Intraguild Predation among Aphidophagous Coccinellids and Lacewings

Transcription

1 Faculteit Bio- ingenieurswetenschappen Academiejaar Intraguild Predation among Aphidophagous Coccinellids and Lacewings Christophe Noppe Promotor: Prof. dr. ir. Patrick De Clercq, Prof. dr. J.P. Michaud Tutor: ir. Brecht Ingels Masterproef voorgedragen tot het behalen van de graad van Master in de bio- ingenieurswetenschappen: Milieutechnologie

2 21 januari 2011 De auteurs en de promotoren geven de toelating deze scriptie voor consultatie beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting de bron te vermelden bij het aanhalen van resultaten uit deze scriptie. De promotoren De auteur Prof. dr. ir. Patrick De Clercq Christophe Noppe Prof. dr. J.P. Michaud

3 Preface This thesis was made possible in part by the financial support of the EU- US Atlantis Student Exchange Program that promotes the exchange of scientific and cultural experiences among students. I extend my sincere gratitude to all of those involved with this organization for providing the opportunity for such a valuable experience to myself and all others participating in the exchange program. A very special thanks goes to my two mentors, Prof. De Clercq and Dr. Michaud, for providing advice, support, and dedicating extensive time and effort to the development of this thesis. Having the opportunity to conduct research at the Kansas State University Agricultural Research Station in Hays, Kansas was a truly unique and life- changing experience. I am grateful to the many people who contributed to my positive experience in Hays. Christophe Noppe Gent, 2011

4 Table of Contents Preface Table of Contents List of abbreviations... i Summary...ii Samenvatting... iii I. Introduction...1 II. Literature Study...2 II.1. Intraguild Predation Theory...2 II.1.1. Defining Intraguild Predation... 2 II.1.2. Intraguild Predation vs. Predation vs. Competition... 2 II.1.3. General Theoretical Considerations... 3 II.1.4. Theoretical Considerations on the Level of the Individual... 4 II.1.5. Theoretical Considerations on the Population and Community Levels... 4 II.2. Intraguild predation and Biological Control...8 II.2.1. Introduction... 8 ìi.2.2. Intraguild Predation Theory and Biological Control... 8 II.2.3. Intraguild Predation within the Aphidophagous Guild II.2.4. Intraguild Predation between Coccinellids and Lacewings II.3. The Studied Intraguild Predators and Extraguild Prey II.3.1. The Coccinellid Coleomegilla maculata II.3.2. The Chrysopid Chyrsoperla carnea II.3.3. The Aphid Schizaphis graminum III. Materials and Methods III.1. Breeding of the Intraguild Predators and Extraguild prey III.1.1. Coleomegilla maculata III.1.2. Chrysoperla carnea III.1.3. Schizaphis graminum... 29

5 III.2. Design of a microcosm III.2.1. General Considerations III.2.2. The Conetainer Microcosm III.2.3. Preliminary Testing of the Conetainer III.3. Experiment 1: Intraguild Predation in the Microcosm III.4. Experiment 2: Intraguild Predation in Petri Dishes III.5. Data analysis IV. Results IV.1. Temperature IV.2. Pooling of Data IV.3. Plant Survival in Conetainers IV.4. Biological Control of Aphids in Conetainers IV.5. Starvation and Larval Survival IV.6. IGP Outcome IV.7. Cannibalism IV.8. Development V. Discussion VI. Conclusion Reference List... 52

6 List of abbreviations A. fabae Aphis fabae A. glycines Aphis glycines A. pisum Acyrthosiphon pisum C. carnea Chrysoperla carnea C. coeruleus Curinus coeruleus C. maculata Coleomegilla maculata C. plorabunda Chrysoperla plorabunda C. semptempunctata Coccinella semptempunctata C. rufilabris Chrysoperla rufilabris H. axyridis Harmonia axyridis IGP Intraguild predation M. euphorbiae Macrosiphum euphorbiae NPK Nitrogen, phosphorus and potassium ODK Oviposition- deterring kairomones O. nubilali Ostrina nubilalis O. v-nigrum Olla v-nigrum S. graminum Schizaphis graminum i

7 Summary This thesis concerns the deeper understanding of intraguild predation among Chrysopidae and Coccinellidae. After defining the term intraguild predation, an overview of some general theoretical considerations is provided. These considerations will be more specified in the direction of biological control, biological control among aphidophagous predators and intraguild predation among Coccinellidae and Chrysopidae. Prior to the section in which a relevant experimental study concerning the topic of this thesis is presented, an overview of studies found in the existing literature will be discussed. This overview makes evident that no conclusion can be made about whether or not intraguild predation between Coccinellidae and Chrysopidae has a negative effect on biological control of pestiferous species in crops. With this in mind, two experiments were carried out to answer the questions concerning which of these two predators will dominate during intraguild interactions, whether or not the experimental arena has influences on the outcome of intraguild predation and whether or not intraguild predation influences the biological control of the pest species. From the results of the first experiment in which intraguild predation among these two predators during their larval stages was studied inside a microcosm, the conclusion could be drawn that no significant negative effect of intraguild predation on biological control had taken place. In addition to this conclusion, the chrysopid Chrysoperla carnea dominated intraguild predation confrontations with Coleomegilla maculata in less cases than was expected. To verify this result, a second similar experiment was carried out in Petri dishes. Within these dishes the larvae of C. carnea demonstrated a strong domination over C. maculata larvae. Combining the results of two experiments suggested that a more complex arena did have an influence on the outcome of intraguild predation. ii

8 Samenvatting In dit werkstuk wordt dieper ingegaan op de intraguild predatie tussen Coccinellidae en Chrysopidae. Na te verduidelijken wat het begrip intraguild predatie inhoudt, worden enkele algemene theoretische beschouwingen hieromtrent overlopen. Voorts worden deze beschouwingen verder verdiept in de richting van de biologische bestrijding, zijn aphidofage tak en de intraguild predatie tussen Coccinellidae and Chrysopidae. Alvorens over te gaan naar het gedeelte waarin de experimentele ondervindingen in het kader van deze masterproef worden voorgesteld, wordt een overzicht gegeven van relevant onderzoek uit de literatuur betreffende het onderwerp van deze masterscriptie. Hieruit blijkt dat er geen éénduidig besluit kan worden genomen of interaguild predatie tussen Coccinellidae en Chrysopidae al dan niet negatieve gevolgen heeft voor het onder controle houden van pestssoorten in gewassen. Met dit in gedachte, werden twee experimenten opgesteld die enige verduidelijking zouden kunnen brengen omtrent de vragen wie van deze twee predatoren de andere het meest domineert, of de experimentele arena de uitkomst van intraguild predatie beïnvloedt en of intraguild predatie het controleren van de pestssoort al dan niet negatief beïnvloedt. Uit de eerste proefopstelling, waarin intraguild predatie tussen deze twee predatoren in larvaal stadium werd onderzocht in een microcosmos, bleek dat er geen significante negatieve effecten van intraguild predatatie op de biologische controle kon worden afgeleid. Bovendien bleken de Chrysopidae Chrysoperla carnea de Coccinellidae Coleomegilla maculata minder te domineren dan werd verwacht. Om dit te verifiëren werd een analoge proefopstelling in petrischalen uitgevoerd. Hieruit bleken de larven van C. carnea overduidelijk te domineren. Deze verschillen met de eerste proefopstelling tonen aan dat een experimentele arena die beter aanleunt bij de omstandigheden van natuurlijke ecosystemen een invloed heeft op de uitkomst van intraguild predatie. iii

