. 3). The predator group did not include G. bullatus or N. alternatus, and

those two species were analyzed separately.

The predator population changed between sampling months (Fig. 2 a, b) and had the

highest increase in July (F7, 32 = 1.8709, P < 0.0001, all between main effect). Predators were

more commonly collected in 2011(Fig. 2b) than 2010 (Fig. 2a) (F1, 32 = 2.3836, P < 0.0001, year main effect). Predator densities increased in plots that were not sprayed for aphids (F1, 32 =

0.1705, P = 0.0259, main effect aphids). As predator density increased in plots not sprayed for aphids, it decreased in plots not sprayed for CPB in July (F1, 32 = 0.1628, P = 0.0292, CPB x

aphid interaction). The predator population changed over the field season especially in July

where the highest population occurred (F2, 31 = 1.6380, P < 0.0001, time main effect). Predator

densities changed in plots not sprayed for aphids with the peak population in July (F2, 31 =

0.8930, P < 0.0001, time x aphid interaction).

The parasitoid population changed between sampling months (Fig. 2 c, d) especially in

July (F7, 32 = 2.2329, P < 0.0001, all between main effect). Parasitoids were more commonly

collected in 2011(Fig. 2d) than 2010 (Fig. 2c) (F1, 32 = 1.3073, P < 0.0001, year main effect).

Parasitoid densities increased in plots that were not sprayed for aphids with a population peak in

July (F1, 32 = 0.5170, P = 0.0003, main effect aphids). Parasitoid densities changed by experiment year with the more found in 2011 than 2010 in plots that were not sprayed for aphids (F1, 32 =

0.2545, P = 0.0075, year x aphid interaction). Parasitoid densities changed over the field season

with an increase in July and decrease in August (F2, 31 = 5.9917, P < 0.0001, time main effect).

The parasitoid population increased in July for both years with a higher population in 2011 (F2, 31

= 1.8050, P < 0.0001, time x year interaction). Parasitoid densities increased in July and

decreased in August in plots not sprayed for aphids (F2, 31 = 0.6074, P = 0.0006, time x aphid

interaction). Parasitoid densities changes by year over time and in plots not sprayed for aphids,

increased by year from 2010 to 2011 and highest in July for both years (F2, 31 = 0.4908, P =

0.0021, time x year x aphid interaction).

Arachnid population changed between sampling months (Fig. 2 e, f) with increases in

July and August (F7, 32 = 0.6401, P = 0.0174, all between main effect). Arachnids were more

commonly collected in 2011 (Fig. 2f) than 2010 (Fig. 2e) (F1, 32 = 0.5152, P = 0.0003, year main

effect). The arachnid population increased over the field season, higher densities in July

andAugust (F2, 31 = 2.1010, P < 0.0001, time main effect). The arachnid population increased

over the field season and by year (F2, 31 = 0.3088, P = 0.0154, time x year interaction). Arachnid densities increased in plots not sprayed for CPB over time for each year (F2, 31 = 0.2301, P =

0.0403, time x year x CPB interaction).

The herbivore population changed between sampling months with an increase in July for

both years (Fig. 3 a, b) and a decrease in August for 2011 (F7, 32 = 1.3481, P = 0.0001, all

between main effect). Herbivores were more commonly collected in 2011 (Fig. 3b) than 2010

(Fig. 3a) (F1, 32 = 0.9063, P < 0.0001, year main effect). Herbivore densities increased in plots

that were not sprayed for aphids (F1, 32 = 0.2222, P = 0.0119, main effect aphids). The herbivore

population changed over the field season with high densities in July decreasing in August (F2, 31

= 2.2151, P < 0.0001, time main effect). The herbivores collected changed by sampling date with

an increase in July and differences between the two years (F2, 31 = 0.3582, P = 0.0087, time x

year interaction). Herbivore densities changed in plots not sprayed for aphids by sampling date

(F2, 31 = 0.2269, P = 0.0420, time x aphid interaction).

Aphid population changed (Fig. 3 c, d) between sampling months with an increase in July

(F7, 32 = 1.7997, P < 0.0001, all between main effect). Aphids were more commonly collected in

2011 (Fig. 3d) than 2010 (Fig. 3c) (F1, 32 = 0.6049, P < 0.0001, year main effect). Aphid densities

increased in plots that were not sprayed for aphids (F1, 32 = 0.8380, P < 0.0001, main effect aphids). Aphid densities changed by year with more found in 2011 than 2010 in plots not sprayed for aphids (F1, 32 = 0.3668, P = 0.0017, year x aphid interaction). Aphid densities

increased in July and then decreased in August for 2010 and 2011 (F2, 31 = 1.7441, P < 0.0001,

time main effect). The aphids collected changed by sampling date and increased by year (F2, 31 =

0.2903, P = 0.0193, time x year interaction). Aphid densities increased in plots not sprayed for

aphids in July and decreased in August (F2, 31 = 0.8930, P < 0.0001, time x aphid interaction).

Aphid densities generally increased by year, over time and in plots not sprayed for aphids (F2, 31

= 0.4958, P = 0.0024, time x year x aphid interaction).

Colorado potato beetle population changed (Fig. 3 e, f) between sampling months (F7, 32 =

0.5890, P = 0.0260, all between main effect). Colorado potato beetles were more commonly

collected in 2010 (Fig. 3e) than 2011(Fig. 3f) (F1, 32 = 0.3650, P = 0.0017, year main effect).

Colorado potato beetle density increased over the sampling season especially in August (F1, 32 =

0.5796, P = 0.0008, main effect time). Colorado potato beetle density increased by sampling date

and decreased in 2011 (F1, 32 = 0.3603, P = 0.0085, time x year interaction).

The two most abundant predators (G. bullatus and N. alternatus) were in separate

categories from the other predators in order to compare their densities in different treatments

(Fig. 4). The G. bullatus population increased between sampling months (F7, 32 = 1.1451, P =

0.0005, all between main effect). G. bullatus were more commonly collected in 2011(Fig. 4b)

than 2010 (Fig. 4a) (F1, 32 = 0.6230, P < 0.0001, year main effect). Geocoris bullatus densities

increased in plots that were not sprayed for aphids (F1, 32 = 0.4393, P = 0.0007, main effect

aphids). Geocoris bullatus densities generally increased over the field season (F2, 31 = 3.3993, P <

0.0001, time main effect). Geocoris bullatus densities increased by sampling date and year (F2, 31

= 0.4489, P = 0.0032, time x year interaction). (F2, 31 = 0.3803, P = 0.0068, time x aphid

interaction).

The N. alternatus population increased between sampling months (F7, 32 = 2.5492, P <

0.0001, all between main effect). Nabis alternatus were more commonly collected in 2011 (Fig.

4d) than 2010 (Fig.4c) (F1, 32 = 1.3111, P < 0.0001, year main effect). Nabis alternatus densities

increased more in plots that were not sprayed for aphids (F1, 32 = 0.1511, P = 0.0015, main effect

aphids) but did also increase in plots not sprayed for CPB (F1, 32 = 0.1511, P = 0.0352, main

effect CPB). Nabis alternatus densities changed by experiment year with more found in 2011 than 2010 in plots not sprayed for aphids (F1, 32 = 0.1298, P = 0.0499, year x aphid interaction).

The N. alternatus population increased in all treatments with higher numbers in 2011, but more

N. alternatus were found in plots not sprayed for aphids (F1, 32 = 0.1821, P = 0.0217, year x CPB

x aphid interaction). Nabis alternatus densities increased over the field season (F2, 31 = 1.4406, P

< 0.0001, time main effect). Nabis alternatus densities changed by sampling date and year (F2, 31

= 0.2910, P = 0.0191, time x year interaction). Location of N. alternatus in plots not sprayed for aphids increased over the season (F2, 31 = 0.8588, P < 0.0001, time x aphid interaction). Nabis

alternatus were found in plots not sprayed for aphids more often over time and in 2011 (F2, 31 =

0.5006, P = 0.0019, time x year x aphid interaction).

Insects with other diets included detritivores, pollinators and unknown feeding habits

(miscellaneous insects) were displayed together (Fig. 5). Detritivores were more commonly collected in 2011 (Fig. 5b) than 2010 (Fig. 5a) (F1, 32 = 0.3681, P = 0.0017, year main effect).

The pollinator population changed between sampling months (F7, 32 = 1.3526, P = 0.0001, all

between main effect). Pollinators were more commonly collected in 2011 (Fig. 5d) than 2010

(Fig. 5c) (F1, 32 = 0.8706, P < 0.0001, year main effect). More pollinators were collected in plots

not sprayed for CPB in 2011 than 2010 (F1, 32 = 0.1338, P = 0.0467, year x CPB interaction). The

pollinator population increased over the sampling season for 2011 and peaked in July for 2010

(F1, 32 = 0.6607, P = 0.0004, main effect time).

Insects in the miscellaneous group had an unknown diet or varied diet. The miscellaneous

insect population changed between sampling months peak in July for 2011 and August for 2010

(F7, 32 = 1.0002, P = 0.0012, all between main effect). Miscellaneous insects were more

commonly collected in 2011 (Fig. 5f) than 2011(Fig. 5e) (F1, 32 = 0.8986, P < 0.0001, year main

effect). The miscellaneous insect density changed over the sampling season increased in July and

decreased in August for 2011 and increased over time for 2010 (F1, 32 = 0.5921, P = 0.0007, main effect time). The miscellaneous insect group changed by sampling date and year with an increase in August for 2010 and increase in July for 2011 (F1, 32 = 0.8724, P < 0.0001, time x year

interaction).

3.2. Visual observations of aphids and CPB

Both aphids and CPB were counted in plots to see where more of each target pest was

found. The aphid population changed between sampling months (Fig. 6) with an increase in July and decrease in August (F7, 32 = 2.9291, P < 0.0001, all between main effect). Aphids were more

commonly observed in 2011(Fig. 6b) than 2010 (Fig. 6a) (F1, 32 = 1.4311, P < 0.0001, year main

effect). Aphid densities increased in plots that were not sprayed for aphids (F1, 32 = 0.9625, P <

0.0001, main effect aphids). Aphid densities changed by experiment year with more observed in

2011 than 2010 in plots not sprayed for aphids (F1, 32 = 0.5116, P = 0.0003, year x aphid

interaction). Aphid densities changed over the field season with an increase in July and decrease

in August (F2, 31 = 1.7506, P < 0.0001, time main effect). The aphids observed changed by

sampling date and year with increase in July and higher number in 2011 than 2010 (F2, 31 =

1.3106, P < 0.0001, time x year interaction). Aphid densities changed in plots not sprayed for

aphids by sampling date with increase in July and decrease in August (F2, 31 = 0.7677, P =

0.0001, time x aphid interaction). Colorado potato beetle population decreased in July and

increased in August (F7, 32 = 1.1929, P = 0.0003, all between main effect). Colorado potato

beetles were more commonly observed in 2010 (Fig. 7a) than 2011(Fig. 7b) (F1, 32 = 0.4109, P =

0.0010, year main effect). Colorado potato beetle densities increased in plots that were not

sprayed for CPB (F1, 32 = 0.2343, P = 0.0100, main effect CPB). Colorado potato beetles density

was higher in plots not sprayed for CPB and lower in plots sprayed for CPB for both years, and

the overall CPB population was much higher in 2010 than 2011 (F1, 32 = 0.1592, P = 0.0310, year

x CPB x aphid interaction).

