CONSERVATION OF INSECT NATURAL ENEMIES IN HETEROGENEOUS VEGETABLE LANDSCAPES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Janet Louise Lawrence, M.S.

* * * * *

The Ohio State University 2004

Dissertation Committee: Approved by Dr. Casey Hoy, Adviser

Dr. Clive Edwards

Dr. Parwinder Grewal Adviser Dr. Larry Phelan Department of Entomology ABSTRACT

The position taken is that populations of natural enemies occupying both soil and foliar habitats are regulated by lower trophic levels. A series of hypotheses were tested on the influence of lower trophic levels on population persistence of natural enemies with divergent life history traits; the entomopathogenic nematode, Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) and the larval parasitoid, Diadegma insulare Cresson (Hymenoptera: Ichneumonidae). Initial studies on the entomopathogenic nematode, were aimed at understanding their ecology in vegetable landscapes and thereafter investigations were conducted to determine the influences of insect host availability and plant diversity on population densities and the mechanisms underlying their effects. Investigations for D. insulare, focused on its responses to densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) and flowering coriander.

Several strains of entomopathogenic nematodes that were able to infect and reproduce in common vegetable insect pests were recovered along grassy banks adjacent to cultivated areas of a vegetable production system. Soil moisture was the principal factor associated with the presence of these endemic nematode populations. Persistence of H. bacteriophora populations differed in plots with different insect host availability, but persistence differed among strains and was related, in part, to the abilities of the

ii nematodes to survive under test site conditions. For those strains with high survival rates, populations increased with increasing host availability, whereas no such effects were observed for populations with low survival rates. Higher nematode population densities were observed in plots with more diverse plants and insect herbivores. A correlation was found between nematode population density and chrysomelid beetle abundance, suggesting that persistence was at least partially as a result of nematodes recycling within these insects. D. insulare aggregated twice as much in areas with high compared with low populations of diamondback moth. Parasitism was greater when flowering coriander was present, but this effect was only observed at low population densities. In summary, bottom-up effects of plant and herbivore communities on natural enemies above and below-ground were similar. Population persistence for the two natural enemies was higher with increasing insect hosts and plant diversity; but the effects occurred at different spatial and temporal scales.

iii DEDICATION

To my mother, Enid Dorothy Lawrence

iv ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my major advisor, Dr Casey Hoy for his guidance in shaping this project, constant support and infinite patience. To have had the opportunity to work with a true mentor and someone whose mind I greatly admire was indeed a privilege. To Dr Clive Edwards, who encouraged me to enroll at the

Ohio State University and whose support has been tremendous, especially during my time in Columbus, I am grateful. Special thanks to Dr Larry Phelan for his thought provoking queries that challenged me to think more deeply and clearly about this project.

Grateful thanks to Dr Parwinder Grewal, for always taking the time to give suggestions, generously opening his lab facilities to me and reintroducing me to the “tiny creatures” I had left behind years ago.

There are really no words to express my gratitude to Mike Dunlap for his logistical support over the years and his friendship. Mike worked tirelessly, to ensure that

I had all I needed to execute this project. My appreciation to Dr Mustapha Jallow for his willingness to help with the many field sampling expeditions, Dr Krishna Prasad for assisting with GIS software and Ms Nuris Acosta for taxonomic support. Thanks to the multitude of summer workers that provided youthful exuberance during challenging sampling times, namely: Niccoli Fioritto, Timothy Taush, Joe Dunlap, Benjamin Dunlap,

Kyle Kauffman, Kate Williams, Charles Gilbert, Jeshua Cressap, Katie Gerber, and

v Jeremy Jewel. Thanks also to Rick Callendar and the field staff of the OARDC Muck

Crops Branch for their assistance, in particular during trying times of flooded field plots.

Special thanks to: Catherine Herms for constantly being available to help me to understand the workings of the GPS, Bonnie Slovboda for the last minute poster demands and the library staff for their assistance.

My deep appreciation to Mabel Kirchner and Shirley Holmes for their encouragement and support. To the many friends who boosted my spirits and helped me to feel at home in Ohio, I am grateful.

I would like to thank the Department of Entomology for supporting my graduate education here at The Ohio State University. Appreciation also to the Ohio Vegetable and

Small Fruit Research, Ohio Department of Agriculture and United States Department of

Agriculture Specialty Crop Block Grant and Development Program for consistently funding our research proposals to execute this work.

Finally, I am truly indebted to my mom, Enid and sister, Phillipa who have been my sources of inspiration, encouragement and strength at all times.

vi VITA

December 11th, 1965……………………………. Born in Kingston, Jamaica

1985 – 1989 ...……………………………...... B.S. University of the West Indies. ………………………………………………… Kingston Jamaica

1990 - 1991 …………………………………… Research Associate, Caribbean …………………. Agricultural Research and Development …………………………………………………..Institute (CARDI). Kingston Jamaica

1991 - 1994 ……………………………………. M.S. University of Florida. Gainesville, …………………………………………………..Florida

1994 - 2000 …………………………………… Scientist. Caribbean Agricultural ………………………………………………….Research and Development Institute ………………………………………………….(CARDI). Kingston Jamaica

2000 - 2004…………………………………… Graduate Teaching Assistant and …………………………………………………Research Assistant. The Ohio State …………………………………………………University

FIELDS OF STUDY

Major Field: Entomology

Integrated Pest Management

vii TABLE OF CONTENTS

Page

Abstract ……………………………………………………………………………… ii

Dedication …………………………………………………………………………… iv

Acknowledgments …………………………………………………………………… v

Vita …………………………………………………………………………………... vii

List of Tables ………………………………………………………………………... x

List of Figures ……………………………………………………………………….. xi

Glossary ……………………………………………………………………………... xv

Chapters:

1. Conservation of insect natural enemies in heterogeneous vegetable landscapes 1 Introduction …………………………………………………………………. 1 Literature review ……………………………………………………………. 4 Thesis ………………………………………………………………………... 34

2 Distribution and persistence of endemic entomopathogenic nematodes In heterogeneous vegetable landscapes in Huron County, Ohio ……….………… 39 Introduction ……………………………………..…………………….….….. 39 Methodology ………………………………………………………………… 40 Results ….…………….………………….….……………………………….. 47 Discussion………………….………………….……………………………… 58

3. Life-history characteristics of endemic entomopathogenic nematode strains isolated from the vegetable production area in Huron County, Ohio ...... 66 Introduction ...... ……………………………………………………….….….. 66 Methodology ………………………………………………………………… 67 Results ...... …………….………………….………………………………….. 71 Discussion ..……………….………………….……………………………… 76

viii 4. Influence of host availability on the regulation of the entomopathogenic nematode, Heterorhabditis bacteriophora ...... 82 Introduction…………………………………………………………….….….. 82 Methodology ………………………………………………………………… 83 Results .....….………….………………….………………………………….. 88 Discussion....……………….………………….……………………………… 91

5. Bottom-up effects of plant communities on the persistence of two strains of The entomopathogenic nematode, Heterorhabditis bacteriophora ...... 101 Introduction ...... ……………………………………………………….….….. 101 Methodology ………………………………………………………………… 102 Results ...... …………….………………….………………………………….. 106 Discussion ...... …………….………………….……………………………… 113

6. Bottom-up factors regulating the persistence of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) in heterogeneous vegetable cropping systems ……...... 118 Introduction…………………………………………………………….….….. 118 Methodology ………………………………………………………………… 120 Results………………….………………….………………………………….. 122 Discussion………………….………………….……………………………… 123

7. Conservation biological control in vegetable systems: synthesis, possible strategies and future direction…………………………………………..…………. 135

Bibliography ………………………………………………………………………… 145

ix LIST OF TABLES

Table Page

2.1 Characteristics of crop and soil sections surveyed in the vegetable production area, Celeryville (Huron County, Ohio) in 2000. …………...... 42

2.2 Soil type and disturbance regime of habitats within the vegetable landscape, Celeryville (Huron County, Ohio) in 2002 .…………... 44

2.3 Composition and abundance of arthropod populations along grassy banks adjacent to cultivated areas in Celeryville (Huron County, Ohio) in July 2001……………………………………………………………...... 49

2.4 Multiple regression analysis of infective juvenile densities and abiotic (i.e., temperature, moisture) and biotic (i.e., macro and micro-arthropods) factors. …………………………………………………… 59

3.1 Reproductive potential (infective juveniles, IJ’s) of endemic and exotic strains of H. bacteriophora and S. feltiae in black cutworms, Agrotis ipsilon (Lepidoptera: Noctuidae) and onion maggots, Delia antiqua (Diptera: Anthomyiidae). …………………… 75

4.1 Influence of host supply on the persistence of infective juvenile populations: treatment structure. ………………………………………………………….. 86

5.1 Composition and abundance of insect populations colonizing high and low plant diversity plots, June-October 2003 ……………………………………. 107

x LIST OF FIGURES

Figure Page

1.1 Conceptual model of bottom-up regulation of entomopathogenic nematodes (EPN) by crop patch and landscape components and the subsequent top-down regulation of soil pests within a heterogeneous landscape. ……………………...... 35

1.2 Conceptual model of bottom-up regulation of the parasitoid, Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) by crop patch and landscape components and the subsequent top-down regulation of the diamondback moth, Plutella xylostella L. 36 (Lepidoptera: Plutellidae).………………………………………………...

1.3 Current research status of bottom-up regulation of entomopathogenic nematodes (EPN) by lower trophic groups and the subsequent top-down regulation of soil pests. …………………………………………...... 37

1.4 Current research status of bottom-up regulation of the parasitoid, Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) by lower trophic groups and the subsequent top-down regulation of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae)..…… 38

2.1 Distribution of entomopathogenic nematodes within the vegetable production area in Celeryville, (Huron County, Ohio) in 2000 and 2001 …………………...... 48

2.2 Temporal variation in entomopathogenic nematode population densities across sites along grassy banks adjacent to the cultivated areas in Celeryville (Huron County, Ohio), May-October 2003. ...………………. 50

2.3 Spatial and temporal variation of entomopathogenic nematodes (IJ’s infective juveniles) along grassy ditch banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May-October 2003. ……………… 51

2.4 Spatial and temporal distribution of entomopathogenic nematode populations within sites along grassy banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May-October 2003. ………………… 52 xi

2.5 Composition and abundance of arthropod populations at sites along grassy ditch banks adjacent to cultivated areas, Celeryville (Huron County, Ohio), May-October 2003. ………………...... 54

2.6 Mean soil temperatures across sites monitored along grassy ditch banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May-October 2003. ………………...... 55

2.7 Soil moisture (%) at sites along ditch banks adjacent to cultivated area, Celeryville (Huron County, Ohio), May-October 2003. ………...... 56

2.8 Soil moisture (%) at sites at which entomopathogenic nematodes were detected and not detected along grassy banks adjacent to cultivated area, Celeryville (Huron County, Ohio) (May-October 2003). ………… 57

3.1 Infectivity of endemic and exotic strains of H. bacteriophora and S. feltiae to the black cutworm, Agrotis ipsilon Hufner (Lepidoptera: Noctuidae) ………………………………………………… 72

3.2 Infectivity of endemic and exotic strains of H. bacteriophora and S. feltiae to the onion maggot, Delia antiqua Meigen (Diptera: Anthomyiidae). ………………………………………………... 74

3.3 Exponential decay rates of endemic entomopathogenic nematode strains, Heterorhabditis bacteriophora and Steinernema feltiae in the absence of an insect host. ...………...... ……………………… 77

4.1 Season I (2001) – Influence of host supply on the persistence of Heterorhabditis bacteriophora, exotic strain (Lewiston). ..……………... 89

4.2 Season I (2001) – Influence of carrot weevil larvae, Listronotus oregonesis (CW) and onion maggots, Delia antiqua (OM) on the persistence of Heterorhabditis bacteriophora, 90 exotic strain (Lewiston). ……………….…………………………………

4.3 Season II (2002) – Influence of host supply on the persistence of Heterorhabditis bacteriophora, exotic strain (HP88). …………………... 92

xii 4.4 Season II (2002) – Influence of host supply on the persistence of Heterorhabditis bacteriophora, endemic strain. ……………………… 93

4.5 Season II (2002) – Influence of carrot weevil larvae, Listronotus oregonesis (CW) and onion maggots, Delia antiqua (OM) on the persistence of Heterorhabditis bacteriophora, exotic strain (HP88). ……………………………………………………... 94

4.6 Season II (2002) – Influence of carrot weevil larvae, Listronotus oregonesis (CW) and onion maggots, Delia antiqua (OM) on the persistence of Heterorhabditis bacteriophora, endemic strain. …………. 95

5.1 Composition and abundance of insects populations colonizing high and low plant diversity plots, June-October 2003. …………………….... 108

5.2 Diversity indices for insects colonizing high and low plant diversity plots, June-October 2003. ……………………………………………………..... 109

5.3 Mean population abundance of chrysomelid beetles colonizing high and low plant diversity plots, June-October 2003. ………………………….... 111

5.4 Persistence of an exotic (A) and endemic (B) strain of Heterorhabditis bacteriophora in relation to plant diversity, June-October 2003. ……….. 112

6.1 Number of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) visiting collard plots infested with high and low population densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae). ……………………………………………….. 124

6.2 Time for observing the first sighting of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) in collard plots with high and low population densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae). ………………………... 125

6.3 Retention time of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) in plots of collards infested with high and low population densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae). ……………………….. 126

xiii

6.4 Frequency of visits of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) to collard plots infested with high and low population densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae). ………………………………………………. 127

6.5 Parasitism of Diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) by Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) in plots with collards and collards and flowering coriander in Celeryville, Ohio. ……………... 128

6.6 Correlation between number of visits of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) to plots of collards and plots with collards and flowering coriander with high and low densities of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) ……………………………………………….. 129

7.1 Current research status of bottom-up regulation of entomopathogenic nematodes by lower trophic groups and the subsequent top-down regulation of pests ...... …...... ………………………. 143

7.2 Current research status of bottom-up regulation of the parasitoid, Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) by lower trophic groups and the subsequent top-down regulation of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) ...... …………………………………………… 144

xiv GLOSSARY

Abundance Number of organisms.

Bottom-up regulation Populations of organisms at high trophic levels of the food chain (natural enemies) are regulated by populations of organisms occupying lower trophic levels (herbivores and plants) of the food chain.

Community Total number of species in an area.

Conservation biological Preservation of natural enemies within a selected area by control manipulating the cropping practices and/or the physical structure of the habitat so that resources such as food, shelter and hosts are provided for natural enemy populations to persist and effect pest regulation.

Endemic entomopathogenic Nematode strain that is native to the area in which it is nematode being applied.

Entomopathogenic nematode Nematodes of the genera Heterorhabditis and Steinernema that feeds on insects (obligate parasite). The only free- living stage is the infective juvenile (IJ), which seeks the insect host.

Entomopathogenic nematode Entomopathogenic nematode culture that is characterized strain by morphological and/or physiological traits that are distinct from other members of the species and therefore given a special designation. E.g., Heterorhabditis bacteriophora, HP88 strain, H. bacteriophora Lewiston strain. For the purpose of this dissertation, I will refer to a nematode culture isolated from a single documented location as a strain.

Exotic strain Nematode strain that is not native to the area in which it has been applied.

xv Habitat An area of similar land use type. E.g., cultivated area consisting of different types of crop patches.

Host quality Nutritional status of a host-plant or host-insect that is used for food and/or oviposition by the entomopathogenic nematode or parasitoid.

Insect natural enemy Organism that feeds on insects to obtain nutrients to complete its life cycle, to the detriment of the insect.

Landscape Mosaic of different habitats, with an extent of hectares to kilometers.

Mortality rate Proportion of organisms dying per unit time

Parasitoid An insect that develops parasitically in or on a host (usually another insect) and consumes and/or kills the host upon completing development.

Persistence Sustained and spatially uniform presence and abundance of organisms within an area over time.

Population All individuals of a species in a particular habitat

Population density Number of individuals per unit area.

Predator An animal that kills and consumes other animals (prey).

Recycling Reproduction of entomopathogenic nematodes in an insect host, such that the free-living stage persists in a host.

Top-down regulation Populations of organisms at lower trophic levels of the food chain (herbivores and plants) are regulated by populations of organisms occupying higher trophic levels (natural enemies) of the food chain.

Trophic cascade Indirect effects of one trophic level on lower levels or upper levels. E.g., the indirect effect of plants on natural enemies via herbivores. xvi CHAPTER 1

CONSERVATION OF INSECT NATURAL ENEMIES IN

HETEROGENEOUS VEGETABLE LANDSCAPES

INTRODUCTION

Vegetable cropping systems are plagued by a complex of foliar and soil dwelling insect pests that significantly reduce the quality and quantity of harvested crops.

Management of insect pests has been with chemical insecticides and to a lesser extent resistant varieties and cultural practices. Public concerns for food safety, environmental protection, as well as the potential loss (under the food quality protection act, FPQA) of many of the popular organophosphates and carbamates used in vegetable pest management, have prompted the need to identify biologically based pest management alternatives that can be incorporated into vegetable Integrated Pest Management (IPM) programs.

In vegetable production, biologically based practices usually have been inundative releases of large quantities of exotic natural enemies during the growing season, namely entomopathogenic bacteria, Bacillus thuringinensis (Bt), and to a lesser extent egg parasitoids, Trichogramma spp. (Obrycki et al. 1997). Other approaches such as

1 conservation of endemic natural enemies have been less successful due to unfavourable conditions created by frequent pesticide applications, periodic disruption of the soil structure by tillage, frequent plantings and rotation, removal of crop residues, and the destruction of plant structures through harvesting. To survive, natural enemies exploiting these habitats have to disperse to favorable environments (other crop patches, woodlots, hedgerows) before crop patches become unfavorable, or are destroyed (Wissinger 1997,

Smith et al. 1997). Mitigating the impact of these unfavorable conditions by manipulating production practices and enhancing cropping areas with resources required by natural enemies have been proposed to conserve populations (Gurr et al. 2003, Landis et al.

2000, Gurr et al. 1998). Implementing these strategies requires an understanding of the spatial and temporal interactions of natural enemies, their hosts, and plant communities

(Valladares and Salvo 1999, Verkerk et al. 1998).

The ultimate goal for a biological control program for vegetables is sustained regulation of pest populations by natural enemies. A prerequisite for this sustained top- down regulation is having natural enemy populations persist over time. The specific focus of the research presented is to determine the factors affecting the persistence of insect natural enemies in a heterogeneous vegetable landscape. Conservation biological control within vegetable landscapes requires a complex of natural enemies that regulate pests that feed on plant parts above- and below-ground. Research hypotheses were therefore tested on two natural enemies with diverse life histories; a soil-inhabiting entomopathogenic nematode (insect parasite), Heterorhabditis bacteriophora

(Rhabditida: Heterorhabditidae) and a foliar-dwelling larval parasitoid, Diadegma insulare (Hymenoptera: Ichneumonidae). For the entomopathogenic nematode, hosts

2 systems investigated included: onion maggots, Delia antiqua Meigen (Diptera:

Anthomyiidae), carrot weevil, Listronotus oregonensis Le Conte (Coleoptera:

Curculionidae), and black cutworms, Agrotis ipsilon Hufner (Lepidoptera: Noctuidae), all of which have damaging life stages in the soil and limit yields. For D. insulare, a major pest of crucifers, the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) was used as the test insect.

The dissertation is presented in three main parts. An initial review of the literature is presented along with conceptual models of the potential trophic interactions occurring within a vegetable landscape for H. bacteriophora and D. insulare. These models form the basis for the development of the thesis. A series of hypotheses were tested to support the thesis in the succeeding five chapters. Research investigations were conducted largely on the entomopathogenic nematode. Initial research focused on understanding the ecology of endemic entomopathogenic nematode populations and thereafter hypotheses relating to trophic interactions within a vegetable cropping system were tested. For D. insulare, a single study on the interaction of lower trophic groups on population persistence was conducted. The final chapter synthesizes the connection between the data and the conceptual model and also provides possible areas for future research.

3 LITERATURE REVIEW

Scope of Review

This review focuses on the trophic interactions of natural enemies with different life history traits; those that live within foliar environments such as predators and parasitoids and others occupying the soil such as entomopathogenic nematodes (insect parasitic nematode). Predaceous insect species are usually very mobile, generalist feeders that consume several prey during their lifetime. Common generalist predators include carabids, staphylinids, dermapterans, coccinellids, syrphids, nabids, and lacewings.

Conversely, parasitoids belong mainly to the orders Diptera and Hymenoptera and are either specialist or generalist feeders. Parasitic wasps actively seek hosts and depending on the species, lay one egg (solitary) or multiple eggs (gregarious) on or in different stages of the hosts (i.e., egg, larva, or pupa). The immature stages develop by feeding on the host. Examples of common parasitic wasp species found in vegetable systems include: host specific larval parasitoid, Diadegma insulare, generalist egg parasitoid,

Trichogramma spp., and the larval parasitoid Cotesia spp.

Entomopathogenic nematodes share life traits with predators and parasitoids as well as with entomopathogens. These nematodes actively seek insect hosts but have a very limited dispersal range in the soil. Host location occurs by the only free-living stage, the infective juvenile, which enters the host and releases a symbiotic bacteria (i.e.,

Xenorhabdus spp. or Photorhabdus spp.) that kills the host in 48 hours. The infective juvenile feeds on the bacteria as well as the host tissues, and develops and reproduce

4 within cadavers. When the host resources are depleted, nematode reproduction ceases and the offspring develop into environmentally resistant infective juveniles that disperse from the cadaver and seek new hosts (Grewal and Georgis 1998, Kaya and Gaugler 1993).

Literature on natural enemy-host-plant interactions was reviewed at two spatial scales: crop patches within cultivated habitats and, the larger landscape level. In this review, habitat and landscape are not defined from the perspective of the organism (i.e., place or environment where the animal lives) as these definitions are dependent on the individuals’ life history traits, in particular, resource use patterns and dispersal ranges.