9 I. Introduction Various arthropod predators play an important role in controlling herbivorous pest species in agroecosystems and many of them coexist within these systems. Coccinellids and lacewings are two arthropods that demonstrate their importance in this area. Due to the complexity of these agroecosystems, it is not always clear whether or not these predatory arthropods interfere with each other. Possible interference may or may not have a negative effect on the efficiency of controlling the pest species. Whenever this interference takes the form of one of these predatory arhropods preying on the other, this is called intraguild predation. In the case that these two arthropod predators are aphidophagous lacewings and coccinellids, this is called Intraguild predation among aphidophagous lacewings and coccinellids. The scope of this thesis is to investigate the degree of larval intraguild predation among the lacewings and coccinellids and the impact it has on aphid pest suppression. The design of the study was selected to replicate the natural situation more faithfully than has been done in previous studies. Specifically, the predatory insects Coleomegilla maculata and Chrysoperla carnea would be followed for the entire period of their larval development while feeding on growing Schizaphis graminum colonies on Sorghum bicolor in a microcosm ( conetainer ). The following questions were addressed: Which of the two species is the dominant intraguild predator? Does the occurrence of intraguild predation affect the suppression of extraguild prey and the survival of the host plants? Is the outcome of intraguild interactions affected by the presence or absence of a plant in the arena? 1

10 II. Literature Study II.1. Intraguild Predation Theory II.1.1. Defining Intraguild Predation Intraguild predation, hereafter referred to as IGP, was defined by Polis et al. in 1989 as predation among members of the same guild. This occurrence, prior to the publication of their work was formerly known as predation, predatory interference (Case & Gilpin, 1974) or aggression (Mabelis, 1984). The term guild in the definition of IGP and defined by Root (1967) as a group of species that exploit the same class of environmental resources in a similar manner, was later extended by Polis et al. (1989) as all taxa in a community that use similar resources, regardless of their nutrition mode, ecology or taxonomic position. Herein, the species that preys upon another member of the same guild is referred to as the intraguild predator, while the other is called the intraguild prey, and the species that is the common resource is the extraguild prey. II.1.2. Intraguild Predation vs. Predation vs. Competition Since IGP involves a combination of competition and predation it has a far more complex impact on population dynamics than competition and predation each have when considered as isolated factors. IGP not only differs from competition in respect to the direct energetic gain obtained by the predator, but also differs from simple predation due to the reduction of potential scramble competition for the extraguild prey. Whether acts of IGP arise from competitive or predatory interactions is case- specific and sometimes ambiguous, but in most cases IGP is generated from predation, driven by nutritional requirements. Consuming intraguild prey does not come without costs. These costs include the risk of 2

11 being preyed upon by intraguild prey, the risk of being injured, the risk of being contaminated by generalist pathogens and the risk of being poisoned by toxic compounds present in the intraguild prey (Polis et al., 1989; Dixon, 2000; Lucas, 2005). When taking costs into account, benefits must also be considered, which are the elimination of a potential predator, elimination of a competitor, and the acquisition of a protein- rich meal (Polis et al., 1989; Lucas, 2005). Whenever these costs exceed the benefits, IGP is a result of interference competition. II.1.3. General Theoretical Considerations According to Polis et al. (1989), IGP can be categorized according to two important characteristics; symmetry and age structure. Age structure can be categorized further in terms of ontogenetic changes in size or vulnerability. In addition to these criteria, IGP can also be categorized as either symmetric or asymmetric. Asymmetric IGP occurs when one species serves as the intraguild prey in all interactions. Symmetric IGP, frequently referred to as mutual IGP or reciprocal IGP (Janssen et al., 2006), happens when both species prey on each other. This does not imply equal strength or similar chance of winning in IGP confrontations. One species may prey on younger stages of the other, while this could be reverse at another point in time. The most important factors determining the frequency and direction of IGP are body size, degree of specialization and the abundance of extraguild prey; generalist predators preying on smaller intraguild prey are commonly observed. IGP frequency may either increase or decrease with changes in the abundance of extraguild prey. 3

12 II.1.4. Theoretical Considerations on the Level of the Individual Prior to focusing on how IGP may influence population dynamics it is important to consider the main effects of IGP at the level of the individual. IGP, like classical predation, leads to adaptations that are reflected in the morphology, behavior and life cycle of the species involved. These include adaptations of intraguild prey to reduce the risk of IGP, as well as adaptations of intraguild predators to increase success during intraguild encounters. In addition to nutritional and energetic gains, which not only increase the growth rate but also heighten the chance of reproduction and survival of individuals engaging in all cases of predation, individuals succeeding in IGP may benefit by diminishing predation on current and future generations and by experiencing reduced scramble competition for resources. II.1.5. Theoretical Considerations on the Population and Community Levels When considered from the perspectives of population or community level, IGP may alter population dynamics like size and age composition of all species directly or indirectly involved in IGP. For example, IGP may indirectly result in increases or decreases in abundance of the extraguild prey, depending on whether the intraguild predator is a more or less specific/effective predator of this shared resource than the intraguild prey. Another possible consequence is temporal or spatial niche differentiation among guild members, especially those more likely to be intraguild prey, to reduce the costs of interference competition. Most theoretical models of population dynamics (Polis and Holt, 1992; Holt and Polis, 1997; Morin, 1999; Diehl and Feissel, 2000, 2001; Mylius et al., 2001; Kuijper et al., 2003) examine interactions between three species: an intraguild predator, an intraguild prey and an extraguild prey. These models explore the relationship between population density and environmental productivity, which is defined by the population size of a species that the environment can sustain 4

13 indefinitely. This IGP model is divided into three levels of productivity: low (A), intermediate (B,C&D), and high (E) (see Figure 1). Figure 1: Carrying capacity or environmental productivity in function of equilibruim densities of resource, IGP prey and IGP predator (Janssen et al, 2006) Janssen et al. (2006) describe these population equilibria as follows. At low poductivity levels resources are scarce and neither the intraguild predator nor the intraguild prey can prevail. At increased productivity levels, the species that is the most efficient in scramble competition for the extraguild prey can sustain itself with this prey exclusively. If the strongest competitor for the extraguild prey is the intraguild predator, the intraguild prey will be outcompeted, preyed upon and ultimately eliminated from the system. At higher productivity levels, the coexistence of both intraguild predators and intraguild prey is possible, but only when the intraguild prey is the superior competitor for the common 5

14 resource (Polis and Holt, 1992; Holt and Polis, 1997). Comparing the costs and benefits regarding the consumption of extraguild and intraguild prey can reveal whether or not the predator is the dominating interguild predator. It is often assumed that coexistence among intraguild members is only possible when the intraguild predator experiences higher costs and lower efficiency of utilization in the exploitation of the extraguild prey (Rosenzweig, 1966; Levins, 1979; Polis et al., 1989; Polis and Holt, 1992; Rosenheim et al., 1995). If so, the implication is that when the superior intraguild predator is removed from the system, the density of the common extraguild prey will decline. When the intraguild predator is inferior in resource exploitation and productivity further increases, it brings about either coexistence of the three species or a state in which one of the intraguild prey is excluded and only the intraguild predator and the extraguild prey persist. The intraguild predator eliminates the intraguild prey by a combination of IGP and interference competition (Janssenet al., 2006). Outcomes are very sensitive to initial conditions. Introducing an intraguild predator when the intraguild prey and extraguild prey are in equilibrium results in coexistence of all three species. However, when the population of intraguild prey falls below some threshold size in a system where the intraguild predator population is in equilibrium with the extraguild prey population, all the intraguild prey will be eliminated largely due to IGP on vulnerable young stages, aka the priority effect (Polis et al., 1989). At the highest levels of productivity, the intraguild prey is excluded under all initial conditions. The basic population model of IGP can be made more complex by inclusion of additional factors that may lessen the impact of IGP within food webs. These factors, first discussed in this context by Holt and Polis (1997), were identified as age structure (with vulnerable age classes), predator switching behavior, prey defensive behavior, spatial environmental heterogeneity, and food web complexity. Possible effects of age structure include life histories in which the intraguild prey passes through a stage that is invulnerable to IGP, or the intraguild predator has a stage incapable of IGP (Mylius et al., 2001). The effects of prey switching by intraguild predators, although not considered to be of great significance, was 6