3.3. Molecular gut content analysis of two generalist predators

The number of G. bullatus found positive for aphids changed by sampling date and year

with a decrease over time for 2010 (Fig. 8a) and 2011 (Fig. 8b) (F2, 31 = 0.3334, P = 0.0116, time

x year interaction). Geocoris bullatus were detected for aphids in plots not sprayed for aphids

with a decrease slightly over time and slight increase by year (F2, 31 = 0.2282, P = 0.0414, time x year x aphid interaction). The G. bullatus detectability of CPB DNA changed between sampling months (F7, 32 = 1.1105, P < 0.0006, all between main effect). More G. bullatus were detected for

CPB in 2010 (Fig. 8c) than 2011 (Fig. 8d) (F1, 32 = 0.9978, P < 0.0001, year main effect).

Colorado potato beetle DNA in G. bullatus was higher in June and August (F2, 31 = 2.0938, P <

0.0001, time main effect). Geocoris bullatus were found in plots not sprayed for CPB and

increased over time and by year (F2, 31 = 0.3259, P = 0.0126, time x year x CPB interaction)

Geocoris bullatus were positive for CPB in plots not sprayed for aphids and increased over time

and by year, increased as aphid population increased over time and for 2010 (F2, 31 = 0.2152, P =

0.0487, time x year x aphid interaction).

Nabis alternatus detectable for aphid DNA decreased over time in 2011(Fig. 9b) and

peaked in July for 2010 (Fig. 9a) (F2, 31 = 0.4455, P = 0.0033, time main effect). Nabis alternatus

detectable for aphids increased by sampling date for 2010 and decreased for 2011 (F2, 31 =

0.6506, P = 0.0004, time x year interaction).The N. alternatus detected for CPB increased

between sampling months (F7, 32 = 0.6977, P < 0.0112, all between main effect). Nabis alternatus

were more often detected for CPB DNA in 2010 (Fig. 9c) than 2011 (Fig. 9d), which follows the

higher CPB population in 2010 (F1, 32 = 0.5905, P < 0.0001, year main effect). Nabis alternatus

detectable for CPB DNA increased over the field season (F2, 31 = 0.8851, P < 0.0001, time main

effect). Nabis alternatus detectable for CPB increased by sampling date for both years and

decreased in 2011 compared to 2010 (F2, 31 = 0.3238, P = 0.0129, time x year interaction). In

plots not sprayed for aphids, N. alternatus was detected for G. bullatus DNA decreased over time

for 2010 (Fig. 10a) and slightly increased over time for 2011(Fig. 10b) (F2, 31 = 0.2641, P =

0.0265, time x year x aphid).

4. Discussion

Aphids and potato beetles were reduced by the insecticides that targeted them, and both

G. bullatus and N. alternatus reached lower densities in plots where aphids were reduced. The

systemic insecticide flonicamid inhibits the feeding activities of piercing-sucking insects like aphids and whiteflies (Morita et al. 2007, Cloyd 2012). After they insert their stylets into plant tissue, the chemical interferes with the neural regulation of fluid intake through the mouthparts causing the insects to starve (Cloyd 2012). Indeed, we saw significantly lower aphid densities in

plots sprayed with flonicamid (Fig. 6). The insecticidal activity of Bacillus thuringiensis is

mainly due to the Cry and Cyt proteins, and the Cry protein strain tenebrionis is toxic only to

Coleoptera including the Colorado potato beetle (Federici and Bauer 1998). The Cry protein or

endotoxin proteins of the spore-forming bacterium of B. thuringiensis must be ingested, and the

activated toxin molecule binds to glycoprotein receptors on the midgut epithelium membrane

forming pores or lesions that lead to cell lysis and damage the midgut-hemocoel barrier causing insect death (Federici and Bauer 1998). As expected, CPB densities were significantly reduced in plots treated with our Bt sprays (Fig. 7). We found no evidence that potato beetle densities were reduced in plots sprayed with flonicamid, or that aphids were reduced in plots sprayed with Bt, further supporting the selectivity of these two chemicals.

In a laboratory study testing both flonicamid and pymetrozine, development time,

fertility, and parasitism were not negatively affected for a variety of natural enemies including

the hoverfly Episyrphus balteatus, the carabid beetle Bembidion lampros, the parasitoid Aphidius

rhopalosiphi, the ladybird beetle Adalia bipunctata or the rove beetle Aleochara bilineata

(Cloyd 2012). Therefore, we did not anticipate any negative effects of the chemicals on any

insects beyond those that feed on plants using piercing-sucking mouthparts. Of course, both of

our focal predators, G. bullatus and N. alternatus, while primarily predatory, also do some plant

feeding using their piercing-sucking mouthparts (Koss et al. 2004, Tamaki and Weeks 1972a, b).

Thus it was perhaps not surprising that densities of both of these predators were reduced in plots

sprayed with flonicamid (Figure 4). The predator, parasitoid, herbivore, and pollinator groups

were also possibly impacted by flonicamid and had larger populations plots not sprayed aphids

and control plots not sprayed for aphids or CPB. Neither insecticide had an effect on arachnids,

detritivores, and miscellaneous insect groups for either year sampled. The insecticide we used to

reduce potato beetle densities is more selective than flonicamid, impacting only beetles within a

narrow taxonomic range (Federici and Bauer 1998). This is consistent with the lack of non-target

effects across any group of arthropods other than potato beetles (Figure 2, 3, 5), in plots

receiving Bt sprays.

Despite the clear manipulation of prey by both selective insecticides, there were no

significant changes in predation patterns for G. bullatus (Fig. 8) or N. alternatus (Fig. 9, 10). One

possible explanation for this negative result could be the consumption of alternative (non-target) prey items by predators instead of the target pest insect, and the densities of prey other than aphids or Colorado potato beetle were driving the two predators’ hunting behavior.

Another possibility is that the buffer plots in between each experimental plot allowed the predators to freely move among plots, such that individuals were harvesting prey across plots differing in pest-species densities. In an earlier study, we found that aphid and potato beetle prey are generally detectable within predators for less than 24 hours, and frequent movement would be needed to cause no distinct feeding patterns among plots. Predators may maintain relatively inflexible feeding preferences even when background densities of particular prey vary. Indeed, predators sometimes seek nutritional balance through prey mixing (Symondson et al. 2002), and there may be benefits to sticking to a particular, balanced mix of prey. The results presented here in our open-field study contrast sharply with the results of two previous studies where predator movement and prey community composition was constrained within laboratory or field cages

(Koss et al. 2004, Koss and Snyder 2005). Those earlier feeding trials with Nabis spp. and

Geocoris spp. in simple laboratory arenas showed that the presence of aphids reduced their predation rate on Colorado potato beetles, and predators do no reduce their predation rate on aphids when potato beetle eggs fill the role of “alternative prey” (Koss et al. 2004, Koss and

Snyder 2005).

Intraguild predation did occur with N. alternatus consuming G. bullatus, and intraguild predation was highest in June for both years. The insect population was much lower in June than the rest of the summer, so predators may have encountered each other more frequently than prey items. In July and August, a wide variety of prey choices were available to predators in our study including aphids, leafhoppers, Collembola, thrips, CPB, aphids, fungus gnats, vinegar flies, stink bugs, psyllids, whiteflies, seed bugs and stilt bugs. This diversity of prey items may have changed the predators’ diet because generalist predators will sometimes switch to attacking more abundant prey items (Symondson et al. 2002). If we had tested predators for more prey species, t we might have seen an effect of prey other than aphids and Colorado potato beetles on intraguild predation patterns. Intraguild predation may also be important in this predator community: in simple laboratory arenas, N. alternatus (damsel bug) and G. bullatus (big-eyed bug) will feed on each other depending on which predator is in the larger developmental stage (Raymond 2000). In field-cage studies in Washington potato crops, results sometimes suggest the common occurrence of intraguild predation between Nabis and Geocoris (e.g., Koss and Snyder 2005,

Straub and Snyder 2006) whereas others find no evidence for predation among these predators

(e.g., Straub and Snyder 2008). Intraguild predation sometimes reduces a predator’s effectiveness in suppressing pest insects. For example, Mallampalli and colleagues (2002) showed that intraguild predation between the spined soldier bug, Podisus maculiventris Say

(Heteroptera: Pentatomidae), and the twelve-spotted ladybeetle, Coleomegilla maculata Lengi

(Coleoptera: Coccinellidae) reduced overall predation on Colorado potato beetle eggs. A study with Cecidomyiidae, Chrysopidae, and Coccinellidae predators and potato aphids as prey showed that the presence of extraguild prey (aphids) reduced intraguild predation, and intraguild predation decreased as extraguild prey density increased (Lucas et al. 1998). This effect was only

strong at high or low aphid densities (Lucas et al. 1998), and our experiment also had more N.

alternatus detected positive for G. bullatus at the beginning of the season when the prey

population was low.

For many of the functional groups, the highest arthropod population was in the aphid

only plots, which were sprayed with Bacillus thuringiensis tenebrionis. The results showed that

this insecticide was specific to CPB and did not reduce the population of other natural enemies or

pest species. Our study found similar predatory insect and spider species as those reported by

Koss and colleagues (2004), who worked previously in the same region of Washington State.

The dominant predatory hemipteran species were G. bullatus and N. alternatus in our arthropod- community collection. Other generalist predators found in our experimental field were ground beetles, assassin bugs, minute pirate bugs, green and brown lacewings, ladybeetles, dance flies, and spiders. Most of these arthropods were also recorded in a previous study (Koss et al. 2004).

Regardless of the mechanism, the use of flonicamid reduced the generalist predator population and possible biological control of our target pest insects. Flonicamid is a systemic insecticide that causes inhibition of salivation and sap feeding after the insect starts feeding (Morita et al.