Habitat is defined by land use type (e.g., cultivated habitat consisting of different types of crop patches) and landscape is defined as a mosaic of different habitats with an extent of hectares to kilometers. At both scales, the influences of habitat composition and structure on persistence and efficacy of natural enemies were described. Disturbances and changes in the microclimatic conditions in crop patches and habitats were discussed because they impact organisms directly (i.e., disrupt life processes) and indirectly (e.g., loss of food source, or, shelter), these areas are also addressed.

Life Histories of Natural Enemies and Insect Pests in Vegetable Production Systems.

Impact of Disturbances (artificial)

Above-ground

Agro-ecosystem disturbances such as tillage, pesticide applications, field sanitation, and cropping patterns create environments in which herbivores and natural enemies are unable to access resources such as food, shelter, and mates (Landis and

Menalled 1998). Species disperse from these habitats to more favorable environments

5 such as, other crop habitats or interstitices between croplands to obtain resources. The most prevalent form of disturbance in vegetable production is pesticide application which is deleterious to herbivore and natural enemy populations (Hummel et al. 2002). Suttman and Barrett (1979) reported that after an application of carbaryl to oats, arthropod population densities decreased from 1,354 to 475 per m2 and remained below population densities of the unsprayed plots for eight weeks. The reduction in phytophagous insect populations was immediate and greater than that of predator populations. However, long- term effects were greater for predatory species than phytophagous species. Similarly, species richness and abundance of carabid communities were significantly lower in cornfields where chemical fertilizers and pesticides were used (Dritschild and Erwin

1982).

Practices associated with planting affect arthropod communities by exposing them to unfavorable environmental conditions such as desiccation, mechanical destruction, and reduced resources. House and Alzygaray (1989) observed more phytophagous and beneficial arthropod species in no-till corn compared with conventional-tilled corn plots.

Crop residues in no-till plots may have promoted a more trophically balanced system and improved habitat conditions for predators to accumulate (Brust et al. 1985).

Below-ground

The frequency and intensity of agricultural practices severely hamper entomopathogenic nematode survival. Tillage physically disrupts the soil microclimate, impacts potential insect hosts, and exposes nematodes to drying conditions and UV radiation. Brust (1991) observed greater nematode populations in no-till systems with

6 weed communities compared with tilled systems in which the soil was bare. The author attributed these results to the weeds in untilled systems buffering nematodes from environmental extremes, and in addition, the increased organic matter possibly providing a greater diversity of insect hosts for the nematodes to recycle. Shapiro et al. (1999) reported similar findings for Steinernema carpocapse, nematode population densities in corn plots with soybean residues were greater compared with plots in which residues were removed by tillage. They also proposed that the ground cover provided a more conducive environment for nematodes to survive than bare plots. Nematode species respond differently to tillage practices. Millar and Barbercheck (2002) observed that tillage significantly affected S. carpocapse and S. riobrave but not H. bacteriophora.

These observations have been linked to the dispersal activity of the nematodes, H. bacteriophora that tend to disperse to deeper depths within the soil may be less impacted by tillage practices than S. carpocapse that dwells closer to the soil surface (Millar and

Barbercheck 2002).

Broad-spectrum insecticides, surfactants, and other formulation ingredients affect entomopathogenic nematode infectivity and survival (Grewal et al. 1998, Patel and

Wright 1996, Zimmerman and Cranshaw 1990). Infective juveniles vary in their susceptibility to pesticides and may be affected differently by formulations of the same pesticide. Generally, heterorhabditids tend to be more sensitive than steinernematids to pesticides. Increased nitrate levels associated with inorganic fertilizers negatively affect naturally occurring nematodes (Bednarek and Gaugler 1997). However, organic fertilizers such as composted manure and urea may increase populations (Shapiro et al.

1997).

7 Impact of Microclimatic Conditions

Above-ground

Temperature, wind, rain, and light intensity influence entomophagous arthropods

(Cloudsley and Thompson 1962). Temperature is a principal factor affecting longevity, development, and dispersal of natural enemies (Cloudsley and Thompson 1962). Orr et al. (1997) observed that the survival of Trichogramma sp. was significantly less in seed- corn plots with exposed soil and in plots with corn residues compared with plots intercropped with rye grass. The authors attributed these observations to higher soil surface temperatures in plots with corn residues and exposed soil. Suh et al. (2002) also observed that high soil temperatures limited the emergence of several Trichogramma species. Longevity of the ichneumonid parasitoid, Eriborus terebrans, was significantly affected by increasing temperature; optimal longevity occurred at 25°C and was lowest at

35°C. At these high temperatures the wasps exhibited escape behaviors (Dyer and Landis

1996). The combination of temperature and solar radiation also impacted flight activity of two Trichogramma species. An accumulation of solar radiation of <15,000 kj/m2 and temperatures >15°C resulted in low flight activity and a reduction in parasitism of aphid eggs by T. evaensis and T. pretiosum. The threshold for reduced dispersion and flight initiation differed for the two wasps and was related to climatic conditions at the site of origin of the species; T. evanensis is originally from Egypt and T. pretiosum is a nearctic species from Quebec (Fourneir and Bouvin 2000). Response to temperature may differ between sexes. Diadegma insulare females are more sensitive to low temperatures and delay the initiation of foraging activity under these conditions, males are less sensitive to these low temperatures. Differences may be related to the female wasps requiring >15°C

8 to parasitize hosts (Idris and Grafius 1998). Light intensity also affected foraging of D. insulare. Trap catches of males and females were lower in the morning and in the afternoon when light intensity was low but peaked at midday when the intensity was highest; poor visibility may be occurring at light intensities <500 µEm-2s-1 (Idris and

Grafius 1998).

Wind is an important factor for parasitoids with limited control over their flight direction (Idris and Grafius 1998). Under low wind speeds, Trichogramma spp. dispersed in all directions, however, as wind speed increased the wasp species exhibited a more down-wind skewed pattern of flight (Fournier and Boivin 2000). The authors attributed differences between species to the thresholds for parasitoid dispersal and flight initiation; they hypothesized that the original hosts and habitat of the parasitoid were partly responsible. Habitats in which hosts are abundant and uniformly distributed or parasitoids with a wide host range would tend to have higher thresholds as control of flight direction would not be as important as species whose host is highly aggregated. In such cases, control of short-range flight is required to locate patches of resources. Wind thresholds leading to reduced dispersal may, therefore, influence residence time and result in more aggregated parasitism.

The combined effect of wind and rain reduces parasitoid dispersal and the efficiency of host-plant searching and detection (Schworer and Volk 2001). Reduced oviposition is common under these conditions. The reproductive success and patterns of parasitism of the aphid parasitoid, Aphidius rosae, decreased under long periods of wind and rain. Periods of rain prevented foraging, whereas long periods of wind resulted in reduced foraging and changed the distribution of parasitism among aphid colonies

9 (Weisser et al. 1997). The lack of foraging activity during rain may be related to host location cues being masked by water on the surface of aphids (Weinbrenner and Volkl

2002). Occasionally, parasitiods interrupt these periods of inactivity by foraging on sheltered plant structures, thus reducing or eliminating the effects of adverse conditions on oviposition rates (Schworer and Volkl 2001).

Below-ground

Persistence of entomopathogenic nematodes is significantly affected by temperature, UV radiation, and soil moisture and texture (Smits 1996, Barbercheck 1992,

Kaya 1990). Temperature directly affects the survival, infectivity, and reproduction of heterorhabditid and steinernematid nematodes (Baur and Kaya 1998, Mason and

Hominick 1995). In sandy soils at 7% moisture, >90% of nematodes survived after 32 weeks at 15oC; however at temperatures either greater than or less than 15oC, survival decreased (Kaya 1990). More heterorhabditids penetrated hosts at 20oC than at 9oC, and optimal infection occurred between 16oC and 32oC (Westerman 1998). Gouge et al.

(1999) also demonstrated that temperature affected infectivity of both H. bacteriophora and S. riobrave. Depending on the species of nematode and the host, optimal infectivity occurred between 22oC and 28.5°C; but reduced infectivity occurred at temperatures

>35°C for all nematodes. Development and reproduction occurs over a slightly narrower range of temperature than infectivity (Mason and Hominick 1995). The ability of nematodes to survive and infect under various soil conditions is related to their biogeography. Nematode species and strains from tropical locales tend to favor higher temperatures than temperate nematodes (Kaya 1990, Molyneux 1985).

10 Soil moisture and texture interact to affect nematode life functions. Clay soils with small particles, high moisture, and limited oxygen, impairs movement of nematodes, as well as host location and infectivity (Baur and Kaya 1998, Koppenhofer et al. 1995,

Kung et al. 1991, Kaya 1990).

Interactions Between Natural Enemies and Herbivores

Due to the ephemeral nature of crop patches, herbivores and natural enemies occupying cultivated habitats disperse among crop patches within the habitat or among different habitat types throughout the landscape to obtain resources (Dunning et al. 1992).

Movement is affected by intrinsic factors such as population age structure, body size, resource specialization and mobility as well as extrinsic factors including climatic conditions and resource distribution (reviewed by Kennedy and Storer 2000). The dispersal abilities of organisms occupying croplands therefore influence population persistence and herbivore-natural enemy interactions (Roland et al. 2000, Hanski 1989).

Above-ground

Insect hosts of natural enemies are patchily distributed in time and space. The decision to forage for hosts is strongly influenced by the physiological state of parasitoids. Two major physiological states that compete are nutritional needs and reproductive status (Lewis et al. 1998, Stapel et al. 1997, Heimpel et al. 1996). Takasu and Lewis (1995) reported that starved Microplitis croceipes females gave priority to

11 locating sugar sources over hosts. In the presence of sugar rewards, a higher rate of host searching and parasitism was observed, whereas super parasitism occurred when females were starved.

Parasitoids rely on volatiles from host plants and herbivores to locate hosts and tend to restrict their searches to areas where there is a high probability of encountering hosts (Turlings et al. 1998). Both laboratory and field studies demonstrate that parasitoids discriminate between patches of high and low host population densities and tend to aggregate in high host population density environments (Wang and Keller 2003, Waage

1983). Geervliet et al. (1998) observed that Cotesia glomerata and C. rubecula were able to discriminate between infochemicals from plants having high compared with low numbers of the host, Pieris rape, and responsiveness increased with host densities.

Despite the tendency to aggregate, the relationship between parasitism and host population densities varies from positive to negative density dependence to a lack of a relationship (Hassell 2000, 1982, Morrison and Strong 1980). Several factors relating to parasitoid behavior have been proposed for these inconsistencies, including mutual interference and superparasitism (Cronin and Strong 1993), deceleration in the functional response at high host population densities (Umbanhowar et al. 2003, Waage 1983) as well as avoidance (Janssen et al. 1995). The scale of response of parasitoids relative to the scale at which measurements are taken has also been cited as a possible contributory factor for these inconsistencies (Morrison and Strong 1980).

12 Below-ground

Infection of insect hosts by entomopathogenic nematodes occurs by the only free- living stage of the nematode, the infective juvenile. Regardless of the presence or abundance of insect host populations, not all nematodes are infectious at the same time

(Bohan and Hominick 1997, Fan and Hominick 1991). The proportion of nematodes that is infective to a host is dynamic and phased over time; “phased infectivity” (Fairbairn et al. 2000). Two mechanisms have been proposed to account for these observations: (i) an intrinsic mechanism acting within each infective juvenile and (ii) an extrinsic chemical cue arising from infected hosts that prevents nematodes from becoming infective and entering the host.

Nematode species and strains differ in their host location strategies. Strategies vary along a continuum between ambush and cruise foraging (Grewal et al. 1994). Those species that ambush hosts nictate and have the tendency to infect mobile surface-dwelling insects. Nematode species that are mobile and actively respond to host cues (e.g., carbon dioxide, or, frass) tend to locate sedentary hosts (Gaugler et al. 1997). The quality and size of the host is important for maximizing the fitness of penetrating infective juveniles.

Under conditions in which a large number of infective juveniles penetrate the same host, nematode fecundity as well as infective juvenile production declines due to competition for limited nutrients (Selvan et al. 1993).

Within natural habitats, entomopathogenic nematode populations exist as a network of local populations that vary in space and time (Garcia del Pino and Palmo

1997, Glazer et al. 1996, Stuart and Gaugler 1994). Spatial and temporal variation in nematode populations is often linked to the density and distribution of hosts (Baur and

13 Kaya 1998, Lewis et al. 1998, Lewis 2002, Loya and Hower 2002). Under controlled conditions, nematode persistence increases in the presence of hosts and a numerical response to host supply is observed (Kaya and Gaugler 1993, Burlando et al. 1993, Kaya

1990). Under field conditions, however, the influence of host factors has been variable.

Campbell et al. (1998) reported that the patchy distribution of H. bacteriophora two months after application was related to the distribution of Japanese beetle grubs. In contrast, Efron et al. (2001) identified no spatial relationship between entomopathogenic nematodes in citrus orchards and their main host, Maladera matrida, but observed a temporal correlation. Higher population densities of entomopathogenic nematodes occurred during times of peak abundance of M. matrida. Inconsistencies in nematode population densities and the presence of hosts may be linked to the means by which infective juveniles disperse (Efron et al. 2001). Phoretic dispersal by arthropods appears to be important for sustaining viable entomopathogenic nematode populations within landscapes (Baur and Kaya 1998). Localized areas of high population densities of infective juveniles may be related to locations where infected insects died and nematodes emerged (Efron et al. 2001, Baur and Kaya 1998). Spodoptera exigua infected with

Steinernema feltiae dispersed up to 11m from the infection site before dying (Timper et al. 1988). Similarly, Parkman and Frank (1991) reported that nematode infected mole crickets, Scapteriscus spp., remained active for several days after infection and were found in traps several kilometers from the initial release site. Epsky et al. (1988) observed

14 that infective juveniles adhered to the dorsum of mesostigmatid mites suggesting that they could disperse nematodes. In addition, long-range dispersal of infective juveniles may occur via water or farming activities (Kaya 1990).

Interactions of Herbivores and Natural Enemies and Landscape Patterns

Typically, vegetable landscapes are a mosaic of habitats consisting of cultivated lands, weedy verges, hedgerows, shrub lands, and forest fragments. Cultivated habitats are composed of patches that change in composition (i.e., species diversity, richness, and dominance) and structure (i.e., layout, shape, boundaries, and connectivity) both locally and regionally as well as through time within a season and across seasons (review by

Kennedy and Storer 2000, Dunning et al. 1992). This shifting mosaic changes the availability, suitability, and accessibility of resources and the dynamics of pest and natural enemy populations (reviews by Tscharntke and Brandl 2004, Kennedy and Storer

2000).

Crop Patch Composition and Structure

Above ground

Host plant quality. Two major hypotheses have been formulated to explain the impact of host quality on herbivore performance “the plant vigor hypothesis” and “the plant stress hypothesis”. The former hypothesis proposes that plants that grow more vigorously are more susceptible to herbivore attack and the latter hypothesis proposes that physiologically stressed plants become more susceptible to herbivores (Price 1991).

Support for both theories is well documented. Teder and Tammaru (2002) observed that

15 pupal weights of two herbivore species increased with increasing vigor of their host plant,

Typha latifola. Similarly, DeBruyn et al. (2002), reported that the density, survival, and development of leaf-mining flies was better on plants with high nutrient levels and minimum stress compared with low nutritive, drought-stressed plants. Comparable trends were recorded for the silver leaf whitefly, Bemisia agentifolii, the leaf miner, Liriomyza trifolii, and the corn earworm, Heliothis zea (Inbar et al. 2001). Conversely, both

Bjorkman (1998) and DeBruyn (1995) recorded an increase in gall formation for adelgid aphids and the gall-forming fly, Lipara lucens, on stressed plants. The presence of nutrient-rich tissue within galls could be responsible for these observations (DeBruyn

1995).

The role of minerals, in particular nitrogen, in increasing insect growth, development and reproduction has been well documented (reviewed by Awmack and

Leather 2002, Yardim and Edwards 2003, Fox et al. 1990, Hunter and McNeil 1977) but inconsistencies have been reported (Haddad 2000). Busch and Phelan (1999) attributed the variation in part to many studies focusing on manipulating a single mineral while holding others constant rather than looking at mineral ratios. The authors demonstrated interactive effects of nutrient proportions (nitrogen, sulfur, and phosphorus) on the development of the soybean looper and the two-spotted spider mite. For both insects, phosphorus was the most limiting mineral but the ratio of N:S:P necessary for increased growth and development differed between the herbivore species. Beanland et al. (2003) used a similar approach and also demonstrated interactive effects of the micro-nutrients, boron, iron and zinc, on development of three herbivore species: soybean looper,

Mexican bean beetle, and the velvet bean caterpillar. Consistent with the previous study,

16 there were differential responses among species; both the Mexican bean beetle and the soybean looper demonstrated non-linear responses in weight gain and the velvet bean caterpillar a linear response. Interestingly, all species responded best to low levels of boron and the effect of zinc was dependent on the ratio of the other micronutrients.

Noxious chemicals that are repellent, unpalatable, toxic, or interfere with the assimilation of nutrients are often the means by which plants defend themselves from herbivory (review by Awmack and Leather 2002). Four main groups of defensive chemicals have been identified, many of which impact growth and development of many arthropod species. Phytochemicals include cyanogenic glycosides and glucosinolates, terpenoids, phenolics, and nitrogen-containing compounds (review by Awmack and

Leather 2002). Some insects have evolved tactics to overcome plant defenses including selectivity in feeding and oviposition choices, enzymatic metabolism of plant compounds, and sequestration (review by Karban and Agrawal 2002).

Insect parasitoids and predators feed directly on plants as well as on insects. Plant floral resources (pollen and nectars) provide carbohydrates and proteins for many parasitoids. The quality of these resources varies among plant species and affect parasitoid development. Longevity of Bathyplectes curculionis was greater on flowers of dandelion compared with alfalfa and phacelia (Jacob and Evans 2000). Cascading effects of host-plant quality on prey quality and parasitoid fitness have been reported. In the absence of direct adult feeding, the impact of plant quality may be less for entomophagous species than for phytophagous species (Teder and Tammaru 2002, reviewed by Cortesero et al. 2000, Jansson et al. 1991). The development of Diadegma insulare was significantly reduced when its host, diamondback moth, was reared on wild

17 brassica species, Erysimum cheiranthoides L., Thlaspi arvense L., and Brassica incana compared with Brassica crop species. The female-to-male sex ratio of D. insulare was also higher on Brassica species than on non-Brassica species (Idris and Grafius 1996).

Fox et al. (1996), attributed differences in sex allocation of D. insulare to the nutritional status of host-plants; four times as many female wasps emerged from diamondback moth larvae reared on collards grown with high rates of fertilizer compared with collards grown with low rates of fertilizer. Female wasps feeding on larvae that were reared on plants with high levels of nutrients parasitized more diamondback moth larvae (Fox et al.

1990).

Host Plant Architecture. The size and structural complexity of plants affect the abundance and distribution of hosts and consequently natural enemy-host interactions

(reviews by Lawton 1983, Bottrell et al. 1998, Cortesero et al. 2000). Large architecturally complex plants are more likely to be colonized by insects than small simple plants as they provide a greater variety of plant parts for insects to colonize, a range of microhabitats, and an array of shelters to escape predation or parasitism (Raghu et al. 2003, review by Lawton 1983). Increased plant structural complexity limited host- finding success of fire ants, Solenopsis invicta; less diamondback moth larvae were captured on plants with complex architecture (Harvey and Eubanks 2004). Similarly, two species of the egg parasitoid, Trichogramma had differential preferences for cruciferous varieties that varied structurally. Cabbage with the simplest structure was preferred to broccoli and brussel sprouts. As plants aged and height, heterogeneity, and connectivity increased, host discovery rates decreased (Gingas et al. 2003). Similarly, the attack rates by the parasitoid, Leptomastix dactylopii, were negatively correlated with increasing

18 plant size, height, leaf number, leaf surface, and branch number (Cloyd and Sadof 2000).

Legrand and Barbosa (2003) observed that foraging activity of Coccinella septempunctata, a predator of the pea aphid, decreased with increasing morphological complexity of the host plant.

Plant surfaces are embedded with chemicals (e.g., waxes and gums) and physical structures (e.g., pubescence, trichomes, and bracts) that can impede or enhance colonization, movement, feeding, mating, oviposition, and food ingestion by both phytophagous and entomophagous insects (Gingas et al. 2002, reviews by Cortesero et al.

2000, Bottrell et al. 1998, Coll et al. 1997). The preference of the predator, Nabis roseipennis, to oviposit on soybeans over tomato, squash or tobacco was related to a combination of glandular trichomes, plant age, and rind toughness (Pfannenstiel and

Yeargan 1998). Glandular trichomes on the surface of tomato plants impaired the searching and handling time of Podisus nigrispinus females, thus weakening the functional response (LeClercq et al. 2000). Leaf domatia (i.e., small pits, pockets, or cavities located at the junction of leaf veins) or leaf hairs shelter natural enemies from adverse environmental conditions and predation (Norton et al. 2002, review by Cortesero et al. 2000). A positive correlation was identified between the presence of domatia on cotton plants and the number of predatory species including Frankliniella occidentalis,

Orius tristicolor, and Geocoris spp. (Agrawal and Karban 1997). Pubescence on apple leaves (var Erwin Bauer) reduced thrips predation on Tetranychus urticae eggs (Roda et al. 2000).

19 Crop-patch diversity. Plant community diversity is an important determinant of the abundance and species richness of natural enemies and their hosts in crop patches.

Two mutually exclusive, but complementary, hypotheses have been proposed by Root

(1973) to explain ecological mechanisms underlying the response of arthropod species to changes in plant diversity, the “enemies hypothesis” and “resource concentration hypothesis” (Andow 1991, 1990, 1982).

The enemies hypothesis proposes that compared with monocultures, polycultures have a greater abundance of food (i.e., nectar and pollen), prey (especially for generalist enemies), shelter, and microclimates conducive for natural enemies. In these diversified systems, natural enemies are more abundant and top-down regulation of pests occurs to a greater extent than in mono-cropped systems (Altieri 1995, Andow 1990). In a recent review, Langellotto and Denno (2004) conducted a meta-analysis of 43 published studies that covered a wide range of natural enemy taxa and differed by spatial scales (habitat and within-plant), and reported that regardless of scale, increasing habitat complexity increased the abundance of parasitoids and predators. Further, they observed that in 79% of studies predators increased and herbivores decreased, and in 65% of studies reduced damage or increased yields occurred in more complex habitats.