15 considered by Kirvan (2000). If it is assumed that intraguild predators change their source of nutrition in accordance with its availability each source is consumed one at a time, the result is increased persistence of the intraguild predator and this translates into higher productivity. When intraguild predators focus on consuming the most profitable prey in accordance with the optimal foraging theorum (Charnov, 1976), coexistence of intraguild prey and predator is the most likely outcome. A more influential factor in such models is the effect of antipredator behavior. Heitaus (2001) found that this behavior could result in habitat segregation of the intraguild predator and prey under high productivity conditions. The effect of a more complex food web can be theoretically approached by implementing mutual IGP into the basic model (Janssen et al., 2006). This extension of the model predicts no difference from the basic model at low productivity levels, however, at higher productivity levels both intraguild species can exclude each other depending on the priority effect. Janssen et al. (2006) concludes that since neither the basic model nor the extended versions result in increased persistence in food webs with IGP, the onmipresence of IGP in natural food webs can only be explained if it is generally a relatively weak interaction. Thus, theoretical models on the whole tend to imply that strong IGP should rarely occur in nature since, in the long term, most stable equilibria that can arise result in the exclusion of the intraguild prey. However, these models are largely of heuristic value only and are necessarily oversimplifications of natural systems. 7

16 II.2. Intraguild predation and Biological Control II.2.1. Introduction The prevalence of IGP in terrestrial arthropod communities requires consideration of its consequences wherever pest suppression by natural enemies is of importance to agricultural enterprise. Such communities are generally characterized by a small range in size variation among interacting species, which in accordance with the basic theory of IGP, increases the likelihood and frequency of IGP events. Furthermore, most predators in this community are often generalists. Consequently these predators may indirectly have an influence on herbivore population dynamics through IGP on other herbivore suppressors. It has long been a challenge in biological control research to determine whether or not this indirect effect of IGP has positive or negative effects on the suppression of pestiferous herbivores for anthropocentric purposes. ìi.2.2. Intraguild Predation Theory and Biological Control An important disparity between basic IGP theory and theories of biological control in natural systems is that the former considers only three species whereas the latter require consideration at least one more, the host plant. Janssen et al. (2006) begins with the assumption that the host plant or crop has no net effect on the dynamics of the intraguild system. Two cases are distinguished. If the intraguild predator is the superior competitor for the extraguild prey then the intraguild prey is always excluded and the implication is that the intraguild predator is the better biological control agent of the extraguild prey (pest species) so that introduction of the intraguild prey is irrelevant. If, on the contrary, the intraguild predator is the inferior competitor for the shared resource, then the intraguild prey and intraguild predator can only coexist at intermediate levels of productivity (see Figure 2). Thus, when the 8

17 Figure 2: Productivity or carrying capacity of the ecosystem in function of equilibrium prey density (Janssen et al., 2006) intraguild prey is the more efficient predator of the common resource, the intraguild prey alone will result in a higher level of biological pest control than when both predators are simultaneously present. According to this model, IGP has a negative effect on biological control of the pest species at these levels of productivity. At higher levels, similar to the basic theory of IGP, the predator again eliminates the intraguild prey and pest density rises to levels higher than in systems without the intraguild predator. Although agroecosystems are in most cases more simplified than natural ecosystems, the population dynamics of protagonistic species observed in the field often differs in outcome from that predicted by IGP theory, suggesting that current theoretical models are still insufficient to adequately describe the complexity of these systems. Janssen et al. (2006) points to variation in temporal and spatial scales, and food web complexity as possible causes of these discrepancies. For example, the production cycle of many crops may be too short for insect communities to reach any equilibrium state. Thus, transient dynamics, 9

18 rather than final equilibrium states, may be of more importance to biological control outcomes. In spatial terms, pest populations are rarely evenly distributed, are usually clumped, and may therefore consist of several effective subpopulations, each approaching different equilibrium states. In patches where the intraguild predator is present and will suppress the pest population, these pest population densities will be lower than in clusters where no intraguild predators are present. A last reason for the possible cause in the discrepancy between the theoretical models and field conditions is that food webs are far more complex than what is assumed by these models. Most models are based on the interaction of only three species. The presence of more species in biological control systems may result in interactions other than IGP. These additional interactions could make the effects of IGP more difficult to discern. Most models fail to account for antipredator behavior like concealment, avoidance or escape and these behaviors by both extraguild prey and intraguild prey may mitigate the overall importance of IGP to community dynamics. Furthermore, antipredator behavior is typically favored by spatial complexity and the majority of empirical tests of IGP theory typically provide only limited spatial complexity, thus likely magnifying the apparent importance of IGP. II.2.3. Intraguild Predation within the Aphidophagous Guild Guilds of aphidophagous arthropods, which comprise all species preying on or parasitizing aphids, have been the subject of many studies addressing IGP. According to Lucas (2005) this guild forms an ideal group for intraguild interactions for four reasons. The guild is extremely diverse with numerous predators, parasitoids and pathogens (Minks & Harrewijn, 1988). In addition, most aphidophagous species undergo dramatic changes in body size during their development, gradually transforming from very vulnerable insects into high level predators. Since the common resource, aphids, tend to be very patchy in distribution (Shaposhnikov, 1988), their predators tend to follow the same 10

19 distribution, thereby increasing the probability of intraguild interactions. Aphid colonies are also highly ephemeral in occurrence. They begin very small, experience rapid exponential growth, and then crash precipitously once alate forms are produced (Dixon, 1985, 1987). The short period in which these colonies develop (typically 2-3 weeks) creates a narrow time window for the successful development of aphidophagous predators and increases the frequency and intensity of intraguild interactions. Accordingly, IGP among aphidophagous species in the field is frequently observed (Rosenheim et al., 1999) and is a primary source of mortality during the development of most aphidophagaous species (Lucas, 2005). IGP among aphidophagous species can result in three different possible outcomes for aphid biological control (Lucas, 2005); antagonism (interference competition among natural enemies impedes the more effective aphid predators resulting in lower levels of aphid control than could be achieved with these individual species), additivity (IGP has no net negative effect on predators and aphid control improves in direct proportion to the number of predators present) or synergism (IGP favors the more effective aphid predators and thus aphid numbers are reduced more rapidly as a consequence of IGP than in its absence). Thus, both disruptive and regulatory scenarios are possible. Disruptive scenarios result when intrinsically superior aphid predators predominate as intraguild prey with indirect negative effects on aphid control. In regulatory scenarios, IGP generates additive or synergistic effects, either of which may result in improved biological control of aphids. According to Lucas (2005), these effects may arise from either a lack of IGP, or by the intraguild predator regulating the population of the intraguild prey and also the aphid population. II.2.4. Intraguild Predation between Coccinellids and Lacewings Coccinellids are widely recognized as important predators of aphid pests in cereal agroecosystems (Hodek and Honek, 1996; Kannan, 1999). In cereal fields, these aphidophagous beetles frequently co- occur with various lacewing species 11