2007), and both of the predator species used in this study will feed on plant fluids, when other prey items are unavailable and could be killed from feeding on the plants sprayed with flonicamid. Bacterial insecticides like B. thuringiensis tenebrionis are considered by many entomologist and growers to be selective and can be used to combine chemical and biological control (Federici and Bauer 1998). Many selective insecticides including insect growth regulators, selective feeding blockers, and microbials are often considered safe for natural enemies, but even these insecticides can have long term indirect effects on natural enemies

(Cloyd 2012).

Despite the change in pest community structure by manipulation of CPB and GPA, there was no distinct predation pattern or large change in predation rates for consumption of the target prey items. A field cage study showed that potato beetle suppression by a complex of generalist predators was reduced when aphids were also present (e.g., Koss and Snyder 2005), but this did not occur in our study most likely because of other available prey species. The higher insect population did slightly decrease intraguild predation from June to later in the season, but we expected to see a greater drop in intraguild predation as the prey population increased. Geocoris bullatus may be easy for N. alternatus to catch and consume or may be more nutritious than other prey, which is why they were found detectable for G. bullatus throughout the field season for both years of the study. Predators found in plots with fewer Colorado potato beetles and green peach aphids nonetheless commonly consumed these prey items. This suggests that the predators search out both species of prey even when rare, perhaps because they provide a unique nutritional contribution to the predators’ diets. However, these changes in predator and prey community structure did not translate into significant effects on either predator’s likelihood of having eaten aphids or potato beetles, or of N. alternatus eating G. bullatus. This suggests either that the predators’ diets were relatively unaffected by prey availability, or that predators mixed their diets by moving freely among our plots. Our study suggests that, through movement or innate preferences, the diets of generalist predators may remain surprisingly rigid in the face of a mosaic of prey densities.

Acknowledgments

We thank D. W. Crowder and E. Moon for his help with our data analysis. Ben Hieb, Glen

Chase, Jake Gable, Elliott Moon, Courtney Malcom, and Tom Walter provided assistance with field sampling and predator collection. Bailey Cox, Kerri Wheeler and Abbie Estep helped sort arthropods from insect community samples. This project was supported by a USDA AFRI grant.

References

Bailey, K. H., and G. A. Polis. 1987. An experimental analysis of optimal and central place

foraging by the harvester ants, Pogonomyrmex californicus. Oecologia 72: 440–448.

Biever, K.D. and R. L. Chauvin. 1992. Timing of infestation by the Colorado potato beetle

(Coleoptera: Chrysomelidae) on the suppressive effect on field released stinkbugs

(Hemiptera: Pentatomidae) in Washington. Environ. Entomol. 21: 1212–1219.

Blackman, R.L. and V. F. Eastop. 2000. Aphids on the world's crops, an identification and

information guide. Wiley, New York.

Chapman, E. G., Romero, S. A., and J. D. Harwood. 2010. Maximizing collection and

minimizing risk: does vacuum suction sampling increase the likelihood for

misinterpretation of food web connections? Molecular Ecology Resources. 10 (6): 1023-

1033.

Cloyd, R. 2012. Indirect Effects of Pesticides on Natural Enemies, Pesticides - Advances

in Chemical and Botanical Pesticides, Dr. R.P. Soundararajan (Ed.), ISBN: 978-953-51-

0680-7, InTech, DOI: 10.5772/48649. Available from: http://www.intechopen.com/

books/pesticides-advances-in-chemical-and-botanical-pesticides/indirect-effects-of-

pesticides-on-natural-enemies

Federici, B. A. and L. S. Bauer. 1998. Cyt1Aa protein of Bacillus thuringiensis is toxic to

the cottonwood leaf beetle, Chrysomela scripta, and suppresses high levels of resistance

to Cry3Aa. Appl Environ Microbiol. 64(11): 4368–4371.

Greenstone, M. H., Rowley.D. L., Weber, D. C., Payton, M. E., and D. J. Hawthorne. 2007.

Feeding mode and prey detectability half-lives in molecular gut-content analysis: an

example with two predators of Colorado potato beetle. Bulletin Entomological Research.

97: 201-209.

Harwood, J. D., H. J. S. Yoo, M. H. Greenstone, D. L. Rowley, and R. J. O’Neil. 2009.

Differential impact of adults and nymphs of a generalist predator on an exotic invasive

pest demonstrated by molecular gut-content analysis. 11(4): 895-903.

JMP, Version 10. SAS Institute Inc., Cary, NC, 1989-2012.

Koss, A.M., Chang, G.C., and W. E. Snyder. 2004. Predation of green peach aphids by generalist

predators in the presence of alternative, Colorado potato beetle egg prey. Biol. Control.

31: 237-244.

Koss, A. M. and W. E. Snyder. 2005. Alternative prey disrupt biocontrol by a guild of generalist

predators. Biological Control. 32: 243-251.

Landis, D. A., S. D. Wratten, and G. M. Gurr. 2000. Habitat management to conserve natural

enemies of arthropod pests in agriculture. Annu. Rev. Entomol. 45: 175-201.

Lucas, E., Coderre, D., and J. Brodeur. 1998. Intraguild predation among aphid predators:

characterization and influence of extraguild prey density. Ecol.79(3): 1084-1092.

Lundgren, J. G., Ellsbury, M. E., and D. A. Prischmann. 2009. Analysis of the predator

community of a subterranean herbivorous insect based on polymerase chain reaction.

Ecological Applications. 19: 2157–2166.

Mallampalli, N., Castellanos, I., and P. Barbosa. 2002. Evidence for intraguild predation by

Podisus maculiventris on a ladybeetle, Coleomegilla maculata: Implications for

biological control of Colorado potato beetle, Leptinotarsa decemlineata. Biocontrol. 47:

387-398.

Morita, M., T. Ueda, T. Yoneda, T. Koyanagi, and T. Haga. 2007. Flonicamid, a novel

insecticide with a rapid inhibitory effect on aphid feeding. Pest Manag Sci. 2007. 63(10):

969-973.

Mowry, T.M. 2001. Green peach aphid (Homoptera: Aphididae) action thresholds for controlling

the spread of potato leafroll virus in Idaho. J. Econ. Entomol. 94: 1332–1339.

Polis, G. A., C. A. Myers, and R. D. Holt. 1989. The ecology and evolution of intraguild

predation: potential competitors that eat each other. Annual Review of Ecology and

Systematics. 20: 297-330.

Prasad, R. P. and W. E. Snyder. 2006. Polyphagy complicates conservation biological control

that targets generalist predators. Journal of Applied Ecology. 43: 343-352.

Radcliffe, E. B. 1982. Insect pests of potato. Annu. Rev. Entomol. 27: 173-204.

Raymond, B. 2000. Cannibalism and intraguild predation involving three predatory Heteroptera,

Nabis roseipennis, Geocoris punctipes and Orius insidiosus. Dissertation. University of

Kentucky, Lexington, Kentucky, USA.

Rosenheim, J. A.. 1998. Higher-order predators and the regulation of insect herbivore

populations. Annual Review of Entomology. 43:421-447.

Settle, W.H., Ariawan, H., Astuti, E.T., Cahyana, W., Hakim, A.L., Hindayana, D., Lestari, A.S.,

and S. Pajarningsih. 1996. Managing tropical rice pests through conservation of

generalist natural enemies and alternative prey. Ecology. 77: 1975–1988.

Sih, A., Englund, G., and D. Wooster. 1998. Emergent impacts of multiple predators on prey.

TREE. 13(9): 350-355.

Snyder, W. E. and A. R. Ives. 2008. Population dynamics and species interactions. In: Integrated

Pest Management Concepts, Tactics, Strategies and Case Studies (Eds. E. B. Radcliffe,

W. D. Hutchison and R. E. Cancelado). Cambridge University Press, Cambridge, UK.

Pp. 62-74.

Snyder, W. E. and A. R. Ives. 2001. Generalist predators disrupt biological control by a specialist

parasitoid. Ecology. 82(3): 705–716.

Straub, C. S. and W. E. Snyder. 2006. Species identity dominates the relationship between

predator biodiversity and herbivore suppression. Ecology. 87(2): 277-282.

Straub, C. S. and W. E. Snyder. 2008. Increasing enemy biodiversity strengthens herbivore

suppression on two plant species. Ecology 89: 1605-1615.

Symondson, W. O. C., K. D. Sunderland, and M. H. Greenstone. 2002. Can generalist predators

be effective biocontrol agents? Annu. Rev. Entomol. 47: 561–594.

Tamaki, G. and R. E. Weeks. 1972a. Efficiency of three predators, Geocoris bullatus, Nabis

americoferus, and Coccinella transversoguttata, used alone or in combination against

three insect prey species, Myzus persiace, Ceramica picata, and Mamestra configurata,

in a greenhouse study. Environ. Entomol. 1: 258–263.

Tamaki, G. and R. E. Weeks. 1972b. Biology and ecology of two predators, Geocoris pallens

Stål and G. bullatus (Say). Technical Bulletin 1446, US Department of Agriculture.

Figure legends

Fig. 1. maps of field plot experimental design arrangement for 4 treatments (A = aphids present,

ACPB = aphids and Colorado potato beetle, CPB = CPB present, and O = neither species) in (a)

2010 and (b) 2011. Plots were randomized each year using a random number generator.

Fig. 2. Average number of three different types of natural enemies. Predatory insects (a, b), arachnids (c, d), and parasitoids (e, f) found during Dvac suction sampling in the four treatments

(A = aphids present, ACPB = aphids and Colorado potato beetle, CPB = CPB present, and O = neither species) in 2010 (a, c, e) and 2011 (b, d, f).

Fig. 3. Average number of herbivores other than aphids and Colorado potato beetles (a, b), aphids (c, d), and Colorado potato beetles (e, f) found during Dvac suction sampling in the four treatments (A = aphids present, ACPB = aphids and Colorado potato beetle, CPB = CPB present, and O = neither species) in 2010 (a, c, e) and 2011 (b, d, f).

Fig. 4. Average number of the predators: Geocoris bullatus (a, b) and Nabis alternatus (c, d)

found during Dvac suction sampling in the four treatments (A = aphids present, ACPB = aphids

and Colorado potato beetle, CPB = CPB present, and O = neither species) in 2010 (a, c) and

2011 (b, d).

Fig. 5. Average number of detritivores (a, b), pollinators (c, d), and miscellaneous insects (e, f)

found during Dvac suction sampling in the four treatments (A = aphids present, ACPB = aphids

and Colorado potato beetle, CPB = CPB present, and O = neither species) in 2010 (a, c, e) and

2011 (b, d, f).

Fig. 6. Average number of aphids found during visual counts in the four treatments (A = aphids

present, ACPB = aphids and Colorado potato beetle, CPB = CPB present, and O = neither

species) for (a) 2010 and (b) 2011.