Coll and Bottrell (1996) observed that the initial colonization of the parasitoid,

Pediobius faveolatus Crawford, was greater in mono-cropped than in inter-cropped systems; however, emigration was much less in intercropping suggesting that the diversified environment was more suitable for the natural enemy. Both Skovgard & Pats

(1996) and Patis et al. (1997) reported greater egg parasitism of the lepidopteran stem borers, Sesamia calamistis, Chilo parlellus Swinhue, and C. onchalcociliellus Strand, in

20 plots intercropped with maize and cowpea compared with corn monoculture. Greater population density, diversity, and activity of carabid and staphylinid species occurred in plots intercropped with cabbage and multiple species of clover than in mono-crops (Booij et al. 1997). Letourneau (1987) observed that the abundance of parasitoids that attacked the pyrallid, Diaphania hyalinata, was greater in tricultures of squash, maize, and cowpea when compared with squash monocultures, but not when compared with maize monocultures, suggesting that the response of natural enemies was related to host-plant characteristics (chemical and physical).

Entomophagous species rely on volatiles emitted from host plants to locate and select potential prey, the mixing of chemical cues associated with different crop species may reduce searching efficiency of natural enemies in diversified systems (Whitfield

2001,Takabayashi et al. 1998, Vinson 1981, Price et al. 1980). This is especially true for specialist natural enemies that rely on specific cues to locate hosts (Sheehan 1986).

Perfecto and Vet (2003) reported the reverse for two Cotesia spp.; the foraging range for the polyphagous parasitoid, C. glomerata was reduced to a greater extent than the monophagous C. rubeca in mixed plots of brussel sprouts and potatoes. Vegetational textures and architecture as well as high leaf-to-soil ratios within diversified systems may also be contributing to the reduced search efficiency of natural enemies (Cortesero et al.

2000). Sato and Ohsaki (1987) attributed the low searching efficiency of Apanteles glomeratus to its inability to perceive chemical cues in environments in which host plants were shaded by weeds.

21 The resource concentration hypothesis proposes that in diverse patches, food sources are less concentrated and pests (specialists) have a harder time moving and reproducing in these environments (Andow 1982). Generally, there is a tendency for more diversified systems to have fewer pests than simple cropping systems (Ramet and

Ekbom 1996, Elmstrom et al. 1988). Risch et al. (1983) reviewed 150 studies of 198 arthropod species and observed that herbivory was reduced in diversified systems in 53% of the studies, increased in 18%, was variable in 20%, and had no change in 8% of studies.

Support for the resource concentration hypothesis has been widely documented

(Patis et al. 1997, Elmstrom et al. 1988, Risch 1981, Bach 1980). Less thrips damage to leeks was observed when they were intercropped with subterranean clover (Theunissen and Schelling 1997). Weedy bean cultivations reduced colonization of the beetle,

Epilachna varivestis, by inhibiting immigration and hastening emigration (Andow 1990).

Similarly, the specialist beetle, Acalymma vittatum Fab., showed increased within-plot movement and short tenure in diverse habitats (Bach 1980). Flea beetle, Phyllotreta cruciferae Goeze, had impeded movement (Elmstrom et al. 1988), and the Colorado potato beetle, Leptinotarsa decemelineata Say, experienced reduced oviposition in response to host-plant diversity (Horton and Capinera 1987).

Possible mechanisms underlying the resource concentration theory include the masking of host-location cues or confusion, or both, and the repulsion of herbivores to the many visual and chemical cues emanating from complex systems (Andow 1990, Bach

1980). Polyphagous herbivores with low olfactory specificity may be unable to detect subtle quantitative differences in host-plant odors, and are less affected by the mixing of

22 cues than monophagous herbivores, that are very sensitive to host-odor quality and respond to subtle changes in the odor profile. Monophagous species tend to leave these complex systems and search for more suitable habitats (Andow 1991, Sheehan 1986).

Non-host plants may also act as physical barriers and obstruct life functions of herbivores

(Risch 1981, Litsinger et al. 1991). Recently, Finch and Cronin (2000) proposed that the underlying mechanism for reduced pests in polycultures is behavioral rather than a result of physical, chemical, or visual factors. They state that insects do not discriminate between the visual cues of host and non-host plants and land on both. They propose that after accepting a host, the insect makes a series of spiral flights around the plant before landing. In the presence of non-host plants (polycultures), insects will have the tendency to lose orientation to their selected plant at a higher rate than in monocultures; hence less herbivory occurs.

Crop Patch Size. The influence of patch size (plant density) on herbivore abundance differs among species (Idris and Grafius 2001, Maguire 1983, Ralph 1977,

Cromartie 1975). Variability in responses is related in part to the host range of the herbivore. The crucifer specialist, Pieris rapae, was significantly more abundant in large plant patches, whereas generalist herbivores, Frankliniella occidentalis, the flower thrips, and Myzus persicae, the green peach aphid, showed no preferences for crop patches of any particular size (Maguire 1983). However, Bach (1988a) observed contrasting responses among three closely related specialist chrysomelids. Numbers of the striped cucumber beetle, Acalymma vittatum, were greater in smaller patches than in larger ones; the spotted cucumber beetle, Diabrotica undecimpunctata, had larger numbers in intermediate patches and populations of the Western corn root worm, D. virgifera, tended

23 to increase with increasing patch size. The impact of patch size on beetle populations also varied among generations of the same species. Patch-edge effects were in part responsible for the variations among these specialist species; edge plants acted as reflective boundaries, impeding upward flights and retaining beetles in plots thus smaller plots with higher edge-to-area ratios had higher beetle numbers (Bach 1988b). Herbivore abundance commonly increases with decreasing plot size. Pieris rape aggregated in small plots with higher edge proportions because edge plants were closer to wild crucifers that served as adult food sources (Macguire 1983). Likewise, Cappuccino and Root (1992) observed that the tingid, Corythucha marmorata, was more abundant in small patches and females laid more eggs on stems of edge plants than plants inside the plots.

Microclimatic conditions along edges as well as host-plant quality were suggested as contributory factors.

Smaller herbivore populations in small plots may also result from increased emigration. Kareiva (1985) attributed the higher emigration rate of Phyllotreta flea beetles from smaller patches to an increase in boundaries from which beetles could emigrate. A greater risk of predation along patch edges may have also contributed

(Cappuccino and Root 1992, Landis and Hass 1992). For those species not exhibiting strong edge effects and having low emigration rates, immigration could be the dominant factor influencing population density in crop patches (Bowman et al. 2002).

Similar to herbivorous species, the influence of patch size on the abundance of natural enemies is variable. Host-searching ability and parasitism levels of D. insulare were not significantly limited in high population density collard plots; however the total

24 number of Diadegma sp. captured was greater in high population density compared with low population density collard stands (Idris and Grafius 1996). Conversely, colonization of the generalist predator Orius tristicolor in monoculture and polyculture plots was dependent on plant architecture and plant population density rather than on prey populations and diversity (Letourneau 1990).

Below-ground

Crop-Patch Composition. Nematode survival, infectivity, and reproduction can be affected directly or indirectly by plant-community composition. Vegetation (weed mixtures, corn residues) buffers infective juveniles from harsh environmental conditions and improves their survival in the soil environment (Shapiro et al. 1999, Brust 1991).

High organic matter environments may also provide hosts and conditions in which nematodes can recycle (Brust 1991). Within the soil, infective juveniles use cues from plant roots to locate hosts. Van Tol et al. (2001) observed that host location by H. megidis was improved by chemicals released from coniferous plants infested with weevils; these plants were highly attractive to infective juveniles. An abundance of cues emanating from plant roots (e.g., CO2, root exudates), however, can hinder host location (Choo et al.

1989). Similar to above-ground systems, cascading effects of host-plant quality on life- history traits of infective juveniles have been observed (Kunkel et al. 2004, Barbercheck et al. 2003, Kunkel and Grewal 2003). For example, both Barbercheck et al. (1995) and

Eben and Barbercheck (1997), report that the infectivity and reproductive potential of H. bacteriophora in Diabrotica sp. was dependent on the presence and the levels of cucurbitacin present in the host plant consumed by the beetle.

25 Landscape Composition and Structure

Scale

The regulation of pest by predators and parasitoids in vegetable agroecosystems is influenced by landscape patterns at particular spatial and temporal scales. Bommarco and

Banks (2003), conducted a meta-analysis to elucidate the role of scale in determining the relationship between the abundance of phytophagous and entomophagous arthropod species and habitat diversity, and observed that variation in results was related to the scales at which studies were carried out. Experiments conducted in small plots (<16m2) showed a significantly negative relationship between plant diversity and herbivore abundance, those in medium plots (16-256m2) resulted in an intermediate effect for herbivores but an increase in the number of predatory species, and those in large plots

(>256 m2) showed no effect of diversity on herbivores or natural enemies. Differences in results were linked to the life-history traits of the organisms as well as the scale of analysis (Tscharntke and Brandl 2004, Menalled et al. 1999, Jonsen and Fahrig 1997,

Banks 1998). Koricheva et al. (2000), observed that manipulating plant diversity (within habitats) only impacted species that had a narrow host range and were relatively immobile; whereas, highly mobile polyphagous species moved between plots with diminished treatment effects. Further, Chust et al. (2004) reported differences in the scales of response of two mobile insect groups with different body sizes. They reported that the relative abundance of homopterans was influenced by plant diversity at local spatial scales (0.36-2.25 ha), and dipterans were more sensitive to plant diversity at landscape scales (> 250 ha). The extent to which landscape patterns influence ecological processes of biological organisms generally depends on the scale at which these

26 organisms perceive spatial patterns (Chust et al. 2004, Turner 1989, Levin 1992,

Litsinger et al. 1991). Some organisms may view spatial patterns at larger scales and perceive many different habitats as a single habitat, whereas other may only perceive patterns at scales of individual crop patches or individual plants. Landscape patterns cannot be interpreted accurately without reference to the range of scales that are relevant to the target organism and the ecological processes that are being examined; there is no single correct scale to view ecosystems (Levin1992).

Composition, Fragmentation and Connectivity

Increasing landscape diversity increases species richness and abundance of herbivores and natural enemies (Mendalled et al. 2003, Corbett and Rosenheim 1996,

Fahrig and Paloheimo 1988, Cromartie 1975). Examples are available in widely varying ecosystems. Introducing hedgerows of ivy, hawthorn, poplar and ash resulted in an increase in the composition and abundance of herbivorous insects and a complex of entomophagous species that fed on or parasitized psyllids in pear orchards (Rieux et al.

1999). Likewise, the abundance and richness of stem-boring herbivores and their parasitoids in the creeping thistle, Cirsium arvevse, was amplified in non-cropped habitats (Kruess 2003). Marino and Landis (1996) observed higher parasitism of

Pseudaletia unipuncta in complex agricultural landscapes with crop lands intermixed with hedgerows and woodlots compared with simple landscapes consisting primarily of cropped lands; parasitism was 13.1% and 2.4% in the two landscapes, respectively.

Aggregation of phytophagous insect species and natural enemies in complex landscapes is attributed to resources such as food, shelter, and alternate hosts (reviews by

27 Gurr et al. 2003, Landis et al. 2000). Flowering vegetation along crop habitats provide essential nutrients (i.e., nectar, pollen, honeydew) and promotes higher survival, longevity, and fecundity of natural enemies (Cortesero et al. 2000, Baggen and Gurr

1998, Verkerk et al. 1998, Sheehan 1986, Altieri 1995). D. insulare, a key parasitoid of diamondback moth, had high fecundity and longevity when it fed on flowering plant species commonly found in and around crop fields, namely, yellow rocket, Barbarea vulgaris, wild carrot, Daucus carota, and wild mustard, Brassica kaber (Idris and Grafius

1997, 1996). Similarly, Cotesia marginiventris was 4.8 times more likely to survive it fed on sweet alyssum, Lobularia maritime (Johanowicz & Mitchell 2000). Habitats within landscapes may act as refugia or overwintering shelters. Grasslands, woodlots, and field boundaries surrounding farms acted as overwintering sites for carabids, staphylinids, dermapterans, and lacewings and increased their abundance within cereal fields (Thomas et al. 1991, Duelli et al. 1990, Sotherton 1985, Sotherton 1984). Various habitats within the landscape can also serve as a source for alternate prey for natural enemies (review by

Landis et al. 2000) or alternate host plants for phytophagous species (review by Kenndy and Storer 2000). Bianchi and van der Werf (2004) documented that non-cropped habitats in a complex agricultural landscape provided the generalist predator, Coccinella septempunctata, with alternate hosts when its primary aphid host was scarce.

The configuration, isolation, size, and quality of crop habitats within landscapes influence the distribution and persistence of phytophagous and entomophagous species

(Tscharntke at al. 2002). These habitat characteristic are especially more critical for species that travel between different types of habitats to obtain resources (Dunning et al.

1992). Parasitoids generally are more host specific than predators and generally are more

28 affected by habitat loss than their hosts; as a result, disruptions in host-parasitoid interactions are more likely in fragmented landscapes (Tscharntke and Brandl 2004,

Tscharntke et al. 2002). The abundance of stem borers, seed feeders, and parasitoid species attacking these herbivores in red clover, Trifolium pratense patches was correlated negatively with distances between red clover patches. Immigration rate of the seed feeders decreased with increasing distance of crop patches. Top-down regulation of herbivores was reduced by 19-60% in highly isolated patches (fragmented) compared with patches that were close together (Kruess and Tscharntke 1994).

Species with limited dispersal abilities are influenced to a greater extent by habitat fragmentation than highly mobile species (Roland et al. 2000, Banks 1998, Herzig and

Root 1996, With and Crist 1995, Weins 1993, MacDonald and Smith 1990, Fahrig and

Paloheimo 1988). Roland and Taylor (1997) observed that differences in response of four tachinid flies to forest fragmentation were related to their body sizes. They observed that parasitism of the forest tent caterpillar, Malacocoma disstria, by the largest tachinid fly was most strongly correlated when forest structure was measured at a scale of 850m around each site, for medium flies between 212-425m, and the smallest fly at 53m. The effect of fragmentation on population distribution may not occur until some critical level of habitat connectivity is disrupted (With and Crist 1995). Specialized species have higher critical levels of habitat connectivity than polyphagous species, which use a blend of resources.

29 The composition, quality, and configuration of crop and non-crop patches between the focal crop patch (matrix) influences connectivity (Haynes and Cronin 2004,

Taylor et al. 1993, Dunning et al. 1992, Karieva 1985). The matrix may increase the amount of available resources, provide additional resources that are not present in crop patches, or influence the structural connectivity of crop patches and dispersal activity of herbivores and natural enemies (Steffan-Dewenter 2002). The degree to which the matrix facilitates or impedes movement within surrounding patches is a measure of the dispersal efficiency of arthropods (Taylor et al. 1993 Fahrig and Merriam 1985). Roland et al.

(2000) measured that movement of the Alpine butterfly, Parnassius smintheus, was restricted to a greater extent in forest patches than in open meadows of equivalent areas.

Similarly, the distribution of the parasitoid, Anagrus columbi, within patches of prairie cordgrass was reduced when patches were surrounded by mudflats as opposed to native or exotic grasses. The mudflat tended to repel A. columbi consequently the wasps were more abundant in the interior than on the edges of patches. The exotic grass was more permeable and resulted in an even distribution of wasps within patches. More wasps were therefore found in patches embedded in the exotic grass matrix than in the mudflat

(Cronin 2002).

Linear patches of land (corridors) can act as dispersal conduits, aiding the movement between patches; this is particularly important for species whose resource requirements exceed the average patch, or whose patches are isolated and embedded in unsuitable habitats (Rosenberg et al. 1997). Inter-patch movement and densities of

Junonia coenia and Euptoieta claudia, two species of open-habitat butterflies increased in the presence of corridors with the greatest influence occurring at large inter-patch

30 distances (Haddad 1999). Nicholls et al. (2001) also reported greater dispersal of leafhoppers, thrips, and a complex of natural enemies into grape vineyards in the presence of corridors; a gradient was observed with the highest population densities being in the center of fields. Similarly, early-season abundance of populations of Anagrus epos in grape vineyards was associated with French prune trees that were nearby (Murphy et al. 1996); however parasitism was limited only to a few rows located down wind of the trees and there was a gradual decline in numbers of A. epos with increasing distance from the trees (Corbett and Rosenheim 1996). The inclusion of a five-meter wide vegetational strip that included flowering plants running through the field increased the dispersal of A. epos, introduced a complex of predators into the center of fields, and provided nectar sources and alternate hosts for the natural enemy community (Nicholls et al. 2001). The occurrence of increased densities of phytophagous and entomophagous species in connected patches may be related to: (i) a greater frequency of movement between patches, (ii) an increase in the usable area of patches and (iii) interception and re- direction of species from the surrounding landscape into the patches (Haddad and Baum

1999). These factors promote gene flow and recolonization of unoccupied patches and reduce in-breeding and the chances of extinction (Rosenberg et al. 1997).

Conceptual Model

Based on the literature reviewed, conceptual models of the potential trophic interactions occurring in a vegetable landscape were developed for the entomopathogenic nematode and D. insulare (Figures 1.1 and 1.2). For the entomopathogenic nematode, the model proposes that crop patch factors (i.e., size, diversity, and host-plant quality and

31 architecture) as well as landscape elements (i.e., matrix, connectivity and diversity) affect life processes of pest and non-pest herbivores and their subsequent composition and population densities in crop patches. Pest and non-pest herbivores with life stages in the soil serve as hosts for the nematode to infect, feed, and reproduce. Based on the limited dispersal capability of entomopathogenic nematodes, population persistence will be dependent on these pest and non-pest host species dispersing into crop patches in which nematodes are present. Over short ranges, nematodes may use cues from crop plants to locate hosts. Disturbances typical of vegetable systems and microclimatic conditions affect nematode survival directly and also indirectly through lower trophic levels.

For D. insulare similar interactions are proposed; but the scales of the interactions are different. For the nematode, responses occur at scales of cm-m and for the wasp responses occur at scales of m-km. The dispersal capability of D. insulare allows the wasp to emigrate from crop patches during unfavorable conditions thus, compared to the nematode the impact of environmental factors and disturbances on population persistence is less. In contrast to the nematode, the first trophic level directly affects the persistence of D. insulare both at the landscape and crop patch scale. Landscape elements such as the connectivity of the focal patch with other crop patches and habitats directly affect the efficiency at which D. insulare disperses in and out of the focal patch. Crop patch composition also directly influences wasp reproduction, as flowering species in crop and non-crop patches within the focal patch and surrounding crop patches and habitats serve as a source of food.

32 In relation to life processes driving the persistence of these natural enemies, reproductive success within crop patches is key for maintaining their populations. For D. insulare, dispersal is another major life process regulating persistence in crop patches.

Natural enemy persistence in cropping areas would be required for top-down regulation of soil pests by entomopathogenic nematodes and diamondback moth populations by D. insulare, evaluation of top-down regulation of pests is beyond the scope of this project.

Current Research Status

For entomopathogenic nematodes, most of the research has focused primarily on top-down regulation of soil and foliar pests with inundative releases of infective juveniles and the subsequent reduction in crop yields (Figure 1.3). With respect to population ecology, the influence of environmental factors (e.g., soil moisture, temperature, UV radiation), agronomic stresses (e.g., tillage, agri-chemicals), and biotic agents such as soil antagonists on nematode survival, infectivity, and reproduction and the subsequent persistence of populations has been widely addressed. The role of host and plant communities in regulating endemic nematode populations has been studied to a lesser extent. When plant communities have been researched the focus has been on the impact of the physical and chemical properties of crop plants on nematode life processes namely; survival, infectivity, and reproduction. Investigations on the impact of plant communities in regulating nematode population densities through host communities is an area with limited information. For D. insulare, the influence of host population densities and distribution on population persistence within cropping habitats has been examined

(Figure 1.4). In addition, the role of flowering plant species on life processes such as

33 survival and reproduction of the wasp has been widely researched. However, the interaction of these insect host populations and flowering plants on parasitoid population persistence and parasitism is not well documented. Studies have been carried out under laboratory or greenhouse conditions with few studies being performed at scales representative of vegetable operations.

THESIS

Effective top-down regulation of pests in vegetable systems is dependent on persistence of their natural enemies in the cropping area. Natural enemy populations are regulated by lower trophic groups as they provide resources (e.g., shelter, hosts, food) necessary for their survival, development, and reproduction. My research supports bottom-up regulation of natural enemies occupying soil (entomopathogenic nematode) and foliar habitats (D. insulare) by testing hypotheses on the roles of host and plant communities on population persistence. Because of the transience of annual cropping systems, conservation biological control requires the manipulation of habitats to provide natural enemies with hosts and food at the crop patch scale or the landscape scale, or both. From the research generated, a practical habitat manipulation strategy is proposed for conserving entomopathogenic nematodes at a crop patch scale and D. insulare at a landscape scale.

34

EPN (#m-2)

Dispersal Feeding Reproduction Mortality

Landscape

Diversity Connectivity Matrix

Crop patch Herbivore Diversity Quality Shape -2 Architecture (#/m ) Size Edges Environmental & Agronomic Factors

Dispersal Feeding Reproduction Mortality

Figure 1.1. Conceptual model of bottom-up regulation of entomopathogenic nematodes (EPN) by crop patch and landscape components and the subsequent top-down regulation of soil pests within a heterogeneous vegetable cropping system.

35

Parasitoid (#/sqm)

dispersal Feeding Reproduction Mortality

Landscape Diversity Connectivity Matrix

Crop patch Herbivore Diversity Quality Shape Architecture Size (#/sqm) Edges Environmental & Agronomic Factors

Dispersal Feeding Reproduction Mortality

Figure 1.2. Conceptual model of bottom-up regulation of the parasitoid, Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) by crop patch and landscape components and the subsequent top-down regulation of the diamondback moth, Plutella xylostella L (Lepidoptera: Plutellidae) within a heterogeneous vegetable cropping system.