20 (Chrysopidae), another group important aphidophagous group (Hasken and Pheoling, 1995; Kannan, 1999; Dinter, 2002; Frazer, 1988; Boiteau, 1983). The potential for IGP between these two groups of species is high, not only because of their temporal and spatial co- occurence, but also because of their similar feeding habits. (Coderre & Tourneur, 1986; Coderre et al., 1987; Phoofolo & Obrycki, 1998). Given the economic importance of these species and their abundance in cereal fields, IGP among these two groups of generalist predators has often been the subject of empirical investigation. Interactions among lacewings and coccinellids were first investigated in the 1960 s (Bänch, 1964; Ickert, 1968). Subsequent studies on the subject (Sengonca & Frings, 1985; Polis, 1989) appear to have stimulated an increasing number of laboratory investigations examining contests between these insect families. An overview of the available studies is represented in Table 1. This table summarizes information on various aspects of the studies such as the predators used, the duration of the experiments, the spatial scale, the variety of instars placed in combat, and the availability of prey or alternative food sources. The number of replicates in most studies is surprisingly small. The average number of all studies combined is 16 replicates per treatment, with only one study using a number higher than 20 (n=50; Michaud & Grant, 2003). The median is similarily low (n=15). Similarly, the temporal window of predator confrontation tends to be short (30 min to 3 days) in all but the aforementioned study. 12

21 Table 1: Overview of studies on IGP among lacewings and coccinellids Coccinelidae Coccinella septempunctata Coccinella septempunctata Coleomegilla maculata Coleomegilla maculata Coleomegilla maculata Chrysopidae Chrysoperla carnea Chrysoperla plorabunda Chrysoperla rufilabris Chrysoperla rufilabris Chrysoperla rufilabris No. Replicates (per treatment) Temporal scale (maximum) Time until next molt 4 days 30 minutes 24 hours 24 hours Spacial scale Petri dish 9x3 cm Broadbean leaflet in Petri dish One bean plant One potato plant with one leaf One potato plant with one leaf One potato leaf Stages of predators Eggs L1 L2 L3 L4 Pupae L1 L4 L1 L2 L3 L4 Eggs L1 L2 L3 L4 Pupae Eggs L1 L4 Adult Food source Acyrthosiphon pisum No food source Aphis fabae No food source Macrosiphum euphorbiae No food source Reference Sengonca & Frings, 1985 Chang, 1996 Lucas et al., 1997 Lucas et al., 1998 Lucas,

22 Continuation of Table 1 Coccinelidae Coccinella semptempunctata Harmonia axyridis Coleomegilla Coleomegilla maculata Harmonia axyridis Curinus coeruleus Harmonia axyridis Harmonia axyridis Chrysopidae Chrysoperla carnea Chrysoperla rufilabris Chrysoperla rufilabris Chrysoperla carnea Mallada desjardinsi No. Replicates (per treatment) Temporal scale (maximum) 3 days 30 minutes Until predator pupated 3 hours 1 hour Spacial scale Glas vial 3x10 cm One potato plant with one leaf Petri dish 5.5x1.0 cm 4L cylinder with 3 soy bean plants Petri dish 5.5x1.0 cm Stages of predators L3 L4 L1 L2 L3 L4 L1 L2 L3 L2 Adult L2 L3 L4 Food source Acyrthosiphon pisum Ostrina nubilalis No food source Ephestia eggs No food source Aphis. glycines No food source Reference Phoofolo & Obrycki, 1998 Lucas et al., 2000 Michaud & Grant, 2003 Gardiner & Landis, 2007 Nakahira & Arakawa,

23 Since all of the experiments were conducted under laboratory conditions, the physical conditions and spatial scale of experimental arenas differ dramatically from conditions under which IGP occurs in the field. The only semi- natural design was that conducted by Gardiner & Landis (2007) in which interactions were studied for a 30 minute periods in replicates that each contained three plants. In 50 of studies, predators were placed together in Petri dishes or vials containing either no plant material or just one leaf. Experiments in the remaining studies were conducted with two predators facing each other in microcosms that contained only one plant. Since all of the experiments were conducted under laboratory conditions, the physical conditions and spatial scale of experimental arenas differ dramatically from conditions under which IGP occurs in the field. The only semi- natural design was that conducted by Gardiner & Landis (2007) in which interactions were studied for a 30 minute periods in replicates that each contained three plants. In 50 of studies, predators were placed together in Petri dishes or vials containing either no plant material or just one leaf. Experiments in the remaining studies were conducted with two predators facing each other in microcosms that contained only one plant. In the majority of experiments, predatory interactions between coccinellid and lacewing species were induced among different life stages. In three out of 10 publications, predation on eggs by either adults or larvae was tested. In 8 out of 10 of the experiments, IGP between larval stages was induced. In three experiments, predation of of chrysopid larvae by coccinellid adults was tested and only one study examined IGP on pupae. The importance of body size as the determining factor regarding the direction of IGP among lacewings and coccinellids can be derived from the following reports. Sengonca & Frings (1985) concluded that regardless of the presence of aphid prey, Chrysoperla carnea larvae were superior to Coccinella septempunctata larvae of similar vigor. Placing two larvae of different sizes together always resulted in the smaller larva being killed by the bigger larva. Both predators preyed on the eggs of the opposing species. Adults of C. septempunctata attacked only 1st and 2nd instar lacewings. 15

24 Lucas et al. (1997) described the instar- specific defenses of Coleomegilla maculata larvae against the third instar Chrysoperla rufilabris larvae in the absence of prey. The latter species has very similar life history to C. carnea in all respects when used in experiments with a higher humidity range (Tauber & Tauber, 1983). As in accordance to Sengonca & Frings (1985), Lucas et al. (1997) found that mortality of smaller instars was significantly higher than that of third and fourth instar larvae. Lucas et al (1998) tested predation outcomes of various stages (eggs, larvae, pupae and adults) of the same species and concluded that IGP among these species occurred at very high levels. Once again, predator size determined outcomes with the exception of third instar lacewings which were successful against fourth instar C. maculata larvae. This consistency indicates the continued advantage of the C. carnea larvae. During another experiment, Lucas (1998) tested predation among all larval instars and adult C. maculata on the eggs of the C. rufilabris and obtained results in line with those of Sengonca & Frings (1985). Phoofolo & Obrycki (1998) conducted a similar study on the interactions between C. maculata and C. rufilabris. The demise of a lacewing larva as a result of confrontation was only observed in one replicate in which a third instar C. carnea was killed by a fourth instar C. maculata. In all other replicates C. carnea dominated. In a further study, Lucas et al. (2000) reported on interactions between third- instar C. rufilabris and all larval instars of C. maculata where no contests exceeded a 30 min period. Once again, the mortality of coccinellid larvae was significantly higher for first instars compared to later ones and no mortality of chrysopid larvae was observed. During Michaud & Grant s (2003) experiments, C. rufilabris larvae were paired with larvae of three different lady beetle species: Harmonia axyridis, Olla v- nigrum and Curinus coeruleus. With the exception of encounters with H. axyridis, third instar C. rufilabris larvae dominated all contests with second instar coccinellids. When third instar lacewings faced third instar coccinellids, outcomes were more equal, with H. axyridis being the exception as being the dominant predator. Nakahira & Arakawa studied the interaction between larvae of H. axyridis and larvae of the trash- carrying green lacewing, Mallada desjardinsi. The only reported fatalities for second and third instar lacewing 16