Fig.7. Average number of Leptinotarsa decemlineata (Colorado potato beetle or CPB) found

during visual counts in the four treatments (A = aphids present, ACPB = aphids and Colorado

potato beetle, CPB = CPB present, and O = neither species) for (a) 2010 and (b) 2011.

Fig. 8. Proportion of Geocoris bullatus positive for consumption of (a, c) aphids and (b, d)

Colorado potato beetles in the four treatments (A = aphids present, ACPB = aphids and Colorado

potato beetle, CPB = CPB present, and O = neither species) in (a, b) 2010 and (c, d) 2011.

Fig. 9. Proportion of Nabis alternatus positive for consumption of (a, c) aphids and (b, d)

Colorado potato beetles in the four treatments (A = aphids present, ACPB = aphids and Colorado

potato beetle, CPB = CPB present, and O = neither species) in (a, b) 2010 and (c, d) 2011.

Fig. 10. Proportion of Nabis alternatus positive for consumption of Geocoris bullatus in the four treatments (A = aphids present, ACPB = aphids and Colorado potato beetle, CPB = CPB present, and O = neither species) in (a) 2010 and (b) 2011.

Fig. 1. a) 2010

P1 = P2 = P3 = P4 = P5 =

A O ACPB A CPB

P6 = P7 = P8 = P9 = P10 =

CPB A CPB O ACPB

P11 = P12 = P13 = P14 = P15 =

O ACPB A O CPB

P16 = P17 = P18 = P19 = P20 =

ACPB A CPB ACPB O

A = only aphids, ACPB = aphids and CPB, CPB = only CPB, and O = neither CPB or aphids

b) 2011

P1 = P2 = P3 = P4 = P5 =

O ACPB CPB A ACPB

P6 = P7 = P9 = P10 = P8 = A A ACPB CPB O

P11 = P12 = P13 = P14 = P15 =

CPB O ACPB CPB A

P16 = P17 = P18 = P19 = P20 =

A CPB O ACPB O

A = only aphids, ACPB = aphids and CPB, CPB = only CPB, and O = neither CPB or aphids

Fig. 2

a) Predators 2010 b) Predators 2011 20 30 A A ACPB ACPB 25 CPB 15 CPB O O 20

10 15

10 5 5 Averageof# predators found

0 0

c) Parasitoids 2010 d) Parasitoids 2011 20 60 A A ACPB 50 ACPB 15 CPB O CPB 40 O

10 30

20 5 10 Average # of parasitoids found 0 0

e) Arachnids 2010 f) Arachnids 2011 4 12 A A ACPB 10 ACPB 3 CPB CPB O 8 O

2 6

4 1 2 Averageof# arachnids found

0 0 June July August June July August Sampling month Sampling month

Fig. 3

b) Herbivores 2011 a) Herbivores 2010 80 250 A A ACPB ACPB 200 CPB 60 CPB O O 150 40 100

20 50 Averageof# herbivores found 0 0

c) Aphids 2010 d) Aphids 2011 30 60 A A 25 ACPB 50 ACPB CPB CPB 20 O 40 O

15 30

10 20

5 10 Average # of aphids found found aphids of # Average

0 0

c) CPB 2010 f) CPB 2011 8 2.0 A A ACPB ACPB 6 CPB 1.5 CPB O O

4 1.0

2 0.5 Averageof# CPB found

0 0.0 June July August June July August Sampling month Sampling month

Fig. 4

a) Geocoris spp. 2010 b) Geocoris spp. 2011 40 50 A A ACPB ACPB

found found 40 found found 30 CPB CPB O O 30 Geocoris 20 Geocoris 20

10 10 Average # of Averageof#

0 0

c) Nabis spp. 2010 d) Nabis spp. 2011 6 20 A A 5 ACPB ACPB 16 CPB

found found CPB 4 O found O 12 Nabis

3 Nabis 8 2

4 Averageof# 1 Averageof#

0 0 June July August June July August Sampling month Sampling month

Fig. 5

a) Detritivores 2010 b) Detritivores 2011 50 160 A A ACPB ACPB 40 CPB CPB 120 O O 30 80 20

40 10 Averageof# detritivores found 0 0

c) Pollinators 2010 d) Pollinators 2011 2.0 4 A A ACPB ACPB 1.5 CPB 3 CPB O O

1.0 2

0.5 1 Average # of pollinators found pollinators of # Average 0.0 0

e) Miscellaneous insects f) Miscellaneous insects 2011 7 20 A A 6 ACPB ACPB CPB CPB 15 5 O O

4 10 3

2 5

Averageof# misc. insects 1

0 Averageof# misc. insects found 0 June July August June July August Sampling month Sampling month

Fig. 6

a) Aphid visual counts 2010 4 A ACPB 3 CPB O

1 Average # of aphids found found aphids of # Average

b) Aphid visuals counts 2011 12 A 10 ACPB CPB O 8

2 Average # of aphids found aphids of # Average

0 June July August Sampling month

Fig. 7

a) CPB visual counts 2010

A 60 ACPB CPB O 40

Averageof# CPB found

b) CPB visual counts 2011 40 A ACPB CPB 30 O

10 Averageof# CPB found

0 June July August Sampling months

Fig. 8

a) Geocoris positive for aphids 2010 b) Geocoris positive for aphids 2011 0.4 0.5 A A ACPB ACPB CPB 0.4 0.3 CPB O O

positive for aphids for positive 0.3 0.2 0.2 Geocoris 0.1 0.1

0.0 0.0 Proportion of of Proportion aphids for positive Geocoris of Proportion

c) Geocoris positive for CPB 2010 d) Geocoris positive for CPB 2011 0.5 0.20 A A ACPB ACPB CPB 0.4 CPB 0.15 O O 0.3 0.10 0.2

0.05 0.1

0.0 0.00 Proportion of Geocoris positive for CPB for positive Geocoris of Proportion Proportion of Geocoris positive for CPB for positive Geocoris of Proportion June July August June July August Sampling month Sampling month

Fig. 9

b) Nabis positive for aphids 2011 a) Nabis positive for aphids 2010 0.6 1.0 A A ACPB 0.5 0.8 ACPB CPB CPB O O 0.4 0.6

positive for aphids for positive 0.3 0.4 Nabis 0.2

0.2 0.1

0.0 0.0 Proportion of Nabis positive for aphids for positive Nabis of Proportion Proportion of of Proportion

c) Nabis positive for CPB 2010 d) Nabis positive for CPB 2011 1.0 0.4 A A

ACPB ACPB

0.8 CPB CPB O 0.3 O 0.6 positive for CPB for positive 0.2

Nabis 0.4

0.1 0.2

0.0 0.0 Proportion of of Proportion Proportion of Nabis positive for CPB for positive Nabis of Proportion June July August June July August Sampling month Sampling month

100

Fig. 10

a) Geocoris consumption 2010 0.8 A

Geocoris ACPB 0.6 CPB O

positive for for positive 0.4 Nabis 0.2

0.0

Proportion of of Proportion

b) Geocoris consumption 2011 0.6 A Geocoris 0.5 ACPB CPB 0.4 O

positive for for positive 0.3

Nabis 0.2

0.1

0.0

Proportion of of Proportion June July August Sampling month

101

Table 1. Primers used to detect the three different prey species in predators guts for the field experiment.

Prey species tested Primer

Myzus persicae Aphid-414-F: 5’- GGAATTTCATCAATTTTAGGAGCAA-3’

Aphid-565-R: 5’- ACCAGCTAGAACTGGTAGAGATAAAAT-3’

Leptinotarsa Cpb5s-F: 5’- CCTTTTCTCTTGGGCAGTTAT-3’ decemlineata Cpb6A-R: 5’- TTATCCCAAATCCAGGTAGAAT-3’

Geocoris spp. Geo-294-F: 5’- TATCAAGAAGTATAGTAGAAATAGGAGCT-3’

Geo-449-R: 5’- AAATAAAATTAATAGCTCCAAGAATAGAAC-3’

102

Table 2. Total number of insect and spider species collected from insect community sampling for all twenty plots for 2010 and 2011.

Order Family Genus, Genus species Total # collected 2010 2011 Collembola Entomobryidae 414 1041

Odonata Coenagrionidae 0 1

Ephemeroptera unknown 4 11

Psocoptera unknown 1 1

Hemiptera unknown 7 27 Anthocoridae Orius sp. 43 128 Anthocoridae Orius sp. nymphs 2 65 Berytidae 0 2 Lygaeidae 54 48 Miridae nymphs 45 168 Miridae 5 0 Miridae Lygus sp. 41 156 Nabidae Nabis alternatus 149 177 Nabidae Nabis spp. nymphs 30 122 Geocoridae Geocoris bullatus 552 595 Geocoridae Geocoris sp. 0 3 Geocoridae Geocoris spp. nymphs 487 555 Pentatomidae 0 3 Pentatomidae nymphs 9 1 Reduviidae 0 1 Reduviidae nymph 1 5 Aleyrodidae 456 9 Aphididae 110 229 Aphididae alate 19 27 Aphididae Myzus persicae 176 192 Aphididae Myzus persicae alate 33 179 Cercopidae 4 10 Cicadellidae 218 559 Cicadellidae nymphs 86 Cicadellidae Amblysellus spp. 1 2 Cicadellidae Ballana spp. 2 34 Cicadellidae Circulifer spp. (BLH) 52 19 Cicadellidae Dikraneura spp. 16 39 Cicadellidae Empoasca spp. 90 11 Cicadellidae Ceratagallia spp. 10 8 Cicadellidae Extianus spp. 6 2 Cicadellidae Latalus spp. 9 3 Cicadellidae Macrosteles spp. 22 438 Cicadellidae (unknown) 4 3 Psyllidae 4 4 Triozidae Bactericera cockerelli 3 0

Thysanoptera Aeolothripidae Aeolothrips spp. 21 17 Thripidae 347 763

103

Order Family Genus, Genus species Total # collected 2010 2011 Thysanoptera Thripidae Frankliniella occidentalis 98 1327

Chrysopidae 5 10

Neuroptera Chrysopidae larvae 0 2 Hemerobiidae 6 12

Coleoptera unknown 1 2 Anthicidae 6 12 Carabidae Bembidion spp. 0 1 Chrysomelidae 0 6 Chrysomelidae Leptinotarsa decemlineata 2 2 Chrysomelidae Leptinotarsa decemlineata larvae 138 8 Chrysomelidae Oulema sp. 0 5 Coccinellidae 0 16 Coccinellidae Coccinella septumpunctata 1 10 Coccinellidae Hippodamia trecimpunctata 1 0 Coccinellidae Hippodamia convergens 2 34 Curculionidae 24 75 Elateridae 5 22 Lathridiidae 12 18 Staphylinidae 1 4

Diptera unknown 202 374 Chloropidae Thaumatomyia sp. 70 187 Chloropidae 0 14 Culicidae 2 1 Anthomyzidae 1 59 Agromyzidae 3 84 Dolichopodidae 0 4 Drosophilidae Scaptomyza sp. 257 1040 Empididae 56 154 Ephydridae 351 124 Muscidae 173 127 Mycetophilidae 1 9 Sciaridae 887 585 Tephritidae 0 2 Tipulidae 1 16 Chironomidae 61 149 Phoridae 1 13 Lonchopteridae 1 1 Simuliidae 0 1 Sphaeroceridae 1 5

Lepidoptera unknown 15 52 Lepidoptera larvae unknown 3 12

Hymenoptera unknown 413 685 Braconidae Diaeretiella rapae 74 144 Apidae 0 2

104

Order Family Genus, Genus species Total # collected 2010 2011

Halictidae 0 3 Hymenoptera Formicidae 32 0 Vespidae 2 0

Opiliones unknown 0 2 Araneae unknown 100 172

105

CHAPTER 3: Density of green peach aphid, Myzus persicae, alters behavior of the predators

Geocoris bullatus and Nabis alternatus

Christine A. Lynch and William E. Snyder

Department of Entomology, Washington State University, Pullman, WA 99164, USA

ABSTRACT

Generalist predators may more efficiently hunt prey species that are in greater abundance.