36

EPN (#m-2)

- -

Environmental - Factors + 0 - + 0 - Agronomic Practices

+ 0 - Herbivore - (#m-2)

+ 0 - Landscape Crop Patch

-

Figure 1.3. Current research status of bottom-up regulation of entomopathogenic nematodes (EPN) by lower trophic groups and the subsequent top-down regulation of soil pests. Line thickness is representative of the amount of research conducted in the area; symbols are indicative of the results of the studies: + - increase,- - decrease, 0 – no change.

37

+ - Diadegma insulare (#m-2) -0 -0

Environmental + 0 - + 0 - - Factors

+ - Agronomic Factors

+ - - Diamondback moth (#m-2)

+ - Landscape Crop Patch

+ - - -

Figure 1.4. Current research status on bottom-up regulation of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae), by lower trophic groups and the subsequent top-down regulation of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae). Line thickness is representative of the amount of research conducted in the area; symbols are indicative of the results of the studies: + - increase,- - decrease, 0 – no change.

38 CHAPTER 2

DISTRIBUTION AND PERSISTENCE OF ENDEMIC ENTOMOPATHOGENIC

NEMATODES IN HETEROGENEOUS VEGETABLE LANDSCAPES

IN HURON COUNTY, OHIO

INTRODUCTION

Entomopathogenic nematodes within the families Steinernematidae and

Heterorhabditidae (Order: Rhabditida) are obligate parasites of insects that have been recovered from soil in all six of the world’s biomes (Nearctic, Neotropical, Palearctic,

Ethiopian, Oriental and Australian) and from five orders and over fifteen families of insects (Stock et al. 1999, Garcia del Pino and Palmo 1996, Peters 1996, Baur and Kaya

1998). These nematodes are potential biological control agents for many soil-dwelling pests, however, low persistence and efficacy of infective juveniles after field releases has limited their wide scale use in vegetable production (Lewis et al. 1998).

Considerable variation exists among nematode species and strains in their ability to survive various environmental conditions, as well as infect and reproduce within insect hosts (Grewal et al. 2002, Baur and Kaya 1998, Georgis and Grewal 1998). Differences in the persistence of species and strains to climatic conditions are correlated with the geographic origin of the nematode (Grewal et al. 1994, Kaya 1990). Understanding the 39 abiotic and biotic factors responsible for the persistence of endemic nematode populations is therefore important for managing field releases of infective juveniles in production areas.

In an effort to understand the factors regulating natural population of entomopathogenic nematodes within the vegetable landscape of Huron County, Ohio, investigations were conducted to determine the natural occurrence of these beneficial nematodes in the production landscape and to identify the abiotic and biotic factors responsible for their persistence. Specifically, soil temperature and moisture as well as the plant and insect communities were monitored with respect to nematode population density.

METHODOLOGY

Surveys for endemic entomopathogenic nematodes

The vegetable production area in Celeryville (Huron County, Ohio) spans approximately 16 km2 and is largely comprised of cultivated areas interspersed with hedgerows, grassy banks, shrub lands, wetlands, farm buildings, and residences.

Generally, the soils within the area are muck soils (moist bulk density of 0.15-0.4 g/cc, pH of 4.5-7.8, and OM content of 40-70%) and silt loams (moist bulk density of 1.3-1.82 g/cc, pH of 4.5-7.3, and clay content of 15-35%) (Ernst and Martin 1994). Surveys were conducted between July and August in 2000 and 2001, and November 2002 to identify the presence of endemic entomopathogenic nematodes

40

Survey of cultivated areas (Summer 2000)

Soil and crop maps were used to demarcate the production area into eight unique sections (Table 2.1). From each section, at least 25 soil samples were collected (n= 220).

The location of each sample was recorded with a Global Positioning System (GPS,

Trimble Navigation Limited, Pro XL 8-channel receiver; Thales GeoSolutions, LandStar

Mark IV Navigator receiver). At each sampling point, three cores (11cm diameter, 20cm depth) of soil were collected (3.5kg), placed in a polyethylene bag and kept in a cooler

(20°C) for transport to the laboratory for processing.

Survey of grassy verges adjacent to cultivated areas (Summer 2001)

Soil along grassy verges adjacent to cultivated areas were sampled at intervals of

55–65m (n= 100). The locations of the sample sites were recorded and soil samples were collected similar to the procedures used in Summer 2000. Arthropod populations were sampled at each location. All vegetation within a 1m radius of the sample point was vacuumed to sample foliar-insect communities (modified leaf blower, STIHL BG72).

Insects collected were placed in polyethylene bags and kept in a cooler (20°C) for transport to the laboratory. Two pitfall traps were established to measure ground dwelling arthropod activity per sample site. Traps consisted of a plastic cup (473ml, Dart

Container Corp., Mason Michigan) in which a paper funnel (20ml conical cup,

Sweetheart Products. Maryland) was placed just below the rim. The cup was covered with a lid with a 6cm hole (10cm diameter, Dart Corp., Michigan). Traps were placed 1m apart and buried so that they were level with the soil surface.

41

Section 1Soil Type Crops typically planted

1 Lenawee silt clay loam Greens, pumpkins, peppers

2 Linwood muck - marginal 2Mixed vegetables

3 Linwood muck Mixed vegetables

4 Linwood muck Potatoes and Onions

5 Carlisle – old marginal muck Mixed vegetables

6 Carlisle – old muck Mixed vegetables

7 Carlisle – new muck Potato and onions

8 Carlisle – new muck Mixed vegetables

1Lenawee silt clay loam: Moist Bulk Density 1.25-1.5 g/cc, pH 5.1-7.3, Clay 30-40%, Organic Matter 5-15% Linwood Muck: Moist Bulk Density 0.15-0.4 g/cc, pH 4.5-7.8, Clay 0%, Organic Matter 40-70% Carlisle Muck: Moist Bulk Density 0.13-0.23 g/cc, pH 4.5-7.3, Clay 0%, Organic Matter >70% (Ernst and Martin 1994) 2 Mixed: vegetables included lettuce, radishes, leeks, and cilantro,

Table 2.1. Characteristics of crop and soil sections surveyed in the vegetable production area, Celeryville (Huron County, Ohio), in 2000.

42 The traps were placed at the sample site at the time of sampling and were retrieved with captured insects after one week. In the laboratory, all samples were placed in the freezer for a minimum of 48 hours after which insects were placed in vials containing alcohol for identification to family.

Survey of cultivated, semi-natural and natural habitats (Fall 2002)

Soil samples were collected from four habitats associated with the vegetable landscape; intensely cultivated cropped areas, grassy banks adjacent to crop cultivated areas, uncultivated shrub-lands, and uncultivated forest (Table 2.2). A total of 30 sites were sampled within each habitat (n=120). Sample sites were recorded and soil samples were collected similar to the procedure used in 2000.

Assessment of entomopathogenic nematode populations

For all surveys, the presence of entomopathogenic nematodes was determined using a modified insect baiting technique (Fan and Hominick 1991). Soil was mixed,

600g were removed, and 200g were placed in each of three plastic containers (470ml Deli plastic containers with lids, DART Container Corp. Mason, Michigan). Ten greater wax moth, Galleria mellonella larvae were incorporated into the soil of each container and samples were incubated in a growth chamber in total darkness, at 25°C, and 88% RH.

One week after incubation, all dead insects were individually placed in modified White traps (White 1927) and placed in growth chambers for an additional 7 to10 days. Traps were observed for the presence of entomopathogenic nematodes.

43

Habitat Disturbance Regime Soil Type Clay OM pH (%) (%)

Intensely cultivated High - weekly Carlyle Muck 0 >70 4.5-7.3 cropped (cilantro) applications of pesticides, tillage, and crop removal.

Grassy banks adjacent Medium - mowing Linwood muck 0 4.5-7.8 to cultivated areas every 2-4 weeks Carlyle muck 0 >70 4.5-7.3 Colwood silt loam 27-35 2-4 4.5-6.0

Uncultivated shrub Low - natural events Bennington silt loam 15-25 2-4 4.5-7.3 Cardington silt loam 12-20 0.5-3 4.5-7.3 Colwood silt loam 27-35 2-4 4.5-6.0

Uncultivated forest Low - natural events Bennington silt loam 15-25 2-4 4.5-7.3 Carlyle muck 0 >70 4.5-7.3 Walkill silt loam 12-25 1-3 5.6-7.3 Reference: Ernst and Martin (1994).

Table 2.2. Soil type and disturbance regime of habitats surveyed within the vegetable landscape of Celeryville (Huron County, Ohio), in 2002.

44 The presence of entomopathogenic nematodes was confirmed by inoculating wax moth larvae with the emerging nematodes and observing cadavers for characteristic nematode symptoms (i.e., color, texture, and smell). Nematode-infected cadavers were then established in White traps and the emerging nematodes were stored at 10°C.

Persistence of endemic entomopathogenic nematode populations (Summer 2003)

Six sites along grassy banks at which nematodes were previously detected and six sites at which they were not detected (survey 2001) were monitored for a growing season to determine the factors affecting persistence of nematode populations (May – October

2003). Sites were relocated with the GPS unit, and at each location four-1m2 quadrats were measured from the sample point. Within each 1m2 area, four cores of soil were collected (2.54cm diameter, 12cm depth, approx 200g) and bagged separately. Samples were placed in a cooler and transported to the laboratory where nematode population densities were estimated. Soil temperature and moisture content as well as the composition and abundance of plant and arthropod species were also documented at each site.

Arthropod populations were assessed by vacuuming the foliage within the sample area, as previously described. Small insects and larvae were extracted from the soil by washing the soil (200g) through a series of graded sieves (US standard sieve series #12

(1.68mm), #18 (1mm), #35 (500µm)). Microarthropods (Collembola, mites and small larvae) also were extracted from the soil (200g) using a heat-gradient apparatus (Tullgren funnel) as described by Edwards (1991). Arthropods collected were preserved in alcohol

(70%) and identified to family for insects and to class for other arthropods.

45 Nematode population density assessment

The standard Galleria bait method was used to determine the population density of infective juvenile nematodes within the soil (Bedding and Akhurst 1975). For each sample (x4 per site), the soil was mixed thoroughly and 100g were placed in a plastic container (470ml Deli plastic containers with lids, DART Container Corp. Mason,

Michigan). Ten wax moth larvae were added and the container sealed and placed in a chamber in total darkness, at 25°C, and 60% RH. After three days of incubation, all wax moth larvae were removed and the soil was re-baited with an additional ten wax moth larvae and incubated for an additional three days. Larvae were then examined and those killed by nematodes (i.e., having characteristic color, smell, and texture) were dissected and the numbers of infective juveniles inside the cadavers were recorded. Live larvae were incubated for an additional three days after which nematode infections and populations were recorded as previously described. If cadavers with nematode-infection characteristics had no observed infective juveniles upon dissection, then a value of one was assigned as the minimum number of infective juveniles that could have been responsible for larval mortality.

Statistical analyses

All data were transformed to achieve normality and equality of variances according to the ladder of powers cited by Fry (1999). Nematode population data was transformed by square root and the arthropod data by log10. In 2001, analysis of variance

(ANOVA) was used to test for significant differences in arthropod populations between sites at which nematodes were detected and those where they were undetectable. In 2003,

46 multiple regression was used to determine the principal factors influencing nematode persistence. Correlations were conducted to determine the relationship between nematode densities at the sites and the variables measured (i.e., soil moisture and temperature, and arthropod population abundance). Multivariate analysis of variance was used to compare changes in moisture levels over time and insect abundance at sites at which nematodes were detected and those at which they were not detected. Multivariate analysis of variance was conducted in SAS and regression models and correlations in MINITAB.

RESULTS

Surveys for endemic entomopathogenic nematodes and arthropods

Entomopathogenic nematodes were detected along grassy verges (adjacent to cultivated areas) but not within the cultivated areas, shrub lands or forests (Figure 2.1).

Entomopathogenic nematodes were detected in 15% and 30% of samples collected in

2001 and 2002, respectively. Heterorhabditis bacteriophora, and Steinernema feltiae were the two species of nematodes recovered. H. bacteriophora was the most frequently identified species, present in over 60% of positive samples. A total of thirty-eight families of insects were recorded along the grassy banks (Table 2.3). The most abundant arthropods were leafhoppers. Total arthropod composition and abundance did not differ significantly between sites at which nematodes were recovered and those at which nematodes were not recovered (F= 3.49; df= 1,48; P= 0.067); mean arthropod density per site was 12.0 (±7) per 1m2.

47

Figure 2.1. Distribution of entomopathogenic nematodes within the vegetable production area of Celeryville (Huron County, Ohio) in 2000 and 2001.

48

Order Family Abundance Order Family Abundance

Cicadellidae Hemiptera Pentatomidae 1 Homoptera 181 Reduvidae 2 Membracidae 10 Nabidae 3 Miridae 8 Coleoptera Chrysomelidae 10 Elateridae 28 Orthoptera Gryllidae 1 Curculionidae 5 Acrididae 2 Scolytidae 1 Tettigoniidae 4 Carabidae 1 Cicindelidae 29 Hymenoptera Formicidae 6 Cydnidae 14 Ichneumonidae 11 Staphylinidae 4 Braconidae 11 Scarabidae 1 Eulophidae 1 Coccinellidae 7 Encyrtidae 6 Diptera Tephritidae 6 Thysanura Thripidae 6 Lonchopteridae 16 Culicidae 11 Dermaptera 2 Drosophilidae 5 Syrphidae 38 Other (Class)Aracnida (spiders) 3 Scatopsidae 3 Isopoda 64 Sepsidae 1 Diplopoda 14 Phoridae 2 Tipulidae 14 Agromyzidae 3 Muscidae 1 Tachinidae 10 Chloropidae 6

Table 2.3. Composition and abundance of arthropod populations along grassy banks adjacent to cultivated areas in Celeryville (Huron County, Ohio) in July 2001.

49

16000

12000 2 8000

IJ's/4m 4000

0

May Jun Jul Aug Sept Oct Months

Figure 2.2. Temporal variation in entomopathogenic nematode population densities across positive sites along grassy banks adjacent to the cultivated areas in Celeryville (Huron County, Ohio), May-October 2003. IJ’s – infective juvenile nematodes.

50

50000

40000 2 30000 IJ's/4m 20000 9A 8B

c 7B 6A

10000 2A 25B

21A 0 17B 16B

15B Sites 12B May July June Aug 10A

Oct Sept Dates

Figure 2.3. Spatial and temporal variation of entomopathogenic nematode populations along grassy ditch banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May-October 2003. IJ’s – infective juvenile nematodes

51

May June 100% 100%

75% 75%

50% 50%

25% 25%

0% 0% 10A 12B 15B 16B 17B 21A 25B 2A 6A 7B 8B 9A 10A 12B 15B 16B 17B 21A 25B 2A 6A 7B 8B 9A

July August

100% area sampled area sampled 100% 2

75% 75%

50% 50%

25% 25%

0% 0% 10A 12B 15B 16B 17B 21A 25B 2A 6A 7B 8B 9A 10A12B 15B 16B 17B 21A25B 2A 6A 7B 8B 9A % IJ’s in each 1m

September October

100% 100%

75% 75%

50% 50% 25% 25% 0% 0% 10A 12B 15B 16B 17B 21A 25B 2A 6A 7B 8B 9A 10A 12B 15B 16B 17B 21A 25B 2A 6A 7B 8B 9A S1 S2 S3 S4 S1 S2 S3 S4

Sites along grassy banks monitored

Figure 2.4. Spatial and temporal distribution of entomopathogenic nematodes within sites along grassy banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May-October 2003. S1, S2, S3 and S4; four-1m2 areas in which nematode population densities were assessed at each site. IJ’s – infective juvenile nematodes

52 Persistence of endemic entomopathogenic nematode populations

In 2003, nematode population densities varied spatially and temporally across sites. Nematodes were detected at all sites that were positive in 2001 and 50% of the sites where they were not detected in 2001 or 2002. Populations fluctuated during the monitoring period with the highest populations occurring in May and the lowest in June; mean nematode population density was 5,274 (±375) per 4m2 (Figure 2.2 and 2.3).

Spatial and temporal variation in nematode population densities also occurred within sites

(Figure 2.4). Both H. bacteriophora and S. feltiae were recovered within sites but this was dependent on the area being sampled and the time of sampling.

In 2003, Dipterans and Homopterans were the most prevalent macroarthropods and mites were the most abundant microarthropods across the sites (Figure 2.5).

Arthropod populations were larger at sites at which nematodes were detected compared with those sites in which nematodes were not detected; mean population densities were

40.25 (± 7.20) and 23.89 (± 6.43) per 4m2, respectively. Populations of Diptera,

Coleoptera and Lepidoptera, (orders in which nematodes have been recovered and/or shown to be moderately susceptible to nematode infection in laboratory and field trials)

(Georgis and Grewal 1998, Peters 1996), were significantly larger at two of the sampling dates at sites at which nematodes were detected (July, September) (P≥ 5.18, df= 1,10; P≤

0.046).

53

Collembola Mites Total Micro Other Lepidopera Homoptera Hemiptera Diptera Hymenoptera Coleoptera Total Macro

composition Arthropod 0 1020304050

2 Mean arthropod abundance/site(4m )

Figure 2.5. Composition and abundance of arthropod populations at sites along grassy ditch banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May- October 2003.

54

30

C) 0 20

10

temp( Soil 0 May Jun Jul Aug Sept Oct

Months

Figure 2.6. Mean soil temperatures across sites monitored along grassy ditch banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May-October 2003.

55

40

30

20

10

(%) Moisture Soil 0

May Jun Jul Aug Sept Oct

Months

Figure 2.7. Soil moisture (%) at sites along ditch banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May-October 2003.

56

60

40

20

0 Soil Moisture (%) May Jun Jul Aug Sept Oct

Months Negativ e f or EPN Pos it iv e f or EPN

Figure 2.8. Soil moisture (%) at sites at which entomopathogenic nematodes (EPN) were detected and not detected along grassy banks adjacent to cultivated areas in Celeryville (Huron County, Ohio), May-October 2003.

57 Plant species composition and abundance did not differ among sites or during the monitoring period. Perennial rye grass, Lolium perenne L., was the most prevalent plant followed by dandelion, Taraxacum officinale Weber, covering >80% and 10-15% of the sample area, respectively. Morning Glory, Ipomoea spp., and chickweed, Stellaria media, occupied <5% of the sample area.

Soil temperatures ranged from 12°C to 23°C during the season (Figure 2.6).

Temperature did not differ at sites where nematodes were detected and those where they were not detected; mean temperatures were 18.26°C (± 1.15) and 17.67 °C (± 0.64), respectively. Soil moisture content was consistently higher at sites where nematodes were present (Figure 2.7); significant differences were observed during July, August, and

October (F≥ 5.96, df= 1, 10; P≤ 0.037) (Figure 2.8). Moisture was the best predictor of nematode persistence along grassy banks in a multiple regression analysis. It is possible that the variables measured in the study are correlated, however, when analyzed individually none of the other variables provided a significant explanation for nematode population persistence along grassy banks (Table 2.4).

DISCUSSION

Soil texture, temperature, moisture, agronomic practices, soil antagonists, and host resources (quantity and quality) are major factors affecting nematode persistence in the soil environment (Baur and Kaya 1998, Barbercheck 1992, Kaya 1990) and could be responsible for the differences in detection of nematodes among habitats.

58

Source df SS MS F P

Regression 4 23365 5841 4.50 0.003

Residual Error 64 83008 1297

Total 68 106373

SE Predictor Coefficient Coefficient T P

Constant -5.79 23.24 -0.25 0.80

Temperature °C 0.30 0.97 0.31 0.76

% Moisture 1.46 0.36 3.97 <0.01

Macro arthropods (Dipterans, 0.09 0.12 0.73 0.47 Coleopterans, Lepidopterans)

Microarthropods (mites and -2.21 0.29 -0.74 0.46 collembola)

R2= 0. 22

Table 2.4. Multiple regression analysis of entomopathogenic nematode population densities in relation to abiotic (i.e., soil temperature and moisture) and biotic (i.e., macro and micro arthropods) factors.

59 Entomopathogenic nematodes were recovered only along grassy banks adjacent to the cropped area. The presence of nematodes along grassy banks could be attributed in part to the thatch buffering nematode populations from environmental stresses and providing optimum soil conditions (low temperatures and adequate moisture) for dauer juvenile survival and infectivity (Glazer et al. 1996, Kaya and Gaugler 1993, Brust 1991).

Grassy banks may act as refuges for pests and non-pests that disperse from the cultivated areas during perturbations associated with the production system. Some of these insects may be serving as hosts in which nematodes recycle and persist. Heterorhabditid nematodes were recovered from a greater percentage of sites than Steinernematid nematodes, which may be related to differences in the dispersal behavior of the nematode species and strains recovered. Garcia del Pino and Palmo (1997) reported that H. bacteriophora tended to disperse deeper in the soil than S. feltiae. As such, S. feltiae populations in the production area may have been closer to the soil surface, affected to a greater extent by harsh environmental conditions, and less detectable.

The absence of nematodes within cultivated areas, shrub lands and forests may be related to a combination of factors. Intense production regimes such as tillage, crop removal, and frequent agrichemical applications typical of the cultivated area may have limited survival of infective juveniles. Tillage physically disrupts the soil microclimate and exposes nematodes to drying conditions and UV radiation and may also be deleterious to insect host populations. Millar and Barbercheck (2002) observed that tillage significantly reduced the survival of S. carpocapse and S. riobrave. Higher nitrate levels associated with inorganic fertilizers and broad spectrum insecticides, surfactants

60 and other formulation ingredients can also affect entomopathogenic nematode infectivity and survival (Grewal et al. 1998, Patel and Wright 1996, Zimmerman and Cranshaw

1990).