25 larvae occurred when they faced fourth instar H. axyridis larvae. Based on these experiments it can be concluded that larger larvae generally dominate smaller ones, whereas chrysopids have the advantage when larvae of equal size are paired. Four studies have investigated whether the presence of prey affected the rate of IGP between these two species. Sengonca & Frings (1985) found that the number deaths decreased dramatically when extraguild prey were available; 28 out of 144 fatalities in comparison 78 out of 144 when aphids were not present. Lucas et al. (1998) studied the effect of increasing aphid density on IGP and concluded that confrontations between lacewings and coccinellids decreased over a 24 hour period in the presence of extraguild prey. However, in some larval combinations a very high density of aphids was required to lower the occurrence of IGP. Phoofolo & Obrycki (1998) studied the effect of Ostrinia nubilalis eggs and Acyrthosiphon pisum on IGP among C. carnea and C. maculata larvae over the course of three days. Most IGP occurred during the first 24 hours in the absence of food. When a large number of aphids (10-20) were present, the rate of IGP was reduced and the intraguild prey were not entirely consumed until 48 hours. With the availablity of five O. nubilalis egg masses per day, intraguild interactions resulted in only 80% mortality of C. maculata during the 72 hour period. When no food was available, the mortality of C. maculata larvae was 100%. When Michaud & Grant (2003) paired larvae of C. rufilabris with larvae of H. axyridis, O. v-nigrum and C. coeruleus with or without food being present in the Petri dishes where the encounters took place. When similar- sized larvae were introduced into the same Petri dish, 122 fatalities among 300 larvae were recorded in the presence of food (eggs of Ephestia kuehniella) whereas 149 fatalities were observed in its absence. In treatments where coccinellid larvae were larger than lacewing larvae, 142 of 150 encounters in the absence of food resulted in the killing of one predator while 117 of 300 larvae were killed in the presence of Ephestia eggs. The above four experiments show that whenever any food source is available, the rate at which IGP occurs among lacewings and coccinellids follows the basic rules of IGP, being the reduction in IGP frequency. 17

26 Since both predators prey on each other, IGP among lacewings and coccinellids can be characterized as mutual or reciprocal. Lucas et al. (1998) proposed an index of symmetry computed as the proportion of replicates in which a given predator was eaten divided by the total number of replicates in which there was occurrence of IGP. The combined outcomes of all of the above- mentioned reports involving C. maculata versus lacewings can be used to calculate global symmetry indices of IGP among green lacewings and this coccinellid (Figure 3). Whereas it is clear that older larvae can dominate younger larvae of the opposing species, when same- age larvae are paired, lacewings prove superior in intraguild encounters, with third instar lacewings killing even most fourth instar C. maculata. Because of age- dependent outcomes, and because both species prey on the other s eggs, population age structure is considered of critical importance to IGP outcomes between green lacewings and coccinellids. Figure 3: Index of IGP symmetry. The IGP symmetry index is the rate of the number of cases in which the species was preyed upon on the total number of IGP contests. (A = adult, P = pupa, L1 = first instar, L2 = second instar, L3 = third instar and L4 is fourth instar 18

27 After pupation, most green lacewings are no longer predatory, but convert to herbivorous diets. The possible IGP interactions are depicted in Figure 4 Figure 4: IGP directions among lacewings and coccinellids. The direction of the arrow indicates that the species is eaten by the species to which it points. Evolution of aphidophagous species under the selective influence of IGP has led to morphological, behavioral and life history adaptations that diminish the risk of being an IGP victim without reducing predation success on primary food sources. Various defensive mechanisms have been described by Engler (1986, 1991) and later modified by Lucas (2005) and these strategies will be discussed as they pertain to lacewings and coccinellids. Encounter rates A first step toward reducing the risk of becoming intraguild prey is to avoid contact with potential intraguild predators and both coccinellids and lacewings have developed avoidance mechanisms. Given that the risk of predation for sessile stages such as eggs and pupae is high, adult females of aphidophagous species are able to detect larval tracks (a.k.a ODK, or oviposition- deterring kairomones) of their own species as well as those of related and unrelated 19

28 aphidophagous competitors. The ability to detect these larval tracks allows them to avoid choosing oviposition locations where the risk of IGP is high (Ružička, 1997; Doumbia et al., 1998; Ružička & Havelka, 1998; Ružička & Zemek, 2003). Prepupal larvae of both coccinellids (Lucas et al., 2000) and chrysopids (personal observations) typically abandon feeding sites within aphid colonies to seek a safer locations for pupation where the risk of IGP is lower. Crypsis Lacewings seem to have developed mechanisms to avoid detection by potential predators. Trash- carrying lacewing larvae like Mallada desjardinsi cover themselves with fragments of plant material and the dead bodies of their prey to lower their rate of detection by visually searching predators. Naked lacewing larvae, such as those of C. carnea, resort instead to behavioral crypsis (personal observation). On plants, these larvae hide in the folds of dead leaves that are similar in color to the larvae's body and pupating larvae may wrap themselves in silk within the same dead plant material. Escape Upon encounter with a potential intraguild predator, a potential intraguild prey may engage in evasive manoeuvers to avoid being caught. Such manoeuvers typically include backing way from the point of contact or dropping from the surface of the plant (Lucas et al. 1997; Yasunda et al., 2001; Michaud & Grant, 2003; Lucas, 2005). Combat Both coccinellids and lacewings have an array of defensive behaviors they may employ when combat cannot be avoided. The defensive behaviors of larval C. maculata when under attack from C. carnea include wriggling and biting (Lucas, 1997). When responding with a counter- attack, larval coccinellids typically grip the subject with their first pair of legs and bite it with their crushing mouth parts. On the other hand, chrysopid larvae can take advantage of their elongated mandibles to pierce and subdue an opponent while remaining out of reach of a counterattack. They may also make use of their flexible body to twist themselves 20

29 beyond the reach of a coccinellid larva (personal observation). While coccinellids, like Curinus coeruleus, rely on their long spines to make attacking by predators more difficult (Michaud, 2003). C. maculata pupae are known for their flipping behavior when disturbed. This flipping behavior closes sclerotized, toothed edges along the junctions of their movable abdominal segments to act as a gin trap capable of snipping off the antennae of investigating insects (Eisner and Eisner, 1992; Hinton, 1955; Lucas et al., 2000). Both lacewings and lady beetles use chemical defenses. Coccinellid larvae (Hindayana et al, 2001; Lucas, 2005), pupae (Edmunds, 1974; Bowers, 1992, Attygalle et al., 1993; Lucas et al., 2000), and adults all use chemical substances containing alkaloids to deter predators. Naked lacewing larvae sometimes excrete droplets containing allomones from their anus in confrontations with predatory arthropods (Szentkiralyi, 2001). Consumption Once subjugated, intraguild prey will make a final attempt to avoid being eaten. An example realted to coccinellids is the coating of their eggs with toxic chemical compounds (Agarwala & Dixon, 1992; Hemptinne et al., 2000a, 200b; Omkar et al., 2004; Lucas, 2005) to discourage the use of their eggs as a food source for the predator. Effects of IGP between coccinellids and lacewings on the population dynamics of the extraguild prey were studied by Chang (1996) and Gardiner & Landis (2007). Chang counted aphids on fava bean plants over a four day period in six microcosms in which one first instar C. plorabuna and one first instar C. septempunctata were feeding. The combination of these predators slowed the population growth of the aphids, but there was no significant difference in final aphid population in comparison to treatments with two predators of the same species. Gardiner & Landis (2007) conducted a microcosm experiment in which he released one adult H. axyridis beetle for three hours in a microcosm containing three V1 soybean plants, three second instar C. carnea larvae as intraguild prey and 45 Aphis glycines aphids as extraguild prey to evaluate the impact of IGP events on the aphid s population. He concluded that the IGP interactions in which 1.07±0.28 out of three second instar larvae of C. carnea in 8 21