Competition from predators of the same or different species may interfere with location and consumption of prey. This study examined patterns of predation and behavior for two species of insect predator, Nabis alternatus and Geocoris bullatus, when Myzus persicae (GPA) density was manipulated on potato, Solanum tuberosum, plants in greenhouse arenas. We examined three different GPA densities (10, 25, or 50 aphids) and three predator combinations (8 G. bullatus, 8 N. alternatus, or 4 of each species). Both species of predators consumed more GPA at when more were available. Foraging activity was highest when single predator species were present, and it was reduced when both predator species were present together. When foraging in the same arenas, G. bullatus and N. alternatus more frequently observed at the middle of the potato plant. More predator observations occurred at the top of the plant in single species treatments. Cannibalism was observed with N. alternatus consuming N. alternatus as was

106

intraguild predation with N. alternatus consuming G. bullatus. Predator behavior and

consumption was affected by predator species composition and aphid density. The risk of

predation presumably made these predators spread out more and stay away from each other when they occurred in the same arena, and predators could forage together without reducing the overall aphid consumption.

Keywords: Generalist predators; Green peach aphid; Damsel bug; Big-eyed bug: GPA

1. Introduction

Generalist predators feed on multiple prey species (Symondson et al. 2002), and their

densities are not tied to a particular prey species (Harmon and Andow 2004). The available prey

species, prey handling times, and changing field microhabitats might change the species of prey

eaten by any individual predator through the season (Symondson et al. 2002). For example,

generalists may already be present in a field feeding on non-pest prey when the pests first invade;

in this way, generalists can form a “first line of defense” (e.g., Settle et al. 1996). Likewise, by

switching among feeding on different pests as each pest becomes abundant, generalists can

impact many different pest species active at different times of the year (Symondson et al. 2002).

Many predators (including generalist predators) also receive nutrients from plant tissues that

include pollen, seeds, floral nectars, extrafloral nectars and honeydew (Lundgren 2009). There is

a limited understanding of how regulation of pests is affected by intraguild competition,

behavioral interference, predation and cannibalism among predators, interactions between

generalists and specialists, and disruption of parasitoid biological control by generalist predators

(Symondson et al. 2002).

107

Omnivory is perhaps most disruptive to biological control when one predator species

feeds on another (Polis et al. 1989, Rosenheim 1998). For example, Mallampalli and colleagues

(2002) showed that intraguild predation between the spined soldier bug, Podisus maculiventris

Say (Heteroptera: Pentatomidae), and the twelve-spotted ladybeetle, Coleomegilla maculata

Lengi (Coleoptera: Coccinellidae) reduced overall predation on Colorado potato beetle eggs. The outcome of intraguild predation is influenced by the size of the predators and feeding specificity of the predators (Lucas et al. 1998). This mainly occurs with generalist predators including predation within the same species (Polis 1981, Polis et al. 1989), and the smaller individual is more likely to be eaten by larger individual (Werner and Gilliam 1984). Predators sometimes retreat during a predator-predator confrontation (Edmunds 1974, Sih 1987), and more mobile predators have a better chance of escaping (New 1991). Nabis spp. (damsel bug) and Geocoris

spp. (big-eyed bug) will feed on each other depending on which predator is in the larger

developmental stage (Raymond 2000). Some studies have shown that the presence of extraguild

prey can reduce intraguild predation, and intraguild predation decreased as extraguild prey

density increased (Bailey and Polis 1987, Lucas et al. 1998). For example, under controlled

conditions intraguild predation was higher for most combinations of Aphidoletes aphidmyza,

Chrysoperla rufilabris, and Coleomegilla maculata lengi at high levels with no prey present, and

intraguild predation was reduced for the same predator combinations with the addition of aphids

(Lucas et al. 1998).

Myzus persicae (Sulzer), the green peach aphid or GPA, is one of the major pests

affecting potatoes in Washington (Biever and Chauvin 1992a, b), and this aphid species is a

vector for the transmission of potato viruses (Radcliffe 1982). Green peach aphids are capable of vectoring over 100 plant viruses (Blackman and Eastop 2000) including the potato leafroll virus

108

(Radcliffe 1982) and potato virus Y (Novy et al 2002). The predator guild on plant foliage in

Washington State potato fields is dominated by omnivorous generalist hemipteran predators,

primarily the big-eyed bug Geocoris bullatus Stål and the damsel bug Nabis alternatus Parshley

(Koss et al. 2004; Tamaki and Weeks 1972a, b). Previous feeding trials in simple laboratory

arenas have shown that the presence of aphids reduced these predators’ predation rate on

Colorado potato beetles (Koss and Snyder 2005). Likewise, a field cage study showed that potato

beetle suppression by a complex of generalist predators was reduced when aphids were also

present (e.g., Koss and Snyder 2005). Intraguild predation may also be important in this predator

community: in simple laboratory arenas, N. alternatus and G. bullatus will feed on each other depending on which predator is in the larger developmental stage (Raymond 2000). In field-cage

studies in Washington potato crops, results sometimes suggest the common occurrence of

intraguild predation between Nabis and Geocoris (e.g., Koss and Snyder 2005, Straub and

Snyder 2006) whereas others find no evidence for predation among these predators (e.g., Straub

and Snyder 2008).

Prior studies have shown that green peach aphids (GPA) are more attractive to Nabis alternatus and Geocoris bullatus than Colorado potato beetles in different stages (Koss and

Snyder 2005, Koss et al 2004). Therefore, we wanted to investigate possible mechanisms of this

conclusion in a greenhouse experiment. Here we investigated the consumption and behavior of

two generalist predators, G. bullatus and N. alternatus, in mixed and single species predator

assemblages at varying densities of GPA. We conducted regularly-timed observations of

predator foraging behavior, and then recorded changes in aphid density at the end of the

experiment.

109

2. Materials and methods

2.1. Field site and planting methods

Predators, Nabis alternatus and Geocoris bullatus, were collected live using a D-vac suction sampler (B&S Model 24 Ventura, CA), from fields of alfalfa, Medicago sativa, and potatoes, Solanum tuberosum, (Ranger and Burbank russet varieties) planted at the Washington

State University Research Farm near Othello, WA, during the 2012 growing season. Mesh D-vac

bags were brought back in coolers with ice packs from the field and were transferred to a cool

incubator (12 °C) back at the laboratory. Predators were later sorted individually into 50 mm x 9

mm BD Falcon Tight-fit lid Petri dishes (Fisher Scientific) with damp cotton dental wicks and

were fed Acyrthosiphon pisum (Harris), pea aphids, if the predators were not going to be used immediately for an experiment. Predators used in experiments were held without aphids for 24-

48 hours before use.

2.3. Rearing potato plants and aphids

Potato plants for the greenhouse microcosm experiments were grown in the greenhouse at

ambient day length and 22–25 °C. Whole certified organic Ranger russet potato seed (Basin

Gold Cooperative, Warden, WA) was planted in decagonal planting containers (McConkey and

Co; Sumner, WA) filled with Sunshine Professional growing mix potting soil (Sungro

Horticulture; Seba Beach, AB Canada). The containers were put in the greenhouse and were

watered. Containers were watered once a week, and potato plants were broken off of the main

110

tuber around 10.16 cm tall and were transplanted into separate 15.24 cm square pots with WIL-

GRO professional products pro balance slow release granular fertilizer (16-16-16-7S, Wilbur-

Ellis Co.; Halsey, OR) sprinkled on top of the Sunshine Professional growing mix potting soil

(Sungro Horticulture; Seba Beach, AB Canada). Plants were watered as needed (three times a

week) before use in experiments at ca. 3 weeks of age or about 15.24 cm tall.

Green peach aphids for both experiments were from a long-term laboratory colony

originally started from GPA collected on potatoes near Prosser, WA USA. This colony was

maintained under the same conditions and in the same greenhouse as the potato plants, but the

aphid colonies were kept in 60 x 60 x 60 cm fine mesh BugDorms (Megaview Science; Taiwan,

China) in a separate room from the potato plants. A pea aphid, Acyrthosiphon pisum (Harris),

colony was originally started from pea aphids collected on alfalfa at the WSU Research Farm

near Othello, WA, in 2011 was used to feed predators being held until use in feeding trials; pea

aphids were reared on pea plants, Pisum sativum. Potato and pea plants in the aphid colonies

were watered 3 times a week.

2.4. Manipulation of aphid density and predator behavior in greenhouse

Nabis alternatus and G. bullatus feeding behavior was observed in greenhouse

microcosms using three densities of green peach aphid and three predator combinations. Twenty-

seven 37.85 liter (10 gallon) glass aquariums, covered with a wire-mesh lid, served as our

experimental units; one potato plant in a 15.24 cm tall square pot was placed inside each

aquarium. In between the top of the aquarium and the wire-mesh lid was a piece of cotton fabric, which decreased the predators’ escape from the aquarium. The experimental design included

111

three aphid densities (10, 25, or 50 aphids per plant) and three predator species-compositions (8

G. bullatus, 8 N. alternatus, or 4 G. bullatus and 4 N. alternatus). These factors were

manipulated within a fully-crossed design, leading to 9 unique treatment combinations. There

were 3 replicates of each combination within each experimental block, and four blocks of this

experiment were run over the summer of 2012 on 25-29 June, 1-6 July 1-6, 16-20 July, and 13-

17 August. In total across the four blocks, this yielded 12 replicates of each treatment for a total

of 108 experimental units (3 aphid densities x 3 predator communities = 9 predator-aphid

combinations x 3 replicates of each per block x 4 blocks = 108 experimental units). A random

number generator was used to determine the location within the greenhouse for each replicate for

each replicate witnin a block.