A lack of detectable nematodes in undisturbed forests and shrub lands could be related to soil texture or biotic antagonists, or both (Table 2.1). The soil collected from the shrub areas tended to have high levels of clay. Small soil particle sizes, high moisture, and limited oxygen characteristic of clay soils can limit survival and impair active movement of nematodes, host location, and infectivity (Baur and Kaya 1998, Kaya

1990). The lack of detection of nematodes in the forest was surprising, as the environment was shaded, moist and the soil was very rich in organic matter. Nematodes have been detected in similar environments (Glazer et al. 1996, Stuart and Gaugler 1994).

However, the conditions within the forested habitat may have also been favorable for soil antagonists (e.g., nematophagous fungi, collembola, and mites) that prey upon entomopathogenic nematodes (Kaya 1990, Epsky et al. 1988).

Along grassy banks, nematode populations were patchily distributed in space and time and there was a lack of spatial dependence; the presence of nematodes at one site was not linked to the presence of nematodes at neighboring sites. Spatial and temporal heterogeneity of naturally occurring nematode populations has been documented in several studies (Efron et al. 2001, Campbell et al. 1995, 1998, Stuart and Gaugler 1994).

These observations are due to a combination of density-dependent (i.e., host resources, competition of conspecifics or heterospecifics) and stochastic density-independent processes (i.e., disturbances, edaphic factors) (Efron et al. 2001, Baur and Kaya 1998).

61 Moisture was the most important abiotic factor influencing nematode persistence; population densities increased with moisture. Koppenhofer et al. (1995) also recorded higher infectivity with increasing soil moisture. Low soil moisture retards movement of infective juveniles (Kaya and Gaugler 1993, Barbercheck 1992) and impacts nematode survival and host finding (Kung et al. 1991, Kaya 1990). Grant and Villiani (2003) observed reduced survival and infectivity of H. bacteriophora and Steinernema spp. in sandy loam soil in which the moisture levels was just below the plant-wilting point; however, virulence was increased when soils were rehydrated.

In this study, macroarthropod population estimates did not provide a statistically significant explanation for nematode population persistence along the grassy banks. The majority of insects collected were frit flies and leafhoppers, both of which are foliage feeders and are not known to be susceptible to nematode infection. Other insects that could serve as hosts were identified (Coleoptera and Lepidoptera larvae), but they made up less than 1% of total insect samples. The role of insect communities in nematode population persistence should not be minimized, as high densities of suitable hosts are known to be required for the long-term persistence of nematode populations (Burlando et al. 1993, Kaya 1990, Klein 1990). A combination of factors including the sampling techniques used, as well as the means by which nematodes disperse within the cropping area, could have contributed to the lack of a significant correlation. Soil dwelling insect species that may be serving as hosts for infective juveniles to recycle were in low numbers and populations may have not been sampled with sufficient intensity or precision. Insect communities were assessed by foliage vacuuming and soil sieving. All foliage within the 4m2 sample area was vacuumed, whereas, for soil insects assessments

62 within each 4m2 area, sixteen soil cores (2.54cm diameter) were collected and only 25-

40% of this soil was processed to determine insect population densities, (the remainder was used to assess nematode population densities). Hence, potential insect hosts present in the soil at the site and also within the samples may have been missed. In addition, host populations may have been underestimated as nematodes could have been reducing host populations along grassy banks to levels that would be difficult to detect.

The dispersal of nematodes within the cropping area could be a contributory factor for the lack of a relationship with infective juvenile densities and insect host communities (Efron et al. 2001, Baur and Kaya 1998). Long-range dispersal of infective juveniles primarily occurs via the action of other agents such as infected insects, water or anthropogenic factors (Kaya 1990). Localized areas of high densities of infective juveniles may be related to locations where infected insects died and nematodes emerged

(Effron et al. 2001, Baur and Kaya 1998). Timper et al. (1988) observed that Spodoptera exigua infected with Steinernema feltiae could disperse up to 11m from the infection site before dying. Similarly, Parkman and Frank (1991) observed that nematode-infected mole crickets, Scapteriscus spp., remained active for several days after infection and were found in traps several kilometers from the initial release site. Epsky et al. (1988) reported that infective juveniles adhered to the dorsum of mesostigmatid mites and, therefore, could be potential dispersers for the nematode. Grassy banks within the production area are mowed approximately every 2-4 weeks and this mowing could also be responsible for distributing some infective juveniles.

63 Seasonal fluctuations of nematodes may be dependent not only on extrinsic but also intrinsic factors (Lewis 2002). Unfavorable conditions (e.g., high temperature, low moisture, and lack of hosts) may induce infective juveniles to undergo physiological changes and enter into a state of inactivity (Ishibashi and Kondo 1990, Womersley et al.

1990). In addition, even with optimal conditions nematodes can be non-infective (Fan and Hominick 1991). Induction of these physiological changes could reduce detection of nematodes.

The techniques used to collect soil and estimate nematode population densities may have influenced the results. Garcia del Pino and Palmo (1997) observed that S. feltiae and H. bacteriophora, the two species that were recovered in this study, occupied different strata within the soil, but this was dependent on the temperature. During warmer months (summer), H. bacteriophora tended to disperse to depths up to 30cm. Sampling for infective juveniles was conducted to a depth of ca.12-15cm and hence nematodes present at greater depths would have been outside of the sampling area. Nematode population densities were estimated using the Galleria soil baiting technique (Bedding and Akhurst 1975). Nematode species and strains differ in their pathogenicity to insect hosts, and even though Galleria is susceptible to nematode infection, the efficiency at which they are able to extract infective juveniles from the soil may differ among species and strains, resulting in bias. Rueda at al. (1993) used greater wax moth larvae, lesser meal worms, house flies, and crickets to isolate entomopathogenic nematodes from soil collected from plant nurseries and observed that although wax moth larvae were the most efficient in isolating nematodes, other bait insects were able to isolate strains that wax moth larvae could not recover. Even with multiple baitings of the soil, Galleria is only

64 able to recover an estimated 30-40% of infective juvenile nematodes (Curran and Heng

1992, Fan and Hominick 1991). Thus, low persistence could have been due in part to a combination of the bait insect used as well as the efficiency of the extraction technique.

Two species of endemic entomopathogenic nematodes were isolated from the grassy banks adjacent to the cultivated areas surveyed. These beneficial nematodes could be important for developing biological control programs for soil inhabiting pests that limit vegetable yields. Surveys indicated that entomopathogenic nematodes were present only along grassy ditch banks of the production landscape, however surveys represent a snapshot in time with limited resolution. Given the sample sizes for this study, surveys should be repeated over several seasons to determine more precisely the presence and abundance of entomopathogenic nematodes within these habitats. Understanding the influence of edaphic factors and host resources on nematode survival and persistence is key for managing and sustaining populations within production areas (Efron et al. 2001,

Campbell et al. 1995, 1998, Glazer et al. 1996). The study suggested that the occurrence and persistence of beneficial nematodes along grassy banks were related mainly to soil moisture content. Water management is an important component of vegetable production.

Manipulating soil moisture conditions could help to sustain entomopathogenic nematode populations within the production area so that pest populations can be regulated.

65 CHAPTER 3

LIFE-HISTORY CHARACTERISTICS OF ENDEMIC ENTOMOPATHOGENIC

NEMATODE STRAINS ISOLATED FROM THE VEGETABLE PRODUCTION

AREA IN HURON COUNTY, OHIO

INTRODUCTION

The low persistence after inundative releases of exotic strains of entomopathogenic nematodes within cultivated areas has limited their efficacy as biological control agents (Georgis and Gaugler 1991). Understanding abiotic and biotic factors responsible for the persistence of endemic populations as well as the life-history traits of infective juveniles within persistent populations will be important for selecting and maintaining releases of exotic strains within crop patches.

From surveys conducted in the vegetable production area in Huron County, Ohio

(2000-2002), (Chapter 2), several strains of Heterorhabditis bacteriophora and

Steinernema feltiae were observed to persist for several seasons along grassy banks adjacent to cultivated areas. Soil moisture was identified as being a principal factor regulating the persistence of these endemic populations. However, the life-history traits

66 that allow these species to persist is still to be determined. A series of laboratory investigations were conducted to characterize these endemic strains of H. bacteriophora and S. feltiae with respect to their survival rate in the absence of insect hosts and their infectivity and recycling potential in two common vegetable pests, the black cutworm,

Agrotis ipsilon Hufner (Lepidoptera: Noctuidae) and onion maggot, Delia antiqua

Meigen (Diptera: Anthomyiidae). Measurements of these traits were also compared with those of exotic strains of the same species.

METHODOLOGY

Soil

Soil used in bioassays was obtained from the vegetable production area of

Celeryville (Huron County, Ohio). The soil was a mixture of Linwood and Carlisle muck and characterized by a moist bulk density of 0.13-0.4 g/cc, pH of 4.5-7.8, and organic matter content of 40 - >70% (Ernst and Martin 1994).

Insects

Onion maggots were obtained from laboratory cultures initially collected from

Celeryville (Huron County, Ohio). Maggots were maintained in culture in plastic rearing cages (84 x 33 x 64 cm) at 26-28ºC, 40-60 RH%, and 16:8 L: D photoperiod. Water and a mixture of sugar, powdered milk, and yeast (2:1:2) were provided for adults. Cups with moist sand and commercially produced white onions were placed in cages for adults to

67 oviposit. Late-stadium maggots were harvested from onions and placed in Petri dishes lined with filter paper (Whatman #4) with a slice of onion until they were used. Black cutworms and artificial diet were obtained from a commercial source (Mass Rearing

Laboratories, Madrid IA). Larvae were kept in paper cups with the diet at 20ºC and 40%

RH until they were used.

Entomopathogenic nematodes

A total of two exotic strains and six endemic strains of H. bacteriophora and S. feltiae were evaluated. The number of nematodes assayed differed for each test conducted. Endemic nematodes were isolated from the vegetable production area of

Celeryville (Huron County, Ohio) between 2001 and 2002, and the exotic strains were obtained originally from commercial sources. All nematodes were reared in Galleria mellonella larvae using standard in-vivo methods (Dutky et al 1964). Infective juveniles were stored at 9ºC until they were applied. For all studies, the nematodes used were between 7-10 days old.

Infectivity and reproductive potential bioassays

Cutworms

Six grams of muck soil were weighed and placed in a plastic cup (30ml Comet P-

10 CUP with LP-1 lid, Comet Products Inc., Chelmsford, MA, USA). Forty infective juveniles suspended in 500µl of water were applied to the soil. For the control treatment, only water was added. With this volume of water (500 µl), soil moisture was estimated to be 40%; a level for optimum nematode activity in muck soils (Miklasiewicz et al. 2002).

68 Nematodes were mixed into the soil with a toothpick. A single third instar black cutworm was added to each cup along with a small piece of diet (2cm2 cube). Cups were covered with lids in which a hole was made with a probe to allow for gas exchange. Cups were incubated in complete darkness at 26ºC and 60% RH. Infectivity was determined 2, 4, and 6 days after treatment. Two exotic and four endemic nematode strains were evaluated and each strain was replicated four times. A total of six insects were used for each replicate. Three to five cadavers having characteristic nematode symptoms (i.e., color, smell, and texture) were randomly selected and dissected to confirm that death resulted from penetrating infective juveniles. Larvae killed by nematodes were established in white traps (White 1927) and infective juveniles emerging from cadavers after 21to 30 days were counted.

Onion maggots

Ten grams of muck soil were weighed and placed in a plastic cup (30ml Comet P-

10 CUP with LP-1 lid, Comet Products Inc., Chelmsford, MA, USA). Four hundred infective juveniles suspended in 700 µl of water were applied to the soil. For the control treatment, only water was added. With this volume of water (700 µl), soil moisture was estimated to be 40%; a level for optimum nematode activity in muck soils (Miklasiewicz et al. 2002). Nematodes were mixed into the soil with a toothpick and ten onion maggots were added to each cup along with a small piece of onion (2cm2 cube). Cups were covered with lids in which a tiny hole was made with a probe to allow for gas exchange.

The cups were incubated under the same conditions as the cutworms. Infectivity was determined 2, 4, 6, and 8 days after treatment. Three to five cadavers having

69 characteristic nematode symptoms (i.e., color, smell, and texture) were randomly selected and dissected to confirm that death resulted from penetrating infective juveniles. Onion maggots that pupated were incubated to confirm that viable adults emerged. Two exotic and six endemic nematode strains were evaluated and each was replicated six times.

Maggots killed by nematodes were established in white traps (White 1927) and incubated at 26°C at 60% RH in total darkness. Infective juveniles emerging from cadavers after 21 to 30 days were counted.

Infective juvenile survival in the absence of an insect host

Survival rates of infective juveniles in the absence of an insect host were estimated for three endemic nematode strains, H. bacteriophora R1-16A, R1-18C, and a

S. feltiae strain. Ten grams of muck soil were added to diet cups (30ml Comet P-10 CUP with LP-1 lid, Comet Products Inc., Chelmsford, MA, USA) and 100 infective juveniles suspended in 1ml of water were added. Cups were sealed with lids and stored at 26ºC at

60% RH in complete darkness. Soil was baited with G. mellonella larvae 1, 2, 6, 8, 16 days after application of the nematodes. Soil was transferred to a Petri dish (9cm) and ten

G. mellonella larvae were added. Plates were incubated for 48 hours under the conditions previously mentioned. Larvae showing typical nematode symptoms were removed from the soil, dissected and the numbers of infective juveniles penetrating cadavers were counted. The soil was baited twice and each nematode/time interval was replicated five times.

70 Data analysis

Data were transformed to achieve normality and equality of variances according to the ladder of powers cited by Fry (1999). Data were transformed by log10x+1 (number of nematodes), or arcsine √x (proportional mortality). Repeated measures analysis of variance was conducted to determine the differences in mortality among nematode strains over time. Analysis of variance with contrasts was also conducted to compare treatments.

Non-linear regression was conducted to estimate and compare the exponential decay rates of infective juveniles in the absence of hosts. Both SAS and SYSTAT were used for the analyses.

RESULTS

Infectivity

The mean infectivity for cutworms after six days was >90%, with the most infections occurring within four days of exposure to nematodes (Figure 3.1). For each time period evaluated, there were significant differences in infectivity among the nematodes (F≥ 6.72; df= 6,21; P≤ 0.0004). Steinernematid strains infected more larvae than heterorhabditid strains; mean percent mortality after 6 days was 100% and 75% (±

8.9), respectively. Steinernematids also killed cutworm larvae faster than the heterorhabditids; two days after nematode application larval mortality differed significantly between species (F= 25.08; df=1; P< 0.001). Within species, no significant differences in infectivity were observed among the strains (F=0.75; df=1; P=0.388).

71

Heterorhabditis bacteriophora Steinernema feltiae

100 75 50 25

% M ortality 0 HP88 R1-16A R2-16B R1-7B SF R1-18B Control (Exotic) (Exotic)

Nematode strains

2 DAT 4 DAT 6 DAT

Figure 3.1. Infectivity of endemic and exotic strains of H. bacteriophora and S. feltiae to the black cutworm, Agrotis ipsilon Hufner (Lepidoptera: Noctuidae).

72 Onion maggots were less susceptible to nematode infection than the cutworms; mean percent mortality after 6 days was 40.27% (± 4.91) (Figure 3.2). For each time period, significant differences in infectivity were observed among the nematode strains (F≥ 4.07; df=8, 54; P≤ 0.001). The heterorhabditid strain R1-16A, infected a significantly larger number of onion maggots than other heterorhabditid strains and the endemic steinernematid R1-18B, which also infected high numbers of maggots (F≥ 7.63; df= 1;

P≤ 0.0062).

Reproductive potential

All of the nematodes strains recycled in the insects provided; however, the proportion of insects from which dauer nematodes emerged was greater for cutworms

(48.7%) than for onion maggots (27%). No statistically significant differences were observed among strains in the number of infective juveniles emerging from cutworm cadavers (F=0.77; df= 5,49; P= 0.57) or onion maggot cadavers (F=0.19; df= 6, 26;

P=0.97). Mean infective juvenile emergence after 21-30 days was 6,373 (± 1,371) and

4,555 (± 538) for the onion maggot and cutworm, respectively (Table 3.1).

Infective juvenile survival in the absence of an insect host

Infective juveniles recovered from the soil after a day declined to 20% of the number applied. Four and eight days after application, population density estimates for both strains of heterorhabditids were significantly greater than for the S. feltiae strain (F≥

4.96; df=1; P≤ 0.045).

73

100 Heterorhabditis bacteriophora Steinernema feltiae 80

60

% M ortality 40

20

0 HP8 8 R3-10D R3-24D R1-16A R2-16B R1-7B SF R1-18B Control (Exotic) Nem atode s trains (Exotic) 2DAT 4DAT 6DAT 8DAT

Figure 3.2. Infectivity of endemic and exotic strains of H. bacteriophora and S. feltiae to the onion maggot, Delia antiqua Meigen (Diptera: Anthomyiidae).

74

Insect Species Nematode Nematode Number Number of Range Species Strain of IJ’s cadavers (SE)

Black Cutworm, Heterorhabditis HP88 Agrotis ipsilon bacteriophora (exotic) 10 5,182 (± 902) 1,800-9,600 Hufner R1-16A 9 3,000 (± 900) 600-9,000 (Lepidoptera: R1-7B 10 6,690 (± 2,216) 600-18,000 Noctuidae) R2-16B 6 3,900 (± 1,321) 1,200-10,200

Steinernema SF feltiae (exotic) 8 3,075 (± 594) 1,200-6,000 R1-18B 10 4,691 (± 974) 600-10,800

Onion Maggot, Heterorhabditis HP88 Delia antiqua bacteriophora (exotic) 0 * * Meigen R1-16A 6 5,200 (± 1,417) 1200-10,200 (Diptera: R1-7B 4 13,350 (± 1200-44,000 Anthomyiidae) 10,388) R2-16B 1 1,800 1800-1800 R3-10D 6 5,700 (± 1,751) 2400-13800 R3- 24D 5 5,400 (± 1,849) 600-10,800

Steinernema SF feltiae (exotic) 5 4,400 (± 1,548) 1200-9,000 R1-18B 6 6,750 (± 2,377) 600-15,600

Table 3.1. Reproductive potential (infective juveniles, IJ’s) of endemic and exotic strains of H. bacteriophora and S. feltiae in black cutworms, Agrotis ipsilon Hufner (Lepidoptera: Noctuidae) and onion maggots, Delia antiqua Meigen (Diptera: Anthomyiidae). * - no progeny emerged from nematode killed cadavers.

75 sf

Exponential decay rates were estimated from non-linear regression models as -0.27, –

0.21, and -0.27 for the H. bacteriophora R1-16A and R-18C strains and the S. feltiae, respectively (Figure 3.3).

DISCUSSION

All strains of the endemic nematodes were able to survive in the absence of an insect host and infected and reproduced within cutworms and onion maggots at levels comparable to or higher than the exotic strains. Previous studies have demonstrated that

H. bacteriophora and S. feltiae can infect and kill Agrotis spp, and Delia spp. (Chen et al.

2003a and b, Yildrim and Hoy 2003, Levine and Oloumi-Sadeghi 1992, Vanninen et al.

1992, Morris and Converse 1991). Cutworms were more susceptible to nematode infection than the onion maggots. These differences in infectivity of the nematodes to cutworms and onion maggots could be attributed to morphological and physical differences between the insects. Infective juveniles enter insects, through the mouth, anus, spiracles, or in the case of heterorhabditids, via the cuticle (Poinar 1990). Maggots have a smaller mouth opening compared with cutworms so fewer infective juveniles may have entered (Van Sloun 1989 as referenced by Vanninen et al. 1992). In addition, onion maggots possess mouth hooks that are very active and may destroy incoming infective juveniles (Renn 1998). Immune responses of onion maggots to penetrating nematodes could also be a factor. Many insects react to foreign bodies with a series of immune system responses including phagocytosis, nodule formation, and cellular and humoral encapsulation (Dunphy and Thurston 1990). Vanninen et al. (1992) observed pigmented

76 30

20 IJ’s

10

0 0 5 10 15 20 TIME

Heterorhabditis bacteriophora, R1-16A. Decay Rate: -0.27

50

40

30 IJ’s 20

10

0 0 5 10 15 20 TIME

Heterorhabditis bacteriophora, R1-18C. Decay Rate: -0.21

20

15

10 IJ’s

5

0 0 5 10 15 20 TIME

Steinernema feltiae. Decay Rate: -0.27

Figure 3.3. Exponential decay rates of endemic entomopathogenic nematode strains, Heterorhabditis bacteriophora and Steinernema feltiae in the absence of an insect host.

77 spots on D. radicum larvae exposed to steinernematids and suggested that physiological resistance mechanisms such as encapsulation may be protecting the insects from attack.

Similar mechanisms could occur with D. antiqua.

All strains of the nematodes appear to be suitable for infecting the cutworm species examined, although, the most promising strains for onion maggots were H. bacteriophora R1-16A and S. feltiae R1-18B. These nematode strains caused >50% mortality. Differences in infectivity among strains could be related to variation in behavioral and physiological traits of infective juveniles (Kaya 1990).

The study demonstrated that the nematodes reproduced in both onion maggots and cutworms, very few studies have been conducted on the reproductive capacity of entomopathogenic nematodes in these insects. Based on the body weight of the test insects, the reproductive capacities were 103.52 IJ’s mg-1 and 637 IJ’s mg-1 for the cutworm and onion maggot, respectively. Compared with G. mellonella, (the standard susceptible insect used for rearing), these estimates are low, but they are within the range of the reproductive estimates reported for other pest insects. The reproductive capacity of nematodes within G. mellonella is estimated at 1,100 IJ’s mg-1 (Dutky et al. 1964), whereas for carrot weevil the capacity was estimated at 300 IJ’s mg-1 (Belair and Boivin

1985). The higher number of progeny produced in onion maggots is somewhat surprising, but could be related to the rate of kill and the time period at which counts were made.

Those insects that were killed and removed from the soil within two to four days, may have had fewer nematodes entering than those that were killed and removed after six or eight days. For the cutworm, in which the majority of insects died within 4 days, fewer infective juveniles may have entered. Hence, a longer time would be required for host

78 resources to be used and infective juveniles to be produced. Because sampling was not extended beyond 30 days, the numbers of infective juveniles counted may not have been the maximum numbers produced within cadavers. For both insects, the relatively low proportions of cadavers that produced progeny may be related to the death of penetrating infective juveniles as a result of the host immune response.