30 replicates were removed during these three hours didn t affect the biological control of the aphid population by the chrysopid larvae. Questions arise concerning whether the conclusions based on these experiments listed in Table 1 are representative of intraguild events and biological control in more complex field conditions. Chang (1996) cautioned against using highly simplified laboratory arenas to evaluate the potential for negative interactions between two biocontrol agents. Lucas et al. (1998) acknowledged that the complexity of ecosystems may influence IGP outcomes. Although Phoofolo & Obrycki (1998) inferred from their work that C. carnea and C. maculata have the potential to negatively affect one another, uncertainty remains because the experiment lacked the complexity of a natural situation. 22

31 II.3. The Studied Intraguild Predators and Extraguild Prey II.3.1. The Coccinellid Coleomegilla maculata The neartic coccinellid Coleomegilla maculata was first described in 1775 by De Geer who originally placed this species in the genus Coccinella. The species is currently taxonomically classified as follows: Order: Coleoptera Suborder: Polyphaga Superfamily: Cucujoidea Family: Coccinellidae Subfamily: Coccinellinae This lady beetle is commonly known as the '12 spot lady beetle. At least 8 subspecies are distinguished based on small variations in color pattern from which three subspecies Coleomegilla maculata lengi (Timberlake), C. maculata fuscilabris (Mulsant) and C. maculata strenua (Casey) are present in the United States (Gordon, 1985). Broader geographical distribution of these beetles ranges from Canada to Argentina on the American continent. Within the United States, the most widespread subspecies is C. maculata lengi. This subspecies exists in a substantially large quantity in the Midwestern Great Plains and can be found in agricultural crops like alfalfa, maize, potato and sorghum (Obrycki & Tauber, 1985; Kieckhefer and Elliott, 1990; Giles et al., 1994). Adults are pink to red with six black spots on each elytron, the pronotum is pink or yellowish with large triangular black spots. Its black head has a pink or red triangular marking. The body length of adults is approximately 5 to 6 mm. Larvae are dark brown with orange markings. After eclosion, the juvenile stage goes through 4 larval instars before pupating. Just before pupating, the larva stops feeding and attaches itself by the anal pseudopod (Majerus, 1994) to substrates like leaves or other plant parts. Larvae can reach a length of up to 9 mm during the last instar. Eggs are orange in color and spindle- shaped. They measure about 1 mm in length, are usually laid in small clusters some distance from aphid colonies 23

32 (Schellhorn & Andrew, 1999; Hemptinne, 2000). Females lay their eggs in protected sites on leaves or stems of plants and can lay more than 1000 eggs during their adult lifespan. Both larvae and adult beetles are relatively generalist predators that prey on mites, insect eggs and small larvae (Hazzard and Ferro, 1991). More importantly, both stages can be found in cereal fields feeding on a broad range of pest aphids such as pea aphids (Acyrthosiphon pisum), green peach aphids (Myzus persicae), cotton aphids (Aphis gossypii), cabbage aphids (Brevicoryne brassicae), potato aphids (Macrosiphum euphorbiae) and greenbugs (Schizaphis graminum). This species often feeds on pollen and can even complete development on pollen as an exclusive diet (Hodek and Honek, 1996). Adult beetles go in hibernation during mid- autumn and emerge from their overwintering sites in spring. Overwintering aggregations form in empty spaces underneath litter or stones along the borders of crop fields II.3.2. The Chrysopid Chyrsoperla carnea The chrysopid Chrysoperla carnea was described by Stephens in 1836 and is taxonomically classified as follows: Order: Neuroptera Suborder: Hemerobiiformia Superfamily: Osmyloidea Family: Chrysopidae Although C. carnea is commonly called the common green lacewing, this species is in fact a complex of morphologically similar sibling species (Tauber & Tauber, 1973; Henry et al. 2001). For this reason, C. carnea is more accurately referred to as a species of the C. carnea- group. This species complex is commercially available for use in biocontrol programs. It has been released in crop fields worldwide, including Europe (Alrouechdi, 1981; Bozsik, 1991), and North- America (Ridgway & Jones, 1969; Tauber & Tauber, 1975; Chang et al., 1995; Daane et al., 1996, Henry et al., 2001). Adults are light green in color with translucent wings and light golden metallic eyes. Their body length measures between 10 and 15 mm. The wings are fragile with a wingspan of up to 30 mm 24

33 and are equipped with a complex system of green veins. Adults are mostly crepuscular or nocturnal and often hide during the day in shrubs or other vegetation. Females may produce up to 1000 eggs during their life span in optimal laboratory conditions (Rousset, 1984; Tauber et al., 1993; Zheng et al., 1993; Rosenheim, 2001). The eggs are green and are projected on a relatively long stalk of 6-8 mm (Lucas, 1998). Females lay their eggs on leaves and stems nearby aphid colonies. Solitary eggs as well as eggs in clusters can be found. After eclosion, larvae undergo two molts before entering the third and final larval instar. The body color of the larvae is mainly brown with small variations in color ranging from gray to reddish. At their smallest size they measure 1 mm in length and grow to a size of up to 8 mm. They have characteristic large, sickle- shaped mandibles. They spin a spherical cocoon to protect themselves during the pupal stage. This cocoon is spun with silk glands located on the dorsal surface of the larval body. Before adult emergence, the pharate adult leaves its cocoon to find a suitable spot to undergo its final stage of metamorphosis. While adults are mostly herbivorous and feed primarily on pollen, nectar and honeydew, the larvae are highly predaceous. These generalist predators feed on mites, insect eggs, nymph stages of other arthropods and herbivores like aphids. In addition, they may also feed on plant- based resources like extra- floral nectar (Limburg & Roseheim, 2001). Adults undergo diapause during which they gather in large clusters. Hibernation spots include unheated parts of buildings, barns, the underside of tree bark, and leaf litter (Canard & Principi, 1984). They leave their hibernation spots during spring. 25

34 II.3.3. The Aphid Schizaphis graminum The greenbug, Schizaphis graminum, was first described by Rondani in It was originally placed within the genus Aphis, and later in the genus Toxoptera, before finally being moved within the genus Schizaphis. S. graminum has the following taxonomical classification: Order: Homoptera Infraorder: Aphidomorpha Suborder: Sternorrhyncha Superfamily: Aphidoidea Family: Aphididae Subfamily: Aphidinae Although S. graminum is widely believed to be of palearctic origin, this is not known for certain. It is presently found in Europe, North, Central and South America, Africa, the Middle East and Asia (Blackman and Eastop 2000). In North America, this aphid species was first observed by the US Department of Agriculture in 1882 (Hunter, 1909) on wheat and barley plants. S. graminum is a rather small aphid species and is pale green to lime green in color. There is a prominent dark green longitudinal stripe along the dorsal surface of the abdomen of last- instar nymphs and adults. This species is a major pest of graminaceous plants ranging from turf to cereals. Greenbug saliva has enzymatic activity that breaks down cell walls and chloroplasts, leading to chlorosis in host plant tissues (Al- Mousawi et al., 1983). This injury can severely reduce the yield of cereals and will eventually result in plant death if the aphid colony is not controlled. Initially, yellow to reddish chlorotic spots are evident on infested plants. These develop into complete leaf yellowing and later browning, progressing downwards from the tip of the leaf. It was not until 1968 that S. graminum began attacking sorghum fields over extensive areas in the United States and emerged as a major pest of this crop (Harvey & Hackerott, 1969). At least nine greenbug biotypes damaging small grains have since been recognized based on the responses of specific plant genotypes. The greenbug is a holocyclic aphid with cyclical parthenogenesis during spring and summer months. First- instar nymphs reach adulthood after four molts. In autumn, sexual females and winged males mate to produce overwintering eggs that will hatch the following spring. 26