Nabis alternatus and G. bullatus were collected using a D-vac suction sampler the week

before starting each block. The 27 potato plants were watered before being added to each tank

the day before the experiment started, and the fabric and lid was placed on top of each tank after

the single potato plant was placed inside each tank. Thirty-six hours before predator release into the arenas, leaves (or pieces of leaves) housing aphids from the aphid rearing colony were selected that contained 10, 25 or 50 aphids, and the leaves were removed with scissors, each leaf

placed in a separate 59.15 mililiter (2 oz) Souffle cup with lid (Dixie; CA USA), were moved to

the experimental greenhouse, and were placed on top of each potato plant in each microcosm as

appropriate by treatment. Fabric was placed over the top opening of the aquarium and the lid was

placed on top of the fabric immediately after releasing the predators in each tank. Two sterile

specimen cups (VWR; Radnor, PA USA) full of water were placed on both horizontal sides of

each cage lid to prevent predators from escaping. Predators were released, and they were

observed at 0, 2, 4, 6, and 8 hours after release into aquariums. Each observation per tank was 2

112

minutes long, and we recorded predators’ location on plants and behavior. (feeding, moving, mating, and location of plant or tank). A final aphid count was done 24 hours after predators were removed to quantify the change in aphid density from the beginning to end of the experiment. Predators were placed individually into 1.5 ml Eppendorf tubes filled with 95%

EtOH.

2.5. Statistics

We examined the relationship between two generalist predators and aphid prey with three different predator combinations and three different aphid densities. This relationship was assessed using Standard Least Squares ANOVA for aphid counts or predator observations followed by a LS means Tukey HSD post hoc test (P < 0.05) to determine differences between treatments in JMP (JMP, Cary, North Carolina, USA). This method was used to analyze the final aphid density counts, predators observed over time, predator location on potato plant, observed predator behaviors (feeding and mating), and observed intraguild predation. The sum of the predator observations for each species was divided by the number of predators of each species present in each treatment by block, which is represented as per capita in the figures.

3. Results

3.1. Predator aphid consumption in greenhouse

113

Green peach aphids (GPA) were consumed by both species of predators for all four

blocks and at all three aphid density levels (Fig. 1). Consumption of GPA differed by block

(F3,107 = 3.3938, P = 0.0224, block main effect) for block 2 (Fig. 1b) had the lowest overall

aphid consumption and block 3 (Fig. 1c) had the highest overall aphid consumption. More aphids

were consumed when more aphids were present (F2, 107 = 286.3282, P < 0.0001, main effect aphid density), which is expected because of different aphid density levels.

3.2. Predators observed and location on plants

Predators were observed for two minutes immediately after placement in each cage then every other hour over an eight hour period. The number of predators observed at each time point changed by species and treatment (Fig. 2). The sum of the predator observations by each time point for each species was divided by the number of predators of each species present in each treatment, which is represented as per capita in the figures. For G. bullatus (Fig. 2a) predators, the number of predators observed changed between observations (F35, 72 = 9.9511, P < 0.0001, between observations main effect) and for predator species, the single species treatment G was higher than treatments with both species (GN) (F2, 72 = 9.4540, P < 0.0001, predator species main effect). The number of predators observed changed over time, decreasing for G. bullatus in the two species treatment and increasing for single species (F4, 69 = 0.8881, P < 0.0001, time main

effect). Over time G. bullatus observed in the treatment with both predator species (GN) decreased and more G. bullatus were observed in the single species (G) treatment (F8, 138 =

0.6346, P < 0.0001, time x predator species interaction).

114

For N. alternatus (Fig. 2b) predators, the number of predators observed changed between

observations (F35, 72 = 16.8304, P < 0.0001, between observations main effect) and for predator

species with the single species treatment showing more observations than two-species treatment

(F2, 72 = 16.5172, P < 0.0001, predator species main effect). The number of predators observed

changed over time, decreasing for N. alternatus in (GN) both species treatment and increasing for single species (F4, 69 = 0.2357, P = 0.0051, time main effect). Over time, the number of N. alternatus observed changed by block (F12, 183 = 0.7059, P = 0.0162, time x block interaction).

Over time, N. alternatus observed in treatment with both predator species (GN) decreased and N.

alternatus observed in the single species (G) treatment increased (F8, 138 = 0.6346, P < 0.0001,

time x predator species interaction). Over time, N. alternatus observed in the treatment with both

predator species (GN) decreased and more N. alternatus were observed in the single species (G) treatments (F8, 138 = 0.7433, P = 0.0074, time x predator species interaction). (F16, 211 = 0.6341, P

= 0.0074, time x aphid density x predator species interaction).

3.3. Predator behavior in greenhouse

Predator location (Fig. 3, Fig. 4) and behavior (Fig. 5, Fig. 6) on each potato plant were also recorded during the observations over time. The sum of the predator observations by block and for each species was divided by the number of predators of each species present in each treatment, which is represented as per capita in the figures. The number of G. bullatus observed on the top of potato plants (Fig. 3a, c, e, and g) changed by block, and block 1 (Fig. 3a) had more

G. bullatus observed than the rest of the blocks (F3, 107 = 9.7857, P < 0.0001, block main effect).

More G. bullatus were observed in the treatment with both species (GN) for block 1 (Fig. 3 a)

115

than the other three blocks for either the single species treatment (G) or treatment with both

species (GN) (Fig. 3 a) (F6, 107 = 4.3487, P = 0.0008, block x predator treatment interaction). The

number of N. alternatus observed on the top of potato plants (Fig. 3b, d, f, and f) changed by

block, and block 1 (Fig. 3b) had more N. alternatus observed than the rest of the blocks (F3, 107 =

3.2272, P = 0.0274, block main effect).

The number of G. bullatus observed on the middle of potato plants (Fig. 4a, c, e, and g)

changed by predator treatment, and more G. bullatus were in treatments with both species (GN)

than in single species treatments (G) (F2, 107 = 37.9088, P < 0.0001, predator treatment main effect). The number of N. alternatus observed on the middle of potato plants (Fig. 4b, d, f, and f) changed by predator treatment, and higher numbers of N. alternatus were observed in both species treatments (GN) treatments than single species (N) treatments (F3, 107 = 20.0023, P <

0.0001, predator treatment main effect). Both predators were found to feed on both GPA and plant tissue, but there were no significant effects of treatment or predator species.

Geocoris bullatus and N. alternatus were both observed mating in the tanks (Fig. 5).

Mating G. bullatus pairs (Fig. 5a, c, e, g) occurred much more frequently in tanks with only G.

bullatus (G) than in tanks with both species (GN) (F8,99 = 24.2436, P <0.0001, predator treatment

main effect). More G. bullatus were observed mating in the single species treatments (G) than

the treatments with both species (GN), and block 2 had the highest observed mating out of all

four blocks (Fig. 5c) (F6, 107 = 2.6026, P = 0.0244, block x predator treatment interaction).

The number of N. alternatus observed mating (Fig. 5a, c, e, and g) changed by block, and

block 4 (Fig. 5h) had the fewest N. alternatus observed mating out of all four blocks (F3, 107 =

2.8467, P = 0.0435, block main effect). There was less mating observed for N. alternatus in the

treatments with 10 aphids than the treatments with 25 or 50 aphids (F2, 107 = 7.7600, P = 0.0009,

116

aphid density main effect). More N. alternatus pairs were observed mating in single species

treatments (G) then in treatments with both species (GN) (F2, 107 = 15.3800, P < 0.0001, predator

treatment main effect). Nabis alternatus were observed mating more frequently in the treatments

with only N. alternatus (N) in all blocks except for block 1 (Fig. 5b), and there was more mating

observed in the treatments with both species (GN) than the single species treatments (G) (F6, 107 =

2.4467, P = 0.0329, block x predator treatment interaction). Overall, there were fewer mating

events observed at 10 aphid density than 25 or 50 densities, but block 3 was the exception

because mating was more frequently seen in the 10 aphid treatment (F4, 107 = 3.8000, P = 0.0074,

block x aphid density interaction).

Nabis alternatus was observed to consume both G. bullatus and other N. alternatus (Fig.

6). Intraguild predation was not observed in block 1, which is why the figure only shows blocks

2, 3, and 4. More G. bullatus were consumed by N. alternatus (Fig. 6a, b) (F2,107 = 5.3333, P =

0.0069, predator treatment main effect), and consumption of N. alternatus by another N. alternatus (Fig. 6b, c) happened less frequently (F2,107 = 3.5714, P = 0.0332, predator treatment

main effect) than consumption of G. bullatus by N. alternatus. Intraguild predation on G. bullatus was observed in blocks 2 (Fig. 6a) and 3 (Fig. 6b) with more predation observed in block 2 (Fig. 6a) (F2,107 = 3.5714, P = 0.0332, block x aphid density x predator treatment

interaction). Consumption of N. alternatus by another N. alternatus was observed in blocks 3

(Fig. 6b) and 4 (Fig. 6c) and was lower in block 4 (Fig. 6c) (F2,107 = 2.1429, P = 0.0241, block x

aphid density x predator treatment interaction). A small number of both predator species were

found dead at the bottom of a tank, but there were no significant effects of treatment or predator

species. Some of the dead predators were due to the observed intraguild predation.

117

4. Discussion

Both G. bullatus and N. alternatus consumed GPA at all three densities and consumed

higher numbers of aphids when more were available. Previous feeding trials in simple laboratory

arenas have shown that while the presence of aphids reduced these predators’ predation rate on

potato beetles, when potato beetle eggs fill the role of “alternative prey” they do not reduce their predation rate on aphids (Koss et al. 2004, Koss and Snyder 2005). Likewise, a field cage study showed that potato beetle suppression by a complex of generalist predators was reduced when aphids were also present (e.g., Koss and Snyder 2005). However, increasing availability of alternative prey does not reliably improve biological control. For example, Prasad and Snyder

(2006) showed that alternative prey disrupted biological control by ground and rove beetles, because the predators preferred to feed on aphids in Brassica fields rather than the cabbage root maggots that were the main pest. Likewise, generalist predator carabid ground beetles indirectly disrupted biological control of aphids by a specialist parasitoid by reducing the aphid population that could be parasitized, which led to a higher aphid population over time (Snyder and Ives

2001). Aphids may not be the most nutritious prey item for Geocoris punctipes, but it prefers

mobile pea aphids to corn earworm eggs because it focuses its attack on mobile prey (Eubanks

and Denno 2000).