Within two days of application, the number of infective juveniles recovered from the soil was approximately 20% of the number applied. The data are consistent with other studies (Smits 1996) that attribute poor survival of infective juveniles to unfavorable abiotic and biotic conditions. For this study, temperature and moisture conditions were within the range for optimal survival of infective juveniles (Miklasiewicz et al 2002,

Gouge et al. 1999). However, the soil used in the study was not sterilized and other microfauna (e.g., fungi, bacteria, and mites) that are deleterious to infective juveniles could have been present. Studies have demonstrated decreased survival of infective juveniles in unsterilized soils and these observations are attributed to the production of toxins by microorganisms (Kaya 1990).

The low survival estimates for the nematode strains may also be related to the efficiency of the extraction technique. Based on extrapolation from the non-linear regression model, the level of recovery of infective juveniles by G. mellonella was approximately 19.3% of the total applied, hence the survival rate estimates may have been understated. These data are somewhat consistent with the extraction efficiency estimates reported for G. mellonella by Curan and Hueng (1992). They reported recovery rates of infective juveniles from the soil with G. mellonella as being between 30-40%. On the second sample date, the numbers of infective juveniles recovered from all strains

79 were consistently lower than all other days. These observations may be due to the quality of the Galleria larvae used to bait the soil during that sample period. Furthermore, nematode populations display phased infectivity, not all infective juveniles are likely to infect a host at the same time (Fairbairn et al. 2000, Bohan and Hominick 1997). Hence, non-infectious nematodes could have been present when baitings were conducted, resulting in lower recovery rates.

Differences in survival rate estimates among strains could also be related to differences in pathogenicity among the nematode strains to the bait insect. Even though

Galleria is a susceptible host, Rueda et al. (1993) observed that other bait insects such as houseflies, crickets and mealworms were able to recover nematode strains that wax moth larvae could not recover.

The endemic strains of H. bacteriophora and S. feltiae isolated from the cropping area infected and recycled in cutworms and onion maggots. This is a promising prospect for the long-term persistence of these nematodes within cultivated areas. Kaya (1990) outlines three criteria that should be met to maintain entomopathogenic nematodes in cropping areas: (i) the presence of a complex of soil pests for most of the year, (ii) pests that have a high economic threshold and are moderately susceptible to nematode infection, and (iii) favorable abiotic conditions at the site of release (i.e., moderate soil temperature and moisture, low UV radiation and soil antagonists, and sandy soils). At any one time, within vegetable production areas of Ohio, over 30 different crops are grown that are attacked by many pests that are susceptible or moderately susceptible to nematode infection: cutworms, onion maggots, flea beetles, Colorado potato beetles, carrot weevils, and thrips (Miklasiewicz et al. 2002, Ebassa et al. 2001, Berry et al. 1997,

80 Levine and Oloumi-Sadeghi 1992, Klein 1990). Many non-pest species are found in these production areas as well (Chapter 2). The first two criteria of Kaya’s (1990) recommendations are met to some extent, but the last criterion will be the most challenging because current agronomic practices (e.g., tillage and agri-chemicals) used in vegetable systems produce unfavorable microclimatic conditions for infective juveniles to persist. Crop-management strategies to conserve nematodes will therefore have to focus not only on manipulating nematode-host interactions but creating favorable soil conditions. Based on the low long-term survival rates of infective juveniles in the absence of an insect host, the latter will be imperative for sustaining populations of nematodes within production areas.

81 CHAPTER 4

INFLUENCE OF HOST AVAILABILITY ON THE REGULATION OF THE

ENTOMOPATHOGENIC NEMATODE, HETERORHABDITIS BACTERIOPHORA

INTRODUCTION

Because nematodes depend on the presence of suitable hosts to complete their life cycle, their spatial and temporal dynamics may be related in part, to the population density and distribution of insects (Stuart and Gaugler 1994). In natural environments, such correlations are variable and are linked to the life history traits of nematode strains and species (Loya and Hower 2002, Efron et al. 2001, Campbell et al. 1998, Kaya 1990).

Understanding the factors influencing interactions between entomopathogenic nematodes and their hosts in the field is critical for understanding how populations are sustained and how they can be encouraged to persist in cropping systems (Lewis et al. 1998).

The use of ecological principles to develop predictive models can accelerate an understanding of nematode population dynamics. Attempts have been made to complement empirical research on entomopathogenic nematodes with simulation models.

So far, models have been used to evaluate short-term strategies to optimize inundative applications (Fenton et al. 2002, 2001, 2000, Westerman 1998). Recently, a spatially

82 explicit, stochastic model that simulates the population dynamics of Heterorhabditis bacteriophora for a typical cropping period (60 days) after an inundative release was developed to predict the conditions under which nematodes persist in the field (Stuart et al. 2004, in review). Model parameters include various host factors (quantity and quality) and nematode mortality rates in the absence of hosts. With the ultimate aim of identifying strategies to conserve entomopathogenic nematodes within vegetable production systems, this model was used to generate hypotheses relating to the influence of insect host supply on nematode persistence. Model simulations predicted that at the field mortality rate previously estimated at the test site (0.1) (unpublished data), both quantity and quality of hosts would impact nematode persistence. At lower levels of mortality (≤ 0.05), nematode persistence would be largely influenced by host quantity rather than host quality, and at higher mortality rates (≥ 0.15), persistence of nematodes would be less likely; requiring a very large quantity of high quality hosts. In this chapter, the model predictions were tested on two insect host species; carrot weevil, Listronotus oregonesis

LeConte, and onion maggots, Delia antiqua Meigen, and one species of entomopathogenic nematode, H. bacteriophora.

METHODOLOGY

Field evaluations were conducted at the Muck Crops Branch of the Ohio

Agricultural Research and Development Center (OARDC), in Celeryville (Huron

County, Ohio) for two seasons, July 24th – September 5th, 2001 and August 12th - October

83 7th, 2002. Soil type at the field station is characterized as muck soil, a Linwood series with moist bulk density of 0.15-0.4 g/cc, pH of 4.5-7.8, and OM content of 40-70%

(Ernst and Martin 1994).

Entomopathogenic nematodes

H. bacteriophora was from two sources: (i) an endemic strain recovered from

Celeryville (Huron County, Ohio) in 2001, and (ii) exotic strains (Lewiston and HP88), commercially purchased from Integrated Biological Control Systems, (Greendale, IN,

USA). The native strain was cultured using standard in-vivo techniques with Galleria mellonella (Dutky et al. 1964). Infectives juveniles (IJ’s) were harvested from White traps (White 1927) and were 4-7 days old when applied. Nematodes were applied with watering cans at rates between 2.0 and 2.5 billion IJ’s ha-1.

Experimental Design

Laboratory reared onion maggots, Delia antiqua Meigen (Diptera:

Anthomyiidae), and carrot weevils, Listronotus oregonensis LeConte (Coleoptera:

Curculionidae) were supplied at various times during the cropping season, according to the treatment. Treatments were replicated five times and arranged in a completely randomized design. The test arena was a cylindrical clay tile (25cm diameter x 65cm length) inserted into the soil to a depth of 55cm.

84 Insect host introductions

For 2001, late stadium carrot weevil and onion maggots were introduced into tiles and, depending on the insect species, either parsley or green onion plants were established two weeks before insect introduction (Table 4.1). For 2002, parsley infested with carrot weevil eggs was transplanted into clay tiles two weeks before the application of the nematodes, so that hosts were present when the nematodes were applied. Onion maggots were introduced by the same procedure used in 2001.

Depending on the treatment, hosts were either added to tiles at the time of nematode application (early season introduction), three weeks after application (late season introduction) or at both times (continuous introduction). For treatments in which there was a continuous supply of hosts, both insect species were used, one introduced at application and the other three-weeks after application. For these treatments, crop plants in tiles were also rotated to maintain the host present. Tiles were covered with cloth

(Reme 40 x 40cm, glued to the tile) to prevent the escape of test insects and the influx of other insects from the surrounding area.

85

Host Supply Host Type Crop

No Hosts (2002 only) None Either green onions or parsley

Continuous - host introduction at Onion maggots followed by Green onion nematode application and 3 weeks carrot weevil larvae followed parsley after nematode application Carrot weevil larvae Parsley followed by followed by onion maggots green onion

Early Season – at nematode Carrot weevil larvae Parley application Onion maggots Green onion

Late Season – 3 weeks after Carrot weevil larvae Parsley nematode application Onion maggots Green onion

Table 4.1. Influence of host supply on the persistence of infective juvenile populations: treatment structure.

86 Entomopathogenic nematode population assessment

Infective juvenile populations were assessed before the application and at three and six weeks after the application of nematodes. The assessment conducted before nematode application was to verify that endemic and/or residual populations from past applications were not present. For each sample period, four cores of soil (2.54cm diameter, 12cm depth, approx 200g) were removed from each tile and infective juvenile populations were assessed using the standard G. mellonella baiting method (Bedding and

Akhurst 1975). For each sample, the soil was thoroughly mixed and 200g were placed in a plastic container. Ten greater wax moth larvae were added and the container was sealed and placed in a chamber in total darkness, at 25°C, and 60% RH. After three days of incubation, all larvae were removed and the soil was re-baited with an additional ten wax moth larvae and incubated for an additional three days. After removal, larvae were examined and those killed by nematodes (i.e., having characteristic color, smell, and texture) were dissected and the number of infective juveniles found inside was recorded.

Live larvae were incubated for an additional three days after which nematode infection and populations were recorded, as previously described. For cadavers with nematode infection characteristics, in which no infective juveniles were found, a value of one was assigned as the minimum number of infective juveniles that could be responsible for larval mortality. For those cadavers in which a large number of nematodes (adults and infective juveniles) were observed, and reproduction was suspected, a value of one was assigned.

87 Data analysis

All data were transformed (log10 + 1) to meet the assumptions of the analysis of variance (normality and equality of variances). Analysis of variance was conducted to determine the effect of host supply on the persistence of infective juveniles. Treatment means were compared using a set of contrasts for the hypotheses of interest (SAS

Institute). Non-linear regression was conducted on changes in infective juvenile density over time to estimate the daily rates of mortality (SYSTAT).

RESULTS

In 2001, exotic H. bacteriophora, (Lewiston strain) persisted in all treatments up to six weeks after application, mean population density was 1,117 IJ’s m-2 (± 374)

(Figure 4.1). Nematode persistence as measured by population density was significantly influenced by host availability after six weeks (F= 3.62; df= 5, 24; P= 0.014). In treatments in which hosts were added continuously, nematodes persisted at significantly higher population densities compared with treatments in which hosts were added either early or late, only (F= 13.58; df= 1; P= 0.0012). Persistence was not influenced by the host species supplied (F= 0. 22; df= 1; P= 0.646) (Figure 4.2).

88

6000 2 4000 IJ's/m 2000 0 Early Late Continuous Host Supply

Figure4.1. Season I (2001) – Influence of host supply on the persistence of Heterorhabditis bacteriophora, exotic strain (Lewiston). Host supply: Early – hosts added at nematode application, Late – hosts added three weeks after nematode application, Continuous – hosts added at nematode application and three weeks after application. IJ’s – infective juvenile nematodes.

89

6000 Early Late Continuous 2 4000 ij's/m 2000

0 CW OM CW OM CW+OM OM+CW Host Type

Figure 4.2. Season I (2001) – Influence of carrot weevil larvae, Listronotus oregonesis (CW) and onion maggots, Delia antiqua (OM) on the persistence of Heterorhabditis bacteriophora, exotic strain (Lewiston). IJ’s – infective juvenile nematodes.

90 In 2002, both strains of H. bacteriophora (endemic and exotic, HP88), persisted over the six-week period, at a mean density of 643 IJ’s m-2 (±132). Differences between strains in population density at the end of six weeks were not statistically significant (F=

2.08; df= 1; P= 0.15) (Figures 4.3 and 4.4). Both nematodes were similarly affected by the absence of hosts (F≤ 0.01; df= 1; P≥ 0.95); the daily mortality rates were estimated as

0.18 and 0.26 for the exotic (HP88) and the endemic strain, respectively.

Lower population densities of the commercial strain were observed in treatments in which hosts were absent, compared with treatments in which hosts were present (F≥

8.76; df=1; P≤ 0.004). However, no differences were observed in infective juvenile population densities between treatments with a continuous or sporadic supply of hosts

(F= 0.17; df= 1; P= 0.685). Infective juvenile persistence was significantly influenced by host type. For treatments in which hosts were introduced early in the season (at nematode application), persistence was greater with carrot weevil than with onion maggots (F=

10.01; df= 1; P= 0.002) (Figure 4.5). For the endemic isolate, persistence was not influenced by the presence, abundance, or species of hosts introduced (F≤ 0.78; df= 1; P≥

0.37) (Figure 4.6)

DISCUSSION

Long-term maintenance of viable populations of infective juveniles at levels at which effective and sustained pest suppression occur is an ultimate goal for using entomopathogenic nematodes for pest management in vegetable production. Long-term

91

6000 2 4000 IJ's/m 2000

0 No hosts Early Late Continuous Host Supply

Figure 4.3. Season II (2002) – Influence of host supply on the persistence of Heterorhabditis bacteriophora, exotic strain (HP88). Host supply: Early – hosts added at nematode application, Late – hosts added three weeks after nematode application, Continuous – hosts added at nematode application and three weeks after application. IJ’s – infective juvenile nematodes.

92

6000 2 4000 IJ's/m 2000

0 No hosts Early Late Continuous Host Supply

Figure 4.4. Season II (2002) – Influence of host supply on the persistence of Heterorhabditis bacteriophora, endemic strain. Host supply: Early – hosts added at nematode application, Late – hosts added three weeks after nematode application, Continuous – hosts added at nematode application and three weeks after application. IJ’s – infective juvenile nematodes.

93

6000 NoneEarly Late Continuous

4000 2 ij's/m 2000

0 None None CW OM CW OM CW+OMOM+CW Host Type

Figure 4.5. Season II (2002) – Influence of carrot weevil larvae, Listronotus oregonesis (CW) and onion maggots, Delia antiqua (OM) on the persistence of Heterorhabditis bacteriophora, exotic strain (HP88). IJ’s – infective juvenile nematodes.

94

6000 NoneEarly Late Continuous

4000 2 ij's/m 2000

0 None None CW OM CW OM CW+OM OM+CW Host Type

Figure 4.6. Season II (2002) – Influence of carrot weevil larvae, Listronotus oregonesis (CW) and onion maggots, Delia antiqua (OM) on the persistence of Heterorhabditis bacteriophora, endemic strain. IJ’s – infective juvenile nematodes.

95 maintenance, however, requires recycling of nematodes through insect hosts (Lewis et al.

1998). All strains of H. bacteriophora persisted for at least six weeks and infective juveniles were present at densities as high as 4,200 IJ’s m-2 in some treatments.

Nematodes could have been sustained at these levels by infective juveniles recycling within introduced hosts. Direct observations of recycling were not conducted and nematodes found after six weeks could have been residual populations from the inundative release or newly emerged infective juveniles from the hosts supplied, or both.

However, host availability was the only treatment variable and nematode populations after six weeks were significantly higher in treatments to which hosts were added.

Moreover, nematode infected hosts were observed during soil processing suggesting that the infective juveniles entered the introduced hosts and recycled.

Nematode strains differed in their response to host availability. The lack of a significant increase in persistence of one exotic strain, HP88, and the endemic strain in relation to host supply could be related to higher field mortality rates. Model predictions suggested that mortality rates and host factors interact in their effects on long-term persistence of nematodes. At field mortality rates ≥0.15, large quantities of high-quality hosts were predicted to be required to maintain nematodes at levels >10,000 IJ’s m-2.

Field mortality rates were estimated at 0.18 and 0.26 for the exotic (HP88) and the endemic strain, respectively. The quantity of hosts that were introduced in the study may have been insufficient to observe a response in nematode persistence equal to that observed in 2001. This would have been more so for the endemic nematode, which declined at a faster rate than HP88.

96 Differential survival of the strains to the climatic conditions of the test site could be linked to the geographic origin of the nematodes (Grewal et al. 1994, Molyneux

1985). The endemic nematode, which had the lowest persistence and showed no response to the presence of hosts, was isolated from a relatively sheltered environment (grassy field border) in the production area where temperature ranged between 10.3°C - 24.0°C and soil moisture ranged between 24.5% and 63.12% (May – October). During the experiment, the weather was very hot and dry and on several occasions the maximum daily temperature exceeded 30°C. Although the total rainfall was estimated at 15cm there were many weeks without any precipitation. Plots were irrigated but this may not have been sufficient to maintain soil moisture at levels suitable for infective juvenile survival.

Temperature and moisture conditions at the experimental site could have been deleterious to infective juveniles (Smits 1996, Kopenhoffer et al. 1995, Kaya 1990) or they could have induced the nematodes to enter a state of inactivity (Womersley 1990). Under these conditions, the infectivity of insect hosts would be reduced (Baur and Kaya 1998).

Differential infectivity and reproduction among nematode strains in the hosts introduced could also have accounted for the variation in response of the nematodes to host supply. Differential infectivity and reproduction have been reported among species and strains of steinernematids and heterorhabditids in carrot weevil and onion maggot

(Chen et al. 2003, Miklasiewicz et al. 2002, Vanninen et al. 1992, Belair and Boivin

1985).

Gouge et al. (1999) demonstrated that both temperature and host species affected infectivity of H. bacteriophora and S. riobrave. Depending on the nematode and host species, optimal infectivity occurred between 22°C and 28.5°C, but infectivity by all

97 nematodes was reduced at temperatures >35°C. Mason and Homminick (1995) recorded similar observations for five Heterorhabditis spp., and in addition, observed that infectivity and reproduction occurred at different thermal ranges. The authors also report that the number of infective juveniles produced within G. mellonella larvae varied with temperature and nematode strains and not all infected larvae produced progeny.

Differential infectivity of nematode strains in response to soil moisture conditions has also been documented (Koppenhofer et al. 1995, Kaya 1990).

Overall, nematodes persisted at significantly lower levels than the population density at the initial application. Low persistence could have resulted from the quantity and the timing of host introductions. The nematode to host ratio was 10,000-12,000:1. This ratio would have resulted in insufficient hosts to support infection by a large proportion of the infective juveniles applied. Moreover, a large number of infective juveniles may have entered the hosts, resulting in overcrowding within cadavers and reduced progeny production. Density-dependent penetration rates and deleterious effects of overcrowding have been reported. H. bacteriophora and S. carpocapse survived optimally at 100 nematodes per G. mellonella larva; at higher numbers there was a trade off in the number and size of progeny (Selvan et al. 1993). At any one time only a portion of infective juveniles is infectious to hosts present. This phased infectivity is thought to be a result of inhibitory cues produced by infected hosts (Fairbairn et al. 2000, Bohan and Hominick

1996). Hence, only a proportion of the large number of infectives applied could have infected the hosts that were introduced; those becoming infectious later would not have had hosts available.

98 Nematode population densities were estimated using the Galleria soil-baiting technique (Bedding and Akhurst 1975). Although Galleria is very susceptible to nematode infection, differential pathogenicity among nematode species and strains could result in variability in the extraction efficiency of the bait insects. Rueda at al (1993) used greater wax moth larvae, lesser mealworms, house flies and crickets to isolate entomopathogenic nematodes from soil collected from plant nurseries and observed that although wax moth larvae were the most efficient in isolating nematodes, other bait insects were able to isolate strains that wax moth larvae could not recover. Even with multiple baitings of the soil, Galleria is only able to recover an estimated 30-40% of nematodes (Fan and Hominick 1991). Thus, the low apparent persistence could have been due in part to a combination of the test insect used and the efficiency of the extraction technique.

Few successful attempts have been made to conserve entomopathogenic nematodes in annual systems such as vegetables. A complete understanding of nematode- host interactions under field conditions is lacking. This study provided insight into the importance of host availability on nematode persistence. In 2001, H. bacteriophora persisted at higher levels in treatments in which a continuous supply of hosts was available. Such conditions could be created in production systems by rotating crops that are attacked by pests susceptible to entomopathogenic nematode infections. This could be a feasible option in heterogeneous vegetable landscapes in which there are multiple pests to serve as potential hosts for the nematodes to recycle. The low levels of persistence of infective juveniles observed, suggest that apart from manipulating crop communities, it

99 may be more feasible to manipulate non-crop plants and soil conditions to provide additional non-pest hosts in which nematodes can recycle. The persistence of nematode populations also will be dependent, however, on their field mortality rates.

100 CHAPTER 5

BOTTOM-UP EFFECTS OF PLANT COMMUNITIES ON THE PERSISTENCE OF

TWO STRAINS OF THE ENTOMOPATHOGENIC NEMATODE

HETERORHABDITIS BACTERIOPHORA

INTRODUCTION

Maintaining populations of natural enemies within production systems by manipulating lower trophic groups, bottom-up regulation, has been widely demonstrated in arthropod systems (reviews by Langellotto and Denno 2004, Denno et al. 2002, Landis et al. 2000, Gurr 1998, Andow 1991, Sheehan 1986) and to a lesser extent with entomopathogenic nematodes. To date, most of the studies addressing the impact of plant communities on the persistence of entomopathogenic nematode populations have been limited to the effects of microclimatic conditions (Brust 1991, Shapiro et al. 1999), host quality (Kunkel et al. 2004, Kunkel and Grewal 2003, Eben and Barbercheck 1996,

Barbercheck et al. 1995), and host finding (van Tol et al. 2001, Choo et al. 1989).

Understanding the influence of plant factors on abiotic conditions and the spatial and temporal distribution of hosts will be important for designing strategies to conserve entomopathogenic nematode populations (Lewis et al. 1998).

101 The main objectives of this study were to: (i) determine the influence of plant diversity on the persistence of an exotic and an endemic strain of Heterorhabditis bacteriophora and (ii) identify the factors responsible for regulating nematode persistence. Increased plant diversity was hypothesized to improve microclimatic conditions for infective juvenile nematodes and increase the abundance and diversity of insect hosts for nematode reproduction.