35 III. Materials and Methods III.1. Breeding of the Intraguild Predators and Extraguild prey III.1.1. Coleomegilla maculata The stock colony of Coleomegilla maculata lengi was established by collecting 120 adult beetles from fields of cultivated sorghum and sunflower on the Kansas State University Agricultural Research Center in Hays, Kansas. Those used during the spring/summer 2010 experiments were collected during the summer of 2009 and those used during the fall/winter 2010 experiments were collected during the summer of After collection, beetles were isolated individually in Petri dishes (5.5 cm d x 1.0 cm h) for five days to detect any individuals parasitized by Perilitus coccinellae (Hymenoptera: Braconidae). Healthy beetles were then moved into 1L, wide- mouth glass jars containing shredded wax paper as harborage and covered with a fine mesh. These jars were placed inside a Formica Scientific climate- controlled growth chamber set at a 24 (±2 ) C and a 16:8 L:D regime. Water was made continuously available on a cotton wick protruding from a test tube filled with water. Parafilm was wrapped around the cotton roll at the mouth of the test tube to prevent beetles from drowning in the tube. All beetles were fed every two days with frozen eggs of the flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). This colony was designated generation 0. To obtain a new generation of C. maculata, females were removed from the colony and isolated in Petri dishes (as above). Frozen E. kuehniella eggs were provided daily and water was provided on small cubes of sponge. The Petri dishes were placed on trays and held under the same conditions as the stock colony. Egg clusters were directly laid on the surface of the Petri dish and were collected every day by transferring the female beetles into clean dishes. Sponges 27

36 were added to dishes containing eggs and moistened daily to maintain humidity during egg incubation,. The eggs were held until hatching under the same environmental conditions as the adult insects. After eclosion, larvae were reared in Petri dishes (15.0 cm x 1.5 cm), 10 to 15 larvae per dish. The bottom of the Petri dish was lined with a piece of paper towel as substrate to absorb fecal matter and facilitate molting. Larvae were further kept inside these larger Petri dishes until pupation. A small lid from a 5.5 cm dish was lined with a moistened paper towel and placed in the dish to function as a water source. Food was provided daily in the form of frozen E. kuehniella eggs and water was provided on a moistened piece of paper towel. Adults were transferred to glass jars within two days of emergence. III.1.2. Chrysoperla carnea A colony of second- instar C. carnea larvae was shipped by Koppert B. V. (Berkel en Rodenrijs, The Netherlands). Immediately after being unpacked, the larvae were isolated in groups of three in 5.5 cm diameter Petri dishes. All life stages were reared under the same physical conditions as described above for the coccinellids. Larvae were fed frozen Ephestia eggs and no extra water was provided. Since chrysopid larvae are highly cannibalistic, cannibalism among these three larvae sometimes occurred. Once a third- instar larvae showed signs of pupating, characterized by their physogastric, inflated body, the two remaining larvae were isolated in their own dishes for pupation. After pupation, the cocoons were carefully detached from the dishes and transferred to one larger 15 cm Petri dish lined with a section of paper towel. Humidity was maintained by placing a moistened paper towel in a small dish within the 15 cm dish. Following the teneral period, newly emerged adults were carefully picked up by their wings with tweezers and placed in a similar 15 cm Petri dish, per dish, and provided with two food sources. A mix of honey and water was added 28

37 to a 5.5 cm dish on a moistened paper towel and ground pollen was placed in a separate 5.5 cm dish. These new adults were designated generation zero. Mating and oviposition of females took place inside of the 15 cm dishes. Eggs were laid directly on the surface of the plastic dish and were collected every day by first cooling the dishes down for minutes inside a walk- in cold chamber set at 3 C. Once cooled, the adults were transferred to new Petri dishes containing fresh food and the eggs were harvested. Young first- instar larvae were kept in the same Petri dishes in which they hatched. Ephestia eggs were provided as soon as the eggs started to darken, a sign that hatching was imminent. The food source was added immediately prior to hatching to lower the rate of cannibalism by first- instar larvae on other first- instar larvae and also on the unhatched eggs. To start a new generation of lacewings, first- instar larvae were put in groups of three in 5.5 cm dishes within 24 hours after eclosion. The rest of the procedure follows the steps of the breeding procedure of generation zero. III.1.3. Schizaphis graminum A laboratory colony of S. graminum biotype I was established from material collected on the grounds of ARCH in Hays, Kansas in the spring of 2010 and maintained on sorghum seedlings (cultivar P8500 ) in a Percival I- 36VL growth chamber under coolwhite fluorescent lighting set to a photoperiod of 16:8 (L:D). The temperature in the growth chamber was set to a 23 C/21 C diurnal cycle to promote optimal reproduction of the greenbugs (Daniels 1963). Sorghum seeds were planted in metal trays (8 h x 26 w x 36 l cm) filled with soil. Germination of the seeds took place inside a second Percival I- 36VL growth chamber. A total of four trays of seedlings were continuously infested with greenbugs, the older trays being periodically replaced with fresh ones as the sorghum plants succumbed to the aphids. New trays of seedlings were infested by physically dislodging the aphids from the older sorghum plants and sprinkling them on the new tray of seedlings. All trays were watered by hand as required. 29

38 III.2. Design of a microcosm III.2.1. General Considerations Experiments involving complex tritrophic ecological systems in which IGP occurs face pitfalls (e.g. premature death of the plant) that may negatively affect the production of useful data. It is therefore important to consider the main factors for the setup of this kind of experiments. These main factors are: Setting physical conditions to optimize growth of both the plant and the aphid colony. The age of the plant at aphid infestation. The age of aphids to infest, and how many to use The period permitted for aphid colony growth prior to predator introduction. III.2.2. The Conetainer Microcosm The microcosm used for the experiments is termed a conetainer and was first developed by Harvey and Kofoid (1993) and later adapted by Jyoti and Michaud (2005). This configuration consists of a ventilated clear plastic tubular cage that sits on top of a plastic cone holding soil in which plants are grown. The cone (Stuewe & Sons, Corvallis, Oregon) has a diameter of 2.5 cm at the top and is 16.5 cm in length (see Figure 5). The cones were filled with soil, seeds of sorghum (P8500) were planted in the soil and the cones were arranged in a special plastic rack. The rack was then immersed in a water bath until all cones had wicked up moisture to saturation, and the rack was moved to a growth chamber for germination. The plastic cylinder that serves to confine insects on the plants is 30.5 cm in height and has a similar outer diameter to that of inner diameter of the cone. To permit adequate ventilation, each cylinder had four pairs of opposing holes (1.5 cm in diameter) each covered by a small portion of mesh glued on with silicone. A plastic lid sealed the top opening. Sorghum of the P 30