Both species were also observed consuming potato plant tissue. It is not uncommon for

predators to receive nutrients from plant tissues that include pollen, seeds, floral nectars,

extrafloral nectars and honeydew (Lundgren 2009). Mated pairs were observed more frequently

in single species treatments and in higher aphid density treatments. More food resources were

available to the predators in treatments with higher aphid densities. Foraging activity was highest

118

when single predator species were present, and it was reduced when both predator species were

present. Predators of different species also altered their foraging location on plants in the presence of other predator species. Geocoris bullatus and N. alternatus were more frequently observed in treatments with both species at the middle of the potato plant, but more predator observations occurred at the top of the plant and for mating in the single species treatments. Both species of predators moved throughout the cage over the 8 hour observation period, but they spent most of their time still or cleaning their mouthparts of antennae. The risk of predation made these predators spread out more and stay away from each other in the tanks, but it did not stop consumption of the green peach aphid. Predators sometimes retreat during a predator- predator confrontation (Edmunds 1974, Sih 1987), and more mobile predators have a better chance of escaping (New 1991). The outcome of intraguild predation is influenced by the size of the predators and feeding specificity of the predators (Lucas et al. 1998). This mainly occurs with generalist predators including predation within the same species (Polis 1981, Polis et al.

1989), and the smaller individual is more likely to be threatened by a larger predator (Werner and Gilliam 1984).

Some studies have shown that the presence of extraguild prey can reduce intraguild predation, and intraguild predation decreased as extraguild prey density increased (Bailey and

Polis 1987, Lucas et al. 1998). For example, under controlled conditions intraguild predation was higher for most combinations of Aphidoletes aphidmyza, Chrysoperla rufilabris, and

Coleomegilla maculata lengi at high levels with no prey present, and intraguild predation was reduced for the same predator combinations with the addition of aphids (Lucas et al. 1998). In our study, both predator species mainly consumed the extraguild prey (GPA) and were not observed consuming each other very frequently. Another indicator of intraguild predation is dead

119

predators in the aquarium, but dead predators were not seen often and some could have died

from other causes like old age or parasitoids. Geocoris bullatus was not seen consuming other G.

bullatus or N. alternatus, but N. alternatus was observed to consume other N. alternatus and G. bullatus. This is most likely because of size, since the N. alternatus is the larger species as an adult, which is the life stage for both species in the experiment. In simple laboratory arenas,

Nabis alternatus (damsel bug) and Geocoris bullatus (big-eyed bug) will feed on each other depending on which predator is in the larger developmental stage (Raymond 2000). In field-cage studies in Washington potato crops, results sometimes suggest the common occurrence of intraguild predation between Nabis and Geocoris (e.g., Koss and Snyder 2005, Straub and

Snyder 2006) whereas others find no evidence for predation among these predators (e.g., Straub and Snyder 2008). For example, Mallampalli and colleagues (2002) showed that intraguild predation between the spined soldier bug, Podisus maculiventris Say (Heteroptera:

Pentatomidae), and the twelve-spotted ladybeetle, Coleomegilla maculata Lengi (Coleoptera:

Coccinellidae) reduced overall predation on Colorado potato beetle eggs. The outcome of intraguild predation is influenced by the size of the predators and feeding specificity of the predators (Lucas et al. 1998). This mainly occurs with generalist predators including predation within the same species (Polis 1981, Polis et al. 1989), and the smaller individual is more likely to be threatened by a larger predator (Werner and Gilliam 1984). Predators sometimes retreat during a predator-predator confrontation (Edmunds 1974, Sih 1987), and more mobile predators have a better chance of escaping (New 1991).

Even though the results of this study are promising, there are limitations to this experiment. It was difficult to view all of the predators in each tank because the potato foliage was often blocking the view. A solution to this problem would be to remove some of the potato

120

leaflets from the plant before starting this experiment, but fewer refuges could increase intraguild

predation. A stronger predator species effect may occur at very low aphid densities or very high

densities, which were not part of this experiment. A field cage experiment with dispersed, even,

or clumped aphid densities may help explain if predators are more attracted to plants with high or

low aphid densities. Using molecular gut content analysis of aphid DNA inside the predators

would tell us how many of the predators were consuming aphids or each other. Intraguild

predation was not observed very often, but the molecular gut content analysis would give us a

more accurate accounting of predation events.

This study showed that both species of predators consumed GPA at all three densities and

consumed higher numbers as more were available. The predator species combinations did affect

predator behavior, but they did not have a large affect on aphid consumption. Foraging activity

was highest when single predator species were present, and it was reduced when both predator

species were present. Predators of different species also altered their foraging location on plants

in the presence of other predator species. Geocoris bullatus and N. alternatus were more

frequently observed in treatments with both species at the middle of the potato plant, but more

predator observations occurred at the top of the plant and for mating in the single species

treatments. Cannibalism was observed with N. alternatus consuming N. alternatus as was intraguild predation with N. alternatus consuming G. bullatus. Intraguild predation did not have a strong effect on aphid predation, but it did occur because of close contact of these predators.

The risk of predation presumably made these predators spread out more and stay away from each other in the tanks, but it did not stop consumption of the green peach aphid. Future studies would include a field experiment that shows if predator movement is changed by different aphid densities or predator combinations.

121

Acknowledgments

We thank D. W. Crowder for his help with our data analysis. Abbie Estep, Alicia Nutter, Max

Savery, and Tina Miller provided assistance with live predator collection. Abbie Estep ran half of the greenhouse experiment while the rest of us were collecting predators. This project was

supported by USDA AFRI grant.

122

References

Bailey, K. H., and G. A. Polis. 1987. An experimental analysis of optimal and central place

foraging by the harvester ants, Pogonomyrmex californicus. Oecologia 72: 440–448.

Biever, K. D.and R. L. Chauvin. 1992a. Suppression of the Colorado potato beetle (Coleoptera:

Chrysomelidae) with augmentative releases of predaceous stinkbugs (Hemiptera:

Pentatomidae). J. Econ. Entomol. 85: 720–726.

Biever, K.D. and R. L. Chauvin. 1992b. Timing of infestation by the Colorado potato beetle

(Coleoptera: Chrysomelidae) on the suppressive effect on field released stinkbugs

(Hemiptera: Pentatomidae) in Washington. Environ. Entomol. 21: 1212–1219.

Blackman, R.L. and V. F. Eastop. 2000. Aphids on the world's crops, an identification and

information guide. Wiley, New York.

Edmunds, M. 1974. Defence in animals. Longman, New York, New York, USA.

Eubanks, M. D. and R. F. Denno. 2000. Health food versus fast food: the effects of prey

quality and mobility on prey selection by a generalist predator and indirect interactions

among prey species. Ecological Entomology. 25: 140-146.

Harmon, J. P. and D. A. Andow. 2004. Indirect effects between shared prey: predictions for

biological control. Biocontrol. 49: 605-626.

JMP, Version 10. SAS Institute Inc., Cary, NC, 1989-2012.

Koss, A. M., Chang, G. C., and W. E. Snyder. 2004. Predation of green peach aphids by

generalist predators in the presence of alternative, Colorado potato beetle egg prey.

Biological control. 31: 237-244.

Koss, A. M. and W. E. Snyder. 2005. Alternative prey disrupt biocontrol by a guild of generalist

123

predators. Biological Control. 32: 243–251.

Lucas, E., Coderre, D., and J. Brodeur. 1998. Intraguild predation among aphid predators:

characterization and influence of extraguild prey density. Ecol.79(3): 1084-1092.

Lundgren, J. G. 2009. Relationships of Natural Enemies and Non- Prey Foods. Springer,

London, UK.

Mallampalli, N., Castellanos, I., and P. Barbosa. 2002. Evidence for intraguild predation by

Podisus maculiventris on a ladybeetle, Coleomegilla maculata: Implications for

biological control of Colorado potato beetle, Leptinotarsa decemlineata. Biocontrol. 47:

387-398.

New, T. R. 1991. Insects as predators. New South Wales University Press, New South Wales,

Australia.

Novy, R. G., Nasruddin, A., Ragsdale, D. W., and E. B. Radcliffe. 2002. Genetic resistances to

potato leafroll virus, potato pirus Y, and green peach aphid in progeny of Solanum

etuberosum. American Journal of Potato research. 79(1): 9-18.

Polis, G. A. 1981. The evolution and dynamics of intraspeciﬁc predation. Annual Review of

Ecology and Systematics 12: 225–251

Polis, G. A., C. A. Myers, and R. D. Holt. 1989. The ecology and evolution of intraguild

predation: potential competitors that eat each other. Annual Review of Ecology and

Systematics. 20: 297-330.

Prasad, R. P. and W. E. Snyder. 2006. Polyphagy complicates conservation biological control

that targets generalist predators. Journal of Applied Ecology. 43: 343-352.

Radcliffe, E. B. 1982. Insect pests of potato. Annu. Rev. Entomol. 27: 173-204.

Raymond, B. 2000. Cannibalism and intraguild predation involving three predatory Heteroptera,

124

Nabis roseipennis, Geocoris punctipes and Orius insidiosus. Dissertation. University of

Kentucky, Lexington, Kentucky, USA.

Rosenheim, J. A.. 1998. Higher-order predators and the regulation of insect herbivore

populations. Annual Review of Entomology. 43:421-447.

Settle, W.H., Ariawan, H., Astuti, E.T., Cahyana, W., Hakim, A.L., Hindayana, D., Lestari, A.S.,

and S. Pajarningsih. 1996. Managing tropical rice pests through conservation of

generalist natural enemies and alternative prey. Ecology. 77: 1975–1988.

Sih, A. 1987. Predators and prey lifestyles: an evolutionary and ecological overview. Pages 203–

224 in C. W. Kerfoot and A. Sih, editors. Predation: direct and indirect impacts on

aquatic communities. University Press of New England, Hanover, New Hampshire, USA.

Snyder, W. E. and A. R. Ives. 2001. Generalist predators disrupt biological control by a specialist

parasitoid. Ecology. 82(3): 705–716.

Straub, C. S. and W. E. Snyder. 2006. Species identity dominates the relationship between

predator biodiversity and herbivore suppression. Ecology. 87(2): 277–282.

Straub, C. S. and W. E. Snyder. 2008. Increasing enemy biodiversity strengthens herbivore

suppression on two plant species. Ecology 89: 1605-1615.