METHODOLOGY

Experimental Site

The study was conducted at the Muck Crops Branch of the Ohio Agricultural

Research and Development Center (OARDC), in Huron County, Ohio (June

– October, 2004). The soil type at the field station is a muck soil; a Linwood series with moist bulk density of 0.15-0.4 g/cc, pH of 4.5-7.8, and OM content of 40-70%) (Ernst and Martin 1994).

Experimental Design

A factorial experiment was conducted to compare the persistence of two nematode strains in three plant communities (high diversity, low diversity and bare- ground) (3 x 2). Treatments were allocated in a randomized compete block design with four replicates. Test plots (6 x 6m) were surrounded by bare soil (1.8m).

102 Crops

High-diversity plots included a mixture of barley, Hordeum vulgare, red clover,

Trifolium repens L., turnips, Brassica rapa, pig-weed, Amaranthus sp., and purslane,

Portulaca oleracea L. Low-diversity plots consisted of barley only. Seeds were broadcasted over each plot with a hand-held seeder (Earthway Products, Bristol Indiana;

Model Number 2700-A) at 6.8Kg, 8.4kg and 102.2Kg per hectare for turnips, clover and barley, respectively. Bare-ground and low-diversity plots and borders were hand-weeded to ensure that plots were devoid of vegetation or that barley was the only plant species present in low-diversity plots.

Entomopathogenic nematodes

The H. bacteriophora strains evaluated were: (i) a endemic strain from

Celeryville (Huron County, Ohio in 2001), and (ii) a exotic strain, HP88 purchased from

Integrated Biological Control Systems, (Greendale, IN, USA). The native strain was cultured using standard in-vivo techniques with Galleria mellonella (Dutky et al. 1964).

Infective juveniles (IJ’s) were harvested from White traps (White 1927) and were 4-7 days old when applied. Within each plot, four 1m2 areas were demarcated with flags and nematodes were applied to these areas with watering cans at a rate of 2.5 billion IJ’s ha-1.

Entomopathogenic nematode sampling, extraction, and population estimation

Nematode populations were assessed within the four-1m2 areas in which applications were made. Infective juvenile populations were assessed before nematode application and monthly for four months. The assessment before nematode application

103 was conducted to verify that endemic and/or residual populations from past applications were not present. Within each m2 area, four cores of soil were collected (5.08cm diameter, 12cm depth, approx 200g). Samples were kept in a cooler and transported to the laboratory for nematode extraction. Nematode population densities were assessed by the standard G. mellonella baiting method (Fan and Hominick 1991). For each sample, the soil was mixed thoroughly, 200g were placed in a plastic container, and ten greater wax moth larvae were incorporated. The container was sealed and placed in a chamber in complete darkness, at 25°C, and 60% RH. Three days later, all larvae were removed and the soil was re-baited with an additional ten wax moth larvae and incubated for an additional three days. Wax moth larvae were examined and those killed by nematodes

(i.e., having characteristic color, smell, and texture) were dissected and the number of nematodes penetrating was recorded. Larvae that were alive were incubated for an additional three days and nematode infectivity and populations recorded as previously described. For those cadavers with symptoms of nematode infection, but in which infective juveniles were not observed during dissection, a value of one was given as the minimum number of infective juveniles that could be responsible for larval mortality.

Population assessment of insect community

Foliage within the four 1m2 sample areas was vacuumed with a modified leaf blower (STIHL BG72). Insects collected were placed in plastic bags and kept in a cooler for transport to the laboratory. Before processing, samples were placed in the freezer for a minimum of 48 hours, insects were then removed from bags and placed in vials containing alcohol (70%).

104 Ground-dwelling insect activity was measured with pitfall traps. Traps consisted of a plastic cup (473ml, Dart Container Corp., Mason Michigan) in which a paper funnel made from a conical cup (20ml, Sweetheart Products, Maryland) was placed just below the rim. The cup was covered with a lid (10cm diameter, Dart Cooperation, Michigan) in which a hole was made (6cm) in the center. A single pitfall trap was buried in the center of each field plot. Traps were collected after one week and arthropods were placed in vials containing alcohol (70%). Small insects and larvae were extracted from a portion of the soil collected (200-400g), by washing the soil through a series of graded sieves (US standard sieve series #12, 1.68mm; #18, 1mm; #35, 500µm). All insects collected were identified to family and diversity indices were calculated (family diversity, richness, and evenness) according to:

S (i) Shannon-Weiner Index H′ = -Σpilogepi, i=1 (ii) Richness= S-1/logeN

(iii) Evenness J′= H′/H′ max

Where, N= the number of individuals identified, S= the number of families identified, a given family regarded as the ith family; p= the proportion of individuals in the ith family.

Data analysis

All data were transformed to achieve normality and equality of variances according to the ladder of powers cited by Fry (1999). Nematode population data was transformed by square root (SQRT +1) and insect community data including diversity indices were transformed by natural log (LN +1) or square root (SQRT +1). Repeated

Measures Analysis of Variance (ANOVA) was conducted to determine the impact of

105 plant diversity on insect populations (total abundance, species diversity and richness) and plant diversity on nematode persistence. Treatment means were compared using contrasts. Spatial and temporal correlations between nematode densities and insect populations were tested at ά= 0.05. Data were analyzed using SAS statistical software and MINITAB.

RESULTS

Insect community

Insects from nine orders and more than 50 families were collected. The mean insect population abundance per plot was 225.1 (±24.6) (Table 5.1). Diptera and

Homoptera were the most common insects followed by Thysanoptera and Coleoptera; mean population densities per sample period were 117.1 (±16.6), 77.5(±9.94),

16.37(±1.82), and 15.72 (±1.72) per 4m2, respectively (Figure 5.1). Total insect population abundance, family diversity, and richness decreased significantly over the 16 weeks of the study (F≥ 13.27; df= 4, 52, P≤ 0.0038) (Figure 5.2). Early in the season, insect population abundance was significantly larger in high compared with low-diversity plots (F=5.49; df= 1, 13; P= 0.035). Family diversity was significantly greater in high- diversity than low-diversity plots 56 days after planting (F= 12.31; df= 1,13; P= 0.005).

Family richness was greater in high-diversity plots for most of the season, but this difference was only significant 28 days after planting (F=6.41; df=1,13; P= 0.02). With the exception of chrysomelid beetles, plant diversity had no significant influence on the abundance of any other family of insect that could serve as potential hosts for

106

Insect Insect Order Family Order Family

Coleoptera Chrysomelidae *+ Hemiptera Nabidae * Coccinellidae Miridae * Anthicidae Anthocoridae Staphylinidae Tingidae Cucujidae Phalacridae * Homoptera Cicadellidae * Carabidae Delphacidae Aphididae Diptera Chloropidae* Membracidae Otitidae Aleyrodidae Cecidomyiidae Anthomyzidae Hymenoptera Eucoilidae Drosophilidae * Pteromalidae Ceratopogonidae * Mymaridae Anthomyiidae + Ichneumonidae Phoridae Aphidinae Lonchopteridae Eulophidae Scatopsidae Braconidae * Chironomidae Encyrtidae Dolichopidae Scelionidae Sciaridae + Ceraphronidae Sphaeroceridae Psychodidae Thysanoptera Thripidae+ Tipulidae Ephydridae Odonata Coenagrionidae Syrphidae Sepsidae Collembola Entomobryidae * Empididae Sminthuridae Poduridae Orthoptera Gryllidae+ Acrididae

*- most abundant families within the order, + families in which entomopathogenic nematodes have been naturally isolated or identified as being susceptible to moderately susceptible in laboratory and or field studies (Peters 1996, Grewal and Georgis 1998).

Table 5.1. Composition and abundance of insect populations colonizing high and low plant diversity plots, June-October 2003. High-diversity plots – barley, Hordeum vulgarei, red clover, Trifolium repens L., turnips, Brassica rapa, pig-weed, Amaranthus sp., and purslane, Portulaca oleracea, and low-diversity plots - barley alone.

107

Thysanoptera Lepidoptera Hymenoptera Homoptera Hemiptera Insect Orders Diptera Coleoptera 0 20 40 60 80 100 120 140 160 Mean abundance/plot

Low plant diversity High plant diversity

Figure 5.1. Composition and abundance of insect populations colonizing high and low plant diversity plots, June-October 2003. High plant diversity plots – barley, Hordeum vulgarei, red clover, Trifolium repens L., turnips, Brassica rapa, pig-weed, Amaranthus sp., and purslane, Portulaca oleracea, and low plant diversity plots - barley alone.

108 600

450

300

c Total abundance 150

0 28 56 81 113 142 Days afte r planting

6 ) S

4

2 Family Richness IndexFamily ( Richness

0 28 56c 81 113 142 Days afte r planting

2.8 ) H' H' 2.1

1.4

0.7 Family Diversity Index ( Family Diversity

0 28 56 81 113 142 Days after planting

0.8

0.6 ) J'

0.4

Family Eveness ( Eveness Family 0.2

0 28 56 81 113 142

Days after planting

Low plant diversity High plant diversity

Figure 5.2. Diversity indices for insects colonizing high and low plant diversity plots, June-October 2003. High plant diversity plots – barley, Hordeum vulgarei, red clover, Trifolium repens L., turnips, Brassica rapa, pig-weed, Amaranthus sp., and purslane, Portulaca oleracea, and low plant diversity plots - barley alone. Indices: Shannon-Weiner Index H′ = -Σpilogepi, Richness= S-1/logeN, Evenness J′= H′/H′ max . Where, N= the number of individuals identified, S= the number of families identified, a given family regarded as the ith family, p= the proportion of individuals in the ith family.

109 entomopathogenic nematodes (Thripidae, Anthomyiidae, Lepidoptera larvae).

Chrysomelid beetle populations were consistently greater in high-diversity than in low- diversity plots (F= 12.85; df=1,13; P= 0.003) (Figure 5.3).

Entomopathogenic nematode population densities

Infective juvenile population densities decreased during the study; mean infective juvenile density after 16 weeks was 2,641 (±303) per m2 (Figure 5.4). Compared with the exotic strain, significantly lower population densities were observed for the endemic strain; mean densities were 3,729 (±506) and 1,552 (±257) per m2, respectively.

Averaged over time, there was a significant effect of plant diversity on nematode persistence (F≥ 6.35; df= 2, 18; P≤ 0.008). Nematodes applied to bare-ground plots persisted at significantly lower population densities compared with plots with vegetation

(F= 169.0; df= 1; P< 0.0001). In high-diversity plots, the exotic nematode strain persisted at significantly higher population densities than in low-diversity plots (F=4.0; df=1;

P=0.048). For the endemic nematode, no differences in nematode population densities were observed between high and low-diversity plots (F= 1.18; df=1; P=0.28).

Interactions between entomopathogenic nematode populations and insect communities

For the endemic nematode strain, a significant correlation was obtained between thrips populations and infective juvenile densities (Pearson’s correlation coefficient=

0.449, P= 0.01). For the exotic nematode strain, a significant correlation was observed

110

25 20

15 10

population/plot 5 Chrysomelid beetle beetle Chrysomelid 0 28 56 81 113 142 Days after planting

Low plant diversity High plant diversity

Figure 5.3. Mean population abundance of chrysomelid beetles colonizing high and low plant diversity plots, June – October 2003. High plant diversity plots – barley, Hordeum vulgarei, red clover, Trifolium repens L., turnips, Brassica rapa, pig-weed, Amaranthus sp., and purslane, Portulaca oleracea, and low plant diversity plots - barley alone.

111 A

4500

2 3000

IJ's/m 1500

0 28 53 87 116 Days after nematode application

B

4500

2 3000

IJ's/m 1500

0 28 53 87 116 Days after nematode application

Bare-ground Low plant diversity High plant diversity

Figure 5.4. Persistence of an exotic (A), and an endemic (B) strain of Heterorhabditis bacteriophora in relation to plant diversity, June-October 2003. IJ’s- infective juveniles. High plant diversity plots – barley, Hordeum vulgarei, red clover, Trifolium repens L., turnips, Brassica rapa, pig-weed, Amaranthus sp., and purslane, Portulaca oleracea, and low plant diversity plots - barley alone.

112 between chrysomelid beetle populations and infective juvenile population densities in high-diversity plots (Pearson’s correlation coefficient= 0.39, P= 0.027); but not low- diversity plots (Pearson’s Correlation Coefficient= 0.175, P= 0.337).

DISCUSSION

Plant communities increased the persistence of both exotic and endemic strains of

H. bacteriophora. The increased persistence of the nematodes may be related to improved microclimatic conditions and the presence of suitable hosts in which nematodes can recycle. Environmental conditions, such as high temperatures, low moisture, and high

UV radiation are principal factors limiting nematode survival and infectivity (Baur and

Kaya 1998, Mason and Hominick 1995, Barbercheck 1992, Kaya 1990). Gouge et al.

(1999) observed optimal infectivity of H. bacteriophora between 22°C and 28.5°C and reduced infectivity at temperatures >35°C. Several studies have demonstrated that low soil moisture impedes movement of infective juveniles and reduces survival and infectivity (Grant and Villiani 2003, Koppenhofer et al. 1995, Kung et al. 1991). At the study site, periods of high temperatures and low moisture were recorded, with highs between 27°C - 30°C and no precipitation for several consecutive days. The plant canopy may have buffered infective juveniles from these unfavorable conditions. Both Brust

(1991) and Shapiro et al. (1999) reported that the presence of weedy cultivations and soybean residues increased nematode persistence and they attributed these observations to the ground cover protecting infective juveniles from adverse environmental conditions.

113 Recycling of infective juveniles through insect hosts present within the plots could have accounted for the greater persistence of the exotic nematode in high-diversity plots. Direct observations of recycling were not conducted during the study, and infected insects were not isolated while processing samples. However, the following trends suggest recycling through hosts: (i) anthomyiids, thrips and chrysomelids, insects from which nematodes have been naturally isolated or shown to be moderately susceptible to infection, were present in the plots (Ebssa et al. 2001, Grewal and Georgis 1998, Peters

1996, Klein 1990), (ii) chrysomelid beetle populations were most abundant in the high- diversity plots, and (iii) a significant correlation was observed between infective juvenile densities and beetle numbers in high-diversity plots.

Nematode-host interactions may have been masked by biases in the population estimates of both infective juvenile and insect host populations. Infective juveniles disperse primarily through other agents including infected insects and microarthropods

(Kaya 1990, Epsky et al. 1988). Parkman and Frank (1991) found that nematode-infected mole crickets, Scapteriscus spp. dispersed several kilometers from the initial release site and Timper et al. (1988) observed that Spodoptera exigua infected with Steinernema feltiae could disperse up to 11m from the infection site before dying. Therefore, infected insects within plots could have moved outside the sample area to other treatment plots or outside of the experimental area before dying, resulting in nematode populations being underestimated.

The movement of colonizing insects could have further masked host interactions

(Koricheva et al. 2000, Banks 1998, Roland and Taylor 1997, Russell 1989). Field plots were 36m2 with 1.8m separators; very mobile insect host species could have dispersed

114 among plots and distorted population estimates. In addition, the timing and intensity of sampling could have affected insect counts. Plots were sampled early in the mornings when it was cool and dewy, insect activity may have been lower compared with later in the day. Infection and recycling occurring in insects with low population densities may have been undetectable at the sampling intensity used.

The lower persistence of the endemic nematode compared with the exotic nematode may have been due to differences in infective juvenile survival under conditions at the test site and differences in infectivity to the insect community colonizing the plots (Baur and Kaya 1998, Kaya and Gaugler 1993). The geographic origin of nematodes is often responsible for their adaptation to test conditions (Grewal et al. 1993, 1994, Molyneux. 1985). The endemic nematode was recently isolated along grassy banks within the vegetable production area, and so infective juveniles may have been more adapted to the abiotic conditions and the insect host communities associated with this habitat.

Differences in infectivity of the strains to the bait insect, G. mellonella, could have also contributed to the variation in measured persistence. The exotic strain is mass cultured in Galleria and may have been selected for this insect species, hence its recovery rate from the soil could be higher. The endemic strain that was recently isolated may not be as strongly adapted to G. mellonella; hence infective juveniles could be recovered at a lower rate. Rueda et al. (1993) used greater wax moth larvae, lesser mealworms, house flies, and crickets to isolate entomopathogenic nematodes from soil collected from plant

115 nurseries and observed that although wax moth larvae were the most efficient insect for isolating infective juveniles from the soil, other bait insects were able to isolate nematode strains that wax moth larvae could not recover.

Conditions under which the nematodes were cultured could also have accounted for differences in persistence between the strains. Both nematodes were produced in-vivo; however, the endemic nematode was cultured in the laboratory and the exotic nematode was obtained from a commercial source. The in-vivo method of production is sensitive to biological variations (Friedman 1990). Good culturing techniques and storage conditions are therefore necessary for producing viable infective juveniles. Conditions at the commercial facility may have differed from the laboratory conditions under which the endemic nematode was reared with resulting differences in viability.

The generally low persistence of both nematode strains compared with the initial application may be in part due to flooding at the study site. Within seven days of applying nematodes, there was 11.6cm of rain and the plots were submerged for more than 24 hours. Nematodes could have been washed from the sample area to surrounding field plots. The efficiency of the extraction technique also could have contributed to low nematode recovery from the soil samples. Even with multiple baitings of the soil,

Galleria is only able to recover 30-40% of infective juveniles present (Fan and Hominick

1991).

Manipulating cropping environments is a potential management strategy to conserve populations of entomopathogenic nematodes. However, selecting plant species that attract and retain an abundance of non-pest host species in which infective juveniles can recycle will be critical for maximizing bottom-up effects of plant communities on

116 nematode persistence. Retaining insect host species within the cropping area will require an understanding of the spatial and temporal scales at which plant-insect host interactions occur. With the exception of chrysomelid beetles, no consistent increases in insect diversity were observed in relation to increasing plant diversity. The scale of the experiment relative to the dispersal range and the resource specialization of colonizing species could be responsible for these observations (Chust et al. 2004, Banks 1998,

Roland and Taylor 1997, Russell 1989). Koricheva et al. (2000) observed that manipulating plant diversity only impacted species that had a narrow host range and were relatively immobile, however, very mobile polyphagous species were affected to a lesser extent and moved between plots. The predominant insect species in the study were leafhoppers and Diptera, both of which are relatively mobile. Chust et al. (2004) reported that the relative abundance of Homoptera was based on factors at local spatial scales

(0.36-2.25 ha) and Diptera were more sensitive to diversity at landscape scales (> 250 ha). An understanding of the life-history traits of colonizing insects will therefore be important for understanding how habitats need to be manipulated. Ohio vegetable growers currently use cover crops to protect seedlings from wind damage and soil erosion, improve soil fertility, conserve soil moisture, regulate soil temperatures, and improve weed suppression (Woolwine and Regan 2001). This practice could be used in developing strategies for conserving entomopathogenic nematode populations in vegetable production areas.

117 CHAPTER 6

BOTTOM-UP FACTORS REGULATING THE PERSISTENCE OF DIADEGMA

INSULARE CRESSON HYMENOPTERA: ICHNEUMONIDAE) IN

HETEROGENEOUS VEGETABLE CROPPING SYSTEMS

INTRODUCTION

Parasitoids divide their time between foraging for hosts and food resources such as pollen and nectar, to optimize their fitness. Foraging for hosts or food is influenced strongly by the parasitoid’s physiological state. Two major physiological states competing are starvation and the need to oviposit (Lewis et al. 1998, Stapel et al. 1997,

Heimpel et al. 1996). Often, parasitoids prioritize foraging for food resources over hosts, as food increases oviposition and lifespan, and starvation leads to super-parasitism and decreased fitness (Lewis et al. 1998, Takasu and Lewis 1995).

In agricultural cropping systems, host and food resources are patchily distributed in space and time, and organisms disperse among crop patches to satisfy their resource needs (Dunning et al. 1992). Manipulating crop and non-crop areas so that food and host resources required by natural enemies are present is a means of increasing their efficacy and persistence in cultivated areas (Landis et al. 2000, Landis and Menalled 1998, Lewis et al. 1998).

118 Throughout vegetable cropping systems of Huron County, Ohio the larval parasitoid Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) is an important biological control agent of the diamondback moth, Plutella xylostella L. (Lepidoptera:

Plutellidae), a major pest of crucifers (Talekar and Shelton 1993). Increasing the efficacy of D. insulare by including floral resources within crucifer plantings has been widely recommended as longer life-spans and greater fecundity are recorded when these wasps feed on flowering species (Johanowicz and Mitchell 2000, Idris and Grafius 1997, 1996).

Within the production area, few flowering species are present, but many herbs that are cultivated such as coriander, Coriandrum sativum (Umbelliferae) possess floral resources that serve as a good source of nutrition for many parasitiods (Baggen and Gurr 1998).

Incorporating flowering coriander in cropping areas may, therefore, increase the effectiveness of D. insulare and its impact on diamondback moth populations.

Many studies on the foraging behavior of D. insulare have focused on determining the influence of either host factors or floral resources on population persistence under laboratory conditions; few have measured the interaction of these factors in the field. The objectives of this study were to determine the influence of high and low population densities of the diamondback moth and flowering coriander on the persistence of D. insulare. Food resource provided by flowering coriander was hypothesized to increase the persistence of D. insulare, with greater persistence occurring under conditions of high compared with low population densities of diamondback moth.

119 METHODOLOGY

Experimental Site

The experiment was conducted at two Ohio Agricultural Research and

Development Center (OARDC) research farms located in Wooster (Wayne County,

Ohio) and Celeryville (Huron County, Ohio) during July – October 2003.

Experimental Design

Twenty plots (1.5 X 6m) with either two rows of collards (Brassica oleraceae var

Champion) (n=10) or two rows of collards intercropped with a single row of coriander

(Coriandrum sativum var Santo) (n=10) were established over a 4ha area, with a minimum distance of 50m between plots. Crops were planted with a Stanhay precision planter at rates of 36 and 26 seeds per meter for collards and coriander, respectively.

All plants in the plots were scouted monthly for diamondback moth larvae and categorized as high or low population density plots. Forty-eight hours before studies were to be conducted, the plots were sampled and laboratory-reared diamondback larvae

(DBM) were added to high population density plots to ensure that they had more than 40 larvae; and low population density plots had less than 25 larvae. Four treatments were evaluated: (i) mixed cultivations of coriander and collards infested with low population densities of DBM larvae, (ii) mixed cultivations of coriander and collards infested with high population densities of DBM larvae, (iii) collards infested with low population densities of DBM larvae, and (iv) collards infested with high population densities of

DBM larvae. Because flooding destroyed some plots, 3-4 replicates per treatment were evaluated.

120 Behavioral observations

Two sets of observations of D. insulare behavior were made on different days.

For each plot, the number of D. insulare visiting within a 15-minute interval was observed and recorded. For each wasp sighting, the time of the sighting and the time of departure of the wasp from the plot were noted.

Parasitism

Within each plot, sentinel plants were introduced to measure parasitism. Cabbage plants (B. oleracea var Hinnova) (8-10 cm; >5 leaves) were infested with thirty diamondback moth eggs and incubated for 3 days until first instars (mining stage) were present. After incubation, plants were transported to the field in individual cups (1000 ml,

Sweetheart clear plastic) covered with domed lids (11 cm diameter, Sweetheart clear plastic). Plants were removed from the large cups, placed in smaller cups (300 ml,

Sweetheart clear plastic) and suspended in the canopy of collard plants with wooden stakes (23cm). An adhesive (®Tangle foot) was applied to the bases of stakes to minimize predation. Two plants were placed in each plot 1m apart. Plants were collected after 48 hours, placed in cups with lids, and held in the laboratory at 26˚C and 12:12 L:D photoperiod. Fresh cabbage leaves were added to each cup until the larvae pupated.

Pupae within each cup were removed and each was placed in an individual cup (35ml,

Dart Corp.) until either a D. insulare or an adult diamondback moth was observed. Total numbers of emerging D. insulare and diamondback moths on each sentinel plant were recorded.

121 Data analysis

All data were transformed to achieve normality and equality of variances according to the ladder of powers cited by Fry (1999). Data was transformed by √x+1

(number of wasp visits, time for first wasp sighting and retention time) or arcsine √x

(proportion of pupae parasitized). Analysis of Variance (ANOVA) was conducted to determine the influence of host densities and floral resources on the number of wasp visits and parasitism. Treatment means were compared using contrasts. A correlation between wasp visits and parasitism was estimated and tested at ά= 0.05. Data were analyzed using SAS statistical software and MINITAB.

RESULTS

Behavioral observations

A total of 177 wasp visits were recorded; 48 in Wooster and 129 in Celeryville.

For either location, no interactions were observed between diamondback moth population densities and the presence of flowering coriander or the simple effects of flowering coriander; the data were pooled over floral resources. For Celeryville, significantly more wasps visited the high population density plots compared with low population density plots per 15-minute observation interval (F= 5.10; df= 2,13; P= 0.041) (Figure 6.1). The mean time for the first visit to be observed was significantly less for the high population density plots compared with the low population density plots (F=7.59; df= 1,14; P= 0.01)

(Figure 6.2). For Wooster, no statistically significant differences were recorded in the number of wasp visits to plots or the time taken for the first wasp sighting (F≤ 0.31; df=

122 2,12; P≥ 0.38). Mean retention times of wasps in the plots ranged between 0.2-1.77 minutes and for both locations, no significant differences were observed between treatments (F≤ 0.95; df= 2, 12; P≥ 0.40) (Figure 6.3 and 6.4).

Parasitism

In Wooster, none of the larvae introduced into the plots on sentinel plants were parasitized. For Celeryville, mean parasitism within plots was 36.32% (±5.66) and ranged between 0 and 78%. A significant interaction occurred between floral resources and host densities (F= 5.03; df= 3, 12; P=0.044) (Figure 6.5). In low population density plots, parasitism was higher in plots that had flowers compared with those without flowers. For high population density plots however, no significant difference among floral treatments was observed. No statistically significant relationship was observed between search activity (number of visits) and the levels of parasitism (Pearson’s correlation coefficient=

0.07, P= 0.778) (Figure 6.6).

DISCUSSION

Previous laboratory and field studies have demonstrated that Diadegma spp. forage to maximize the rate at which they find suitable hosts and aggregate in patches with high host population densities (Wang and Keller 2003, Waage 1983). Consistent with these studies, in Celeryville, D. insulare search activity was greater in plots with high densities of diamondback moth larvae. Optimality theories suggest that parasitoids

123

16

/ 12

8

D.insulare 15 minutes 15

# 4

0 Celeryville Wooster Locations

High Population Density Low Population Density

Figure 6.1. Number of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) visiting collard plots with high and low population densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae).

124

10

8

(minutes) 6 4

2 D.insualre D.insualre Time for 1st sighting of 0 Celeryville Wooster Locations

High Population Density Low Population Density

Figure 6.2. Time for observing the first sighting of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) in collard plots with high and low population densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae).

125

2

1.5

1 0.5

0

Retention Time(minutes) Celeryville Wooster Locations

High Population Density Low Population Density

Figure 6.3. Retention time of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) in plots of collards infested with high and low population densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae).

126

1

0.8

/ minute/ 0.6 0.4

D. insulare 0.2 # 0 Celeryville Wooster Locations

High Population Density Low Population Density

Figure 6.4. Frequency of visits of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) to collard plots infested with high and low population densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae).

127

80 High Population Low Population Density Density 60

40

20

% Parasitism 0 Coll Coll+Cor Coll Coll+Cor Floral Resources

Figure 6.5 Parasitism of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) by Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) in plots with collards (Coll) and collards and flowering coriander (Coll+Cor) in Celeryville, (Huron County, Ohio).

128

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2 % Parasitism (arcsin trans) (arcsin Parasitism % 0.1

0.0

1 2 3 4 5 Wasp visits (sqrt trans)

Figure 6.6. Correlation between number of visits of Diadegma insulare Cresson (Hymenoptera: Ichneumonidae) to plots of collards (Coll) and plots with collards and flowering coriander (Coll+Cor) with high and low population densities of diamondback moth.

129 will increase the time spent in patches with high host population densities; although slightly higher residence times were observed by wasps visiting high population density plots, they were not significantly different. Detailed behavioral observations to determine patch-leaving rules were not made. However, Waage (1983) also observed no significant differences in patch-allocation time of Diadegma sp. to high and low population density patches of diamondback moth and suggested that regardless of patch profitability,

Diadegma sp. has a fixed search time but discovers high population density plots faster and visits more frequently than low population density patches. Our data tends to support this explanation as: (i) the time for an initial wasp sighting was significantly lower in high compared with low population density plots and (ii) the number of visits per minute was higher for high population density plots. Challenges in the experimental design included: (i) the difficulty of observing fast moving wasps, particularly, if there was more than one wasp at the same time, (ii) the uncertainty that the absence of wasps within a particular observation period was not due to the observer missing the visit, especially if wasps spent an extended period of time on the undersides of leaves, and (iii) the limited number of observations made. In spite of these shortcomings, the data are instructive for understanding the response of D. insulare to host densities.

The lack of differences in the number of wasps visiting high and low population density plots at the Wooster site could be related to low populations or low parasitoid activity, or both. During the first observation period, temperatures averaged 4˚C and it was cloudy. Idris and Grafius (1998) observed that optimal activity for D. insulare in the

130 field occurred between 20˚C and 25˚C and low activity occurred at temperatures <15˚C and light intensities <500µEm-2s-1. Hence, the low numbers recorded may have been due largely to the inactivity of the wasps at the temperatures experienced.

A density-independent relationship was observed between parasitism and host number and Diadegma search activity. In the literature, the relationship between parasitism and host population densities ranges from positive to no significant relationship to negative density-dependence (Hassell 2000, 1982, Morrison and Strong

1980). Several factors relating to parasitoid behaviour have been proposed to explain these inconsistencies, including mutual interference and superparasitism (Cronin and

Strong 1993), deceleration in functional responses at high population densities

(Umbanhowar et al. 2003, Waage 1983), as well as avoidance (Janssen et al 1995). The study was not designed to assess changes in the functional response, and although many times several parasitoids were present within the same area, the population densities of wasps observed were too few for interference to occur. Avoidance is a possible mechanism and has been identified in many parasitoids, including ichneumonid species, as a means to reduce competition and superparasitism (Bernstein and Driessen 1996,

Janssen et al. 1995). Castelo et al. (2003) observed that Venturia canescens

(Hymenoptera: Ichneumonidae) was able to recognize odors from hosts as well as conspecifics, and used a combination of both odors to estimate patch profitability. These wasps displayed avoidance behaviours in patches with 20 conspecifics but readily entered patches with five conspecifics. Similar cues may be used by Diadegma sp. to avoid inter- and intra-specific competition. During the study, large numbers of Cotesia plutellae,

131 another natural enemy of the diamondback moth, were observed. In high population density plots where Diadegma foraged more frequently, the cues from competing species could have been stronger and hence healthy larvae may have been avoided.

Other factors contributing to the lack of a relationship between host numbers and parasitism could have been a combination of predation, dispersal of larvae from sentinel plants, and ambient climatic conditions at the sites. Within each plot, 60 larvae were present on the sentinel plants introduced. For high and low population density plots the total number of pupae (both parasitized and unparasitized) collected from sentinel plants was 32 and 57, respectively. Eggs and larvae of diamondback moth are consumed by a complex of predators including, Chrysoperla lacewings (Reddy et al. 2004, Reedy et al.

2002), carabid beetles, Chlaenius sp. (Suenaga and Hamura 1998), spiders, and coccinellids, Harmonia axyridis (Ferry et al. 2003). During observation periods,

Harmonia populations were very high and consistently observed in plots and sometimes on sentinel plants. These predators as well as other species could have consumed parasitized as well as unparasitized larvae on sentinel plants. Monsterrat et al. (2004) investigated the response of three plant-inhabiting predatory bugs (i.e., mirids and anthocorids) to different populations of prey, and observed that all the predator species had the tendency to leave patches when prey populations were low and spent longer times in patches when prey populations were high. Hence, the significantly lower number of pupae recovered from high population density plots may have been due to predators aggregating in plots as a result of more diamondback moth larvae being available.

132 Movement of the larvae from sentinel plants onto collard plants could have occurred. The sentinel plants used were only 10-12cm high and larvae could have moved across to collard plants to avoid competition. Collards are more susceptible than cabbages to attack by the diamondback moth and therefore may have been more attractive to developing larvae (Mitchell 1997). During the study, >25mm of rain was recorded; larvae could have been washed from plants and resulted in fewer larvae on sentinel plants.

For natural enemies to be effective in annual systems, pests must be suppressed during the latent phase of population growth (Wiedenmann and Smith 1997). Natural enemies must locate pests at low population densities so that, the chances of an increase in populations is reduced or prevented. The data from this study suggest that suppression of diamondback moths at low population densities is achievable for D. insulare. During the study, wasps foraged in plots with low population densities of diamondback moth and under these low population density conditions parasitism was enhanced in the presence of flowering coriander. These observations are promising for managing diamondback moth populations during early colonization of crucifer crop patches by D. insulare.

Bottom-up factors regulated the persistence and efficacy of D. insulare; high population densities of diamondback moth increased the activity of D. insulare in crop patches and flowering coriander increased parasitism of the diamondback moth.

Coriander is grown throughout the cropping season and harvested multiple times, allowing small sections of plantings to flower could be an approach for increasing the persistence of Diadegma sp. and parasitism of diamondback moth. Abundant flowering patches should increase the likelihood of encounters and retain parasitoids in the crop

133 area (Lewis et al. 1998). The configuration, and size as well as the location of coriander cultivations relative to crucifer plantings, will determine their impact on D. insulare parasitism. Understanding the dispersal range of D. insulare will be important for identifying the appropriate scale for manipulating the cropping area. In the current study, plots were spaced >50m apart and spread over a 0.4ha area, suggesting that manipulations of coriander plantings would be needed at scales of m2-Km2.

134 CHAPTER 7

CONSERVATION BIOLOGICAL CONTROL IN VEGETABLE SYSTEMS:

SYNTHESIS, POSSIBLE STRATEGIES AND FUTURE DIRECTION

SYNTHESIS

The results of this study supported my thesis of bottom-up regulation of the entomopathogenic nematode, Heterorhabditis bacteriophora and the lepidopteran parasitoid, Diadegma insulare. As predicted by the conceptual model, population persistence for both natural enemies was influenced directly and indirectly by organisms from lower trophic levels; primary producers and secondary consumers. However, the resource use patterns and the spatial scales at which populations were regulated differed for the two natural enemies. Entomopathogenic nematodes with very limited dispersal ranges responded to manipulations of hosts and plant communities at scales of cm2 - m2.

In contrast, D. insulare with a relatively large dispersal range responded to manipulations at larger scales, >m2 - km2.

Consistent with the conceptual model, the composition of the landscape and crop patches affected the persistence of the natural enemies either directly or indirectly or both. The distribution and persistence of endemic entomopathogenic nematode

135 populations within the vegetable landscape was dependent on the composition of the plant community and the disturbance regimes associated with the habitats (Chapter 2). At a smaller scale, crop patch diversity increased the persistence of H. bacteriophora by increasing the diversity of insect hosts in which infective juveniles recycled (Chapter 5)

(Figure 7.1). Manipulating plant communities to increase persistence of entomopathogenic nematodes has been proposed in the literature, but empirical evidence is sparse. The trophic cascade documented, therefore, provides support for this hypothesis. Research findings in Chapter 6, showed that crop patch factors directly influence population persistence of D. insulare; flowering coriander associated with the crop patch increased parasitism of diamondback moths by D. insulare (Figure 7.2).

The model proposed that for both natural enemies, herbivore communities would influence life processes such as infectivity/feeding and reproduction, and in addition the dispersal of D. insulare into crop patches. Endemic and exotic strains of entomopathogenic nematodes infected and recycled in herbivore communities associated with vegetable crop patches and survived poorly in the absence of an insect host

(Chapters 3 and 4). These data provided further empirical evidence that entomopathogenic nematodes require insect hosts to persist and populations respond numerically to host population densities (Chapter 4). In addition, the data provides insight into the influence of climatic conditions on nematode-host interactions, an area in which information is lacking. Both theoretical model predictions and empirical evidence from studies suggested that dauer survival influences bottom-up regulation of nematode population densities by insect hosts. Nematode species and strains that survive poorly under field climatic conditions persist poorly regardless of the presence or the abundance

136 of host insects. Conversely, if dauer survival is high then nematode population densities increase with increasing host supply. These data explain the repeated reports of low persistence and efficacy of exotic releases of entomopathogenic nematodes under conditions of high host densities. For D. insulare, greater persistence was also seen with increasing population densities of its host, the diamondback moth, persistence in this case was a result of wasps dispersing into crop patches rather than a function of reproduction

(Chapter 6). These data offer further support that D. insulare increases foraging activity in areas of high compared with low populations of diamondback moths. For D. insulare, an interaction between plant resources and insect host abundance resulted in higher wasp population in crop patches. When persistence was measured as a function of parasitism, plant resources interacted with insect host abundance but, only when host population densities were low.

PROPOSED STRATEGY FOR CONSERVING ENTOMOPATHOGENIC

NEMATODES AND D. INSULARE IN VEGETABLE LANDSCAPES

For H. bacteriophora and D. insulare to persist within vegetable landscapes several conditions need to be met. For the entomopathogenic nematode, multigenerational recycling of infective juveniles through insect hosts is key. However, for infective juveniles to persist at high population densities, while pests are maintained at low population densities, recycling must occur to a greater extent in non-pest insects than in pest insects. Strategies to increase the persistence of entomopathogenic nematodes will therefore require: (i) selection of crops and soil management practices that provide

137 large quantities of potential non-pest insect hosts in which infective juveniles can recycle

(ii) retention of these potential non-pest insect hosts in crop patches for the duration of the crop cycle. Retaining D. insulare in the cropping area will require diamondback moth populations and floral resources. Potential strategies to enhance populations of entomopathogenic nematodes and D. insulare include:

Frequent irrigation. Moisture limits the persistence of endemic and exotic populations of entomopathogenic nematodes. Frequent irrigation is a typical practice used in vegetable production that could be employed to regulate soil moisture, so that optimum conditions that allow nematodes to persist during the growing season are provided. Overhead irrigation also reduces oviposition by diamondback moth (McHugh and Foster 1995), possibly to low enough population densities to enhance the impact of the intercropping discussed below.

Crop manipulation. For nematodes to persist, a continuous supply of insect hosts is required. This can be achieved through the careful manipulation of crops during the growing season. Rotating vegetable crops that are attacked by pests susceptible to nematode infection could increase the persistence of populations by providing a sequence of potential hosts for infective juveniles to recycle. For example, rotations of green onions with radishes or turnips could potentially provide onion maggots, cabbage maggots, flea beetles, and cutworms, all of which are susceptible to nematode infections.

Intercropping may be another crop manipulation strategy that could be used for both the nematode and D. insulare. Many vegetable growers use cover crops to protect seedlings from wind damage and soil erosion, improve fertility, retain soil moisture, and moderate soil temperature. These cover cropping practices could be used to shelter

138 nematodes from adverse conditions as well as provide non-pest hosts for recycling. For example, if parsley seedlings were intercropped with barley, applying entomopathogenic nematodes early in the season when carrot weevil adults migrate into fields and oviposit may reduce feeding by early larval instars of the weevil. The barley, could potentially improve the microclimatic conditions for nematodes to persist by moderating soil temperature and moisture. In addition, the barley may provide potential hosts for nematodes to recycle. Thrips, for example, reproduce on barley and are susceptible to entomopathogenic nematode infection, as such, these insects could be a source of hosts in which nematodes can recycle and be present when the second generation of carrot weevils emerge later in the season. Including flowering coriander along crop borders as well as allowing small sections of coriander plantings to flower could be a feasible option to increase the persistence of Diadegma spp. and other parasitoid species of lepidopteran pests in the cropping area. Having a high density of flowering patches (patches every

50m) should increase the likelihood of encounters and retain parasitoids in the crop area.

This is especially important for retaining D. insulare in crop patches when host populations are low.

FUTURE RESEARCH DIRECTION

Entomopathogenic nematodes

The research conducted contributed information towards filling gaps in knowledge on nematode-host interactions; however, further research is warranted to advance the development of conservation biological control strategies for entomopathogenic nematodes. Three critical areas for research are identified; firstly, an

139 important abiotic factor limiting nematode distribution and persistence was identified from surveys conducted in the Ohio vegetable production area, but no conclusive information was obtained regarding the biotic factors. The types and densities of hosts regulating these endemic populations need to be determined. Secondly, nematodes have very limited dispersal capabilities, but they are distributed over large areas.

Entomopathogenic nematodes were consistently recovered along grassy ditch banks in the vegetable landscape. The relative roles of water and arthropod communities in aiding the dispersal of nematode populations and the impact of arthropod dispersal on the genetic structure of endemic nematode populations need to be investigated. Finally, even under favorable climatic conditions and an adequate supply of hosts, nematode persistence is sometimes low. This low persistence may be, in part, related to the interactions of infective juveniles with other soil micro-organisms. Presently, our understanding of the role of entomopathogenic nematodes in soil food webs is deficient.

Diadegma insulare

The current studies provided information on potential interactions of diamondback moth population densities and floral resources of coriander on the persistence of D. insulare populations. However, further information is required to determine the underlying mechanism for the interaction. Possible mechanisms to be investigated include differences in retention times of wasps in cropping areas with flowers compared with areas with no flowers, and the impact of floral resources on the functional response of wasps and the resulting levels of parasitism. Based on the dispersal range of D. insulare and other parasitic wasps and the patchy distribution of host and

140 floral resources in vegetable landscapes, studies need to determine the role of non-crop habitats surrounding cultivated areas in providing parasitoids with these resources and others such as refuges and overwintering shelters. This information will be important for manipulating landscapes to conserve natural enemies in vegetable production areas.

CONCLUSION

Biological control is difficult in annual cropping systems. With the increasing emphasis on food safety and emerging environmental policies, however, conservation biological control has to be the framework upon which integrated pest management programs are developed. This goal may be realized only if there is a change in perspective. Smith et al. (1997) challenge researchers to design and implement biological control programs that fit into the spatial and temporal constraints of annual cropping systems rather than fit the system into a rigid set of existing guidelines. Based on the results of studies presented, increasing the persistence of natural enemies in vegetable- cropping habitats could result from manipulating the patterns of crop patches within these landscapes, so that, resources required by these beneficial organisms are provided at the relevant spatial and temporal scales.

The temporal and spatial character of agricultural landscapes is based on biological and social factors. Developing conservation biological control strategies therefore requires multidisciplinary collaboration as well as strong alliances with stakeholders from the agricultural industry, including growers, economists and policy makers. A holistic approach should assist in reducing these spatial and temporal

141 constraints and improve the chances of developing more effective, practical, and economical conservation biological practices from which to build IPM programs for vegetables.

142

EPN (#m-2)

- -

Environmental - + 0 - Factors + 0 - Agronomic Practices

+ 0 - Herbivore - (#m-2)

+ 0 - Landscape Crop Patch

-

Figure 7.1. Current research status of bottom-up regulation of entomopathogenic nematodes by lower trophic groups and the subsequent top-down regulation of soil pests. Line thickness is representative of the amount of research conducted in the area; symbols are indicative of the results of the studies: + - increase,- - decrease, 0 – no change. Red lines indicate areas where research was conducted.

143

+ - Diadegma insulare (#m-2) -0 -0

Environmental - + 0 - + 0 - Factors

+ - Agronomic Practices

+ + - - Diamondback moth (#m-2)

+ - Landscape Crop Patch

+ - - -

Figure 7.2. Current research status of bottom-up regulation of the parasitoid, Diadegma insulare Cresson (Hymenoptera: Ichneumonidae), by lower trophic groups and the subsequent top-down regulation of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae). Line thickness is representative of the amount of research conducted in the area; symbols are indicative of the results of the studies: + - increase,- - decrease, 0 – no change. Red lines indicate areas where research was conducted

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