Symondson, W.O.C., Sunderland, K.D., and M. H. Greenstone. 2002. Can generalist predators

be effective biocontrol agents?. Annu. Rev. Entomol. 47: 561–594.

Tamaki, G. and R. E. Weeks. 1972a. Efficiency of three predators, Geocoris bullatus, Nabis

americoferus, and Coccinella transversoguttata, used alone or in combination against

three insect prey species, Myzus persiace, Ceramica picata, and Mamestra configurata,

in a greenhouse study. Environ. Entomol. 1: 258–263.

Tamaki, G. and R. E. Weeks. 1972b. Biology and ecology of two predators, Geocoris pallens

125

Stål and G. bullatus (Say). Technical Bulletin 1446, US Department of Agriculture.

Werner, E. E., and J. F. Gilliam. 1984. The ontogenetic niche and species interactions in size-

structured populations. Annual Review of Ecology and Systematics 15: 393–425.

126

Figure legends

Fig. 1. Number of aphids eaten by monocultures of Geocoris bullatus (G) and Nabis alternatus

(N), or a mix of G. bullatus and N. alternatus (GN), when foraging among 10, 25, or 50 green peach aphids per potato plant. The experiment was conducted within four blocks: (a) block 1, (b) block 2, (c) block 3, and (d) block 4.

Fig. 2. Per capita number of times that predators were observed over 8 hours when Geocoris bullatus foraged among only conspecifics (G) or when this species foraged alongside Nabis alternatus (G with N) (panel a,), or when N. alternatus foraged among only conspecifics (N) or alongside G. bullatus (N with G) (panels b). Predators were presented with potato plants initially housing either 10, 25 or 50 green peach aphids.

Fig. 3. Per capita number of times that predators were observed on top of the potato plant when

Geocoris bullatus foraged among only conspecifics (G) or when this species foraged alongside

Nabis alternatus (G with N) (panels a, c, e and g), or when N. alternatus foraged among only conspecifics (N) or alongside G. bullatus (N with G) (panels b, d, f and h). Predators were presented with potato plants initially housing either 10, 25 or 50 green peach aphids, and the experiment was conducted within four blocks: block 1 (a, b), block 2 (c, d), block 3 (e, f) and block 4 (g, h).

Fig. 4. Per capita number of times that predators were observed on the middle of the potato plant when Geocoris bullatus foraged among only conspecifics (G) or when this species foraged

127

alongside Nabis alternatus (G with N) (panels a, c, e and g), or when N. alternatus foraged

among only conspecifics (N) or alongside G. bullatus (N with G) (panels b, d, f and h). Predators

were presented with potato plants initially housing either 10, 25 or 50 green peach aphids, and

the experiment was conducted within four blocks: block 1 (a, b), block 2 (c, d), block 3 (e, f) and

block 4 (g, h).

Fig. 5. Per capita number of times that predators were observed mating when Geocoris bullatus

foraged among only conspecifics (G) or when this species foraged alongside Nabis alternatus (G with N) (panels a, c, e and g), or when N. alternatus foraged among only conspecifics (N) or alongside G. bullatus (N with G) (panels b, d, f and h). Predators were presented with potato plants initially housing either 10, 25 or 50 green peach aphids, and the experiment was conducted within four blocks: block 1 (a, b), block 2 (c, d), block 3 (e, f) and block 4 (g, h).

Fig. 6. Per capita number of times that Nabis alternatus were observed consuming Geocoris

bullatus when G. bullatus was present with N. alternatus (G with N) (panels a and b), or when N. alternatus consumed other N. alternatus among only conspecifics (N) (panels b and c). Predators were presented with potato plants initially housing either 10, 25 or 50 green peach aphids, and the experiment was conducted within four blocks: block 2 (a), block 3 (b), and block 4 (c). There was no intraguild predation for block 1, so it is not shown in this figure.

128

Fig. 1.

b) Block 2 60 a) Block 1 G 50 G GN GN N 40 N

20 No. aphids eaten 10

d) Block 4 60 c) Block 3 G 50 G GN GN N 40 N

20 No. aphids eaten aphids No. 10

0 1 2 3 10 25 50 10 25 50 Aphid density Aphid density

129

Fig. 2.

a) Geocoris bullatus 10G 0.8 10GN 25G 0.7 25GN 50G 0.6 50GN

0.5

0.4

0.3

Percapita observed treatment by 0.2 0 2 4 6 8 10

b) Nabis alternatus 10GN 1.0 10N 25GN 0.9 25N 50GN 0.8 50N

0.7 observed treatment by

0.6 Nabis

0.5

0.4 0 2 4 6 8 10 Averageof# Hours observed

130

Fig. 3.

8 a) Block 1 Geocoris bullatus b) Block 1 Nabis alternatus G N GN GN 6

4 c) Block 2 Geocoris bullatus d) Block 2 Nabis alternatus G N GN GN 3

observed

6 e) Block 3 Geocoris bullatus f) Block 3 Nabis alternatus G N GN GN Per capita # 4

2.0 g) Block 4 Geocoris bullatus h) Block 4 Nabis alternatus

G N GN 1.5 GN

1.0

0.5

0.0 101 252 503 101 252 503 Aphid density Aphid density

131

Fig. 4.

4 a) Block 1 Geocoris bullatus b) Block 1 Nabis alternatus

G N 3 GN GN

4 c) Block 2 Geocoris bullatus d) Block 2 Nabis alternatus

G N GN 3 GN

4 e) Block 3 Geocoris bullatus f) Block 3 Nabis alternatus Per capita # observed G N GN 3 GN

4 g) Block 4 Geocoris bullatus h) Block 4 Nabis alternatus N G GN 3 GN

0 101 252 503 101 252 503 Aphid density Aphid density

132

Fig. 5.

3.0 a) Block 1 Geocoris bullatus b) Block 1 Nabis alternatus

2.5 G N GN GN 2.0

1.5

1.0

0.5

0.0

3.0 c) Block 2 Geocoris bullatus d) Block 2 Nabis alternatus

2.5 G N GN GN 2.0

1.5

1.0

0.5

0.0

observed

3.0 e) Block 3 Geocoris bullatus f) Block 3 Nabis alternatus

2.5 G N Per capita # GN GN 2.0

1.5

1.0

0.5

0.0

3.0 g) Block 4 Geocoris bullatus h) Block 4 Nabis alternatus

2.5 G N GN GN 2.0

1.5

1.0

0.5

0.0 101 252 503 101 252 503 Aphid density Aphid density

133

Fig. 6.

a) Block 2 Intraguild predation by Nabis

0.6 GN Nabis ate Geocoris N Nabis ate Nabis

0.4

0.2

0.0

b) Block 3 Intraguild predation by Nabis alternatus

0.6 GN Nabis ate Geocoris N Nabis ate Nabis

observed 0.4

0.2 Per capita # capita Per 0.0

c) Block 4 Intraguild predation by Nabis

0.6 GN Nabis ate Geocoris N Nabis ate Nabis

0.4

0.2

0.0 101 252 503 Aphid density

134

CONCLUSIONS

Generalist insect predators may search out prey species that are nutritious even when they are uncommon in the environment, such that rates of predation will not necessarily track prey abundance. This study examined patterns of predation and behavior for two species of insect predators, Nabis alternatus Parshley and Geocoris bullatus Stål, in laboratory, greenhouse and field experiments. The laboratory experiment detected the presence of DNA from Leptinotarsa decemlineata (Say) (Colorado potato beetle) and Myzus persicae (Sulzer) (green peach aphid) in both predator species at 0, 1, 2, 4, 6, 8, 16, and 24 hours after feeding, and Nabis alternatus was also tested using the same method for consumption of G. bullatus nymphs. This study helps us know possibly how long field collected predators would test positive for a prey item. For both species of predators, prey DNA is low around 24 hours, so most predators collected from fields and tested positive for prey most likely ate the prey less than 24 hours before collection.

An open field experiment manipulated densities of green peach aphid and Colorado potato beetle using pest specific insecticides to determine if changes in prey community structure were reflected by changing predator feeding patterns. Both predator species consumed Colorado potato beetles and green peach aphids in this study, and N. alternatus also consumed G. bullatus.

However, these feeding relationships were not significantly altered by changes in aphid or potato beetle densities. This suggests either that the predators’ diets were relatively unaffected by prey availability, or that predators mixed their diets by moving freely among our plots. Despite the change in pest community structure by manipulation of CPB and GPA, there was no distinct predation pattern or large change in predation rates for consumption of the target prey items. The higher insect population did slightly decrease intraguild predation from June to later in the

135

season, but we expected to see a greater drop in intraguild predation as the prey population

increased. Geocoris bullatus may be easy for N. alternatus to catch and consume or may be more nutritious than other prey, which is why they were found detectable for G. bullatus throughout the field season for both years of the study. Predators found in plots with fewer Colorado potato beetles and green peach aphids nonetheless commonly consumed these prey items. This suggests that the predators search out both species of prey even when rare, perhaps because they provide a unique nutritional contribution to the predators’ diets. However, these changes in predator and prey community structure did not translate into significant effects on either predator’s likelihood of having eaten aphids or potato beetles, or of N. alternatus eating G. bullatus. Our study suggests that, through movement or innate preferences, the diets of generalist predators may remain surprisingly rigid in the face of a mosaic of prey densities.

A third study in the greenhouse manipulated green peach aphid densities (10, 25, or 50 aphids) and predator community structure (8 G. bullatus, 8 N. alternatus, or 4 of each species) to determine if aphid consumption changed with pest density or predator species composition. This study showed that both species of predators consumed GPA at all three densities and consumed higher numbers as more were available. The predator species combinations did affect predator behavior, but they did not have a large affect on aphid consumption. Foraging activity was highest when single predator species were present, and it was reduced when both predator species were present. Predators of different species also altered their foraging location on plants in the presence of other predator species. Geocoris bullatus and N. alternatus were more frequently observed in treatments with both species at the middle of the potato plant, but more predator observations occurred at the top of the plant and for mating in the single species treatments. Cannibalism was observed with N. alternatus consuming N. alternatus as was

136

intraguild predation with N. alternatus consuming G. bullatus. Intraguild predation did not have a strong effect on aphid predation, but it did occur because of close contact of these predators.

137

ATTRIBUTIONS

Chapter 1

Gretchen B. Snyder: helped run feeding trials

Eric Chapman and James Harwood: taught us feeding trials and ran molecular samples

William E. Snyder: provided grant funding for research and supplies

Chapter 2

Eric Chapman and James Harwood: ran molecular samples

William E. Snyder: provided grant funding for research and supplies

138