39 8500 cultivar was selected as a host plant for S. graminum that represented the herbivore population in the micro- ecosystem. Figure 5: The 'Conetainer' III.2.3. Preliminary Testing of the Conetainer A preliminary experiment was carried out in order to test the conetainer design and evaluate the resilience of sorghum seedlings of different ages infested with different greenbug densities. After filling the cones with soil, three sorghum seeds were planted at a depth of 1 2 cm and all of the cones were placed in plastic racks in a water bath for a period of 24 hours. The racks holding the conetainers were then put on separate plastic trays inside a Percival I- 36VL growth chamber under a coolwhite fluorescent lighting set to a photoperiod of 16:8 (L:D). The temperature cycle was set to follow a 23 C/21 C diurnal 31

40 program. Water was provided by filling the plastic trays regularly with water. The day of germination was considered as day 0 and on day 5, plants in all cones were thinned to two per cone. On day 11, 40 cones were infested with fourth instar S. graminum nymphs from the stock colony. Aphids were brushed from their host plants into a 15 cm Petri dish so that suitable stages could be selected under a stereo microscope and carefully transferred onto plants with a soft camel hair brush. After infestation, each cone was covering with a cylindrical cage. A total of four treatments (n = 10 per treatment) were generated by infesting cones with 4, 8, 10 and 12 fourth instar nymphs, respectively. After infestation, the conetainers were placed in their racks inside a growth chamber under same conditions described above and watered as required. This procedure was repeated with 15- day- old and 19- day- old plants to evaluate the effect of plant age at infestation on the plants tolerance of the aphid colonies. The number of aphids per conetainer was counted non- destructively every three days after infestation until both the plants and the aphid colony expired. The results of this preliminary test are graphically presented in Figure 6. Because of very high variation, no error bars are depicted. The mean number of aphids shows a linear pattern followed by a steep decline from the population peak. This steep decline is caused by collapse of the plant that in turn leads to a crash in aphid numbers. With the exception of higher means for the 19- day- old plants, no clear trends were evident (Figure 6a). Death of the plants occurred within 12 days in all four treatments. Seedlings infested with only four aphids tended to die about three days later than plants infested with more aphids, but there was no apparent effect of plant age at infestation (Figure 6b). The preliminary test indicated that the aphid- plant ecosystem in the conetainer persists for about 12 days given the parameter settings employed and the initial conditions specified. The primary concerns for the main IGP experiment were the life span of plants and the establishment of aphid colonies of sufficient size to permit the complete development of at least one predator. It was deemed essential for the success of the experiment that plants survive beyond the period needed for complete development of the predator larvae. The larval developmental time for C. maculata fed on S. graminum at 24 ± 1 C is 12 days 32

41 (Michaud & Jyoti, 2008) and that of C. carnea fed on Hyalopterus pruni (Homoptera: Aphididae) at 25 ± 1 C is within a comparable range (Althan et al., 2004). With these considerations in mind, it was decided to employ three 30- day- old sorghum plants per cone infested with eight fourth instar nymphs of S. graminum. These would be permitted to establish a colony over a period of three days before introduction of first instar predators. Figure 6a: The mean number of aphids on the plants on days of observations for 11, 15 and 19 day old plants. Figure 6b: The cumulative number of plants that had died on days of observation for 11, 15 and 19 day old plants. 33

42 III.3. Experiment 1: Intraguild Predation in the Microcosm In order to evaluate IGP interactions among C. carnea and C. maculata larvae, a total of six treatments were devised, all consisting of a greenbug colony developing on sorghum plants in conetainer. Treatments were as follows: 1. One first instar C. maculata + one first instar C. carnea larva (n=80) 2. One first instar C. maculata larva (n=20) 3. One first instar C. carnea larva (n=20) 4. Two first instar C. maculata larvae (n=40) 5. Two first instar C. carnea larvae (n=40) 6. No predators (n=20) A total of 250 cones were planted and germinated as above except that 4 instead of 3 seeds were planted in each cone. Upon germination, the best 220 cones were selected for use in the experiment; those with uneven germination or fewer than three plants were discarded. The sorghum plants were fertilized during the third week with the NPK fertilizer Miracle- Gro Water Soluble All Purpose Plant Food (Scotts Miracle- Gro Company, Marysville, Ohio) according to the directions of the producer. On day zero of the experiment, when sorghum plants were 30 days old, all conetainers were infested with eight fourth instar S. graminum nymphs and placed in growth chambers under the same physical conditions as described in the preliminary experiment. Temperature data (mean/max/min) were recorded daily from a digital thermal probe (Thermohygro, Gemplers, Madison, WI) placed inside a conetainer of a bigger size than the ones used as a microcosm in the same plastic rack as the experimental replicates (Michaud & Jyoti, 2008). On day three, third generation predator larvae (< 24 h old) were introduced into the appropriate microcosms by transfering them with a soft camel brush. First- instar larvae were selected only if they showed evidence of having fed on Ephestia eggs during their first day of life. For C. carnea, this was evident in the form of visible pale yellowish gut contents, and for C. maculata the appearance of a slightly distended abdomen. This was done in order to minimize the probability of larval 34

43 mortality during their first day in the microcosm, i.e., before they were able to access prey. Starting on day six, the condition of the plants, the aphid colony, and the predators was evaluated every 48 h. Observations were made non- destructively by observing plants and insects through the transparent cylindrical cages without removing them from the plastic cones. The state of the larvae (alive/dead) and the instar of the larvae were recorded during each observation. Exact aphid numbers were not recorded except in two situations: (1) when fewer that 10 aphids per replicate could be observed (colonies larger than 10 aphids were considered sufficient to sustain the replicate until the next observation) and (2) when the aphid colony exceeded the carrying capacity of the plants and began to abandon them. In this case, a default value of >250 aphids was recorded. Plants were only evaluated as alive or dead; a plant was considered dead when all leaves had turned brown. Whenever a predator pupated, it was removed within 24 h and weighed on an analytical balance. A replicate was terminated when either no predatory larvae remained alive, or when the plant died. The final state of the plant (dead or alive) was determined, and all live aphids remaining were counted. Biological control was deemed successful if predation prevented the aphid colony from reaching the exponential growth stage. In replicates where either the surviving predator or the both predators starved, there were often a few aphids that remained concealed in leaf sheaths; these were counted and recorded, but the colony was considered to have been controlled. Replicates in which a predator died due to unknown causes (i.e., causes other than IGP or starvation following exhaustion of prey) were excluded from analysis. The entire experiment was repeated a second time with half the initial number of replicates and utilizing predatory larvae of the sixth generation. All physical conditions were identical to the first run. 35

44 III.4. Experiment 2: Intraguild Predation in Petri Dishes In order to estimate the relative contribution of the microcosm and the plant to experimental results, an experiment with similar treatments was conducted in Petri dishes (5.5 cm x 1.0 cm) under identical physical conditions. Treatments were as follows: 1. One first instar C. maculata + one first instar larva C. carnea (n=100) 2. One first instar C. maculata larva (n=40) 3. One first instar C. carnea larva (n=40) 4. Two first instar C. maculata larvae (n=60) 5. Two first instar C. carnea larvae (n60) As in the earlier experiment, all larvae were first instar (< 24 h old) and were selected only if they showed evidence of having fed on Ephestia eggs. To test for any possible effect of food source on IGP outcomes, half of the replicates in each treatment were fed ad libitum on frozen Ephestia eggs (refreshed daily) while the other half were provided with 30 to 50 S. graminum of various developmental stages every 24 hours. A source of water was not supplied in the dishes (Michaud & Jyoti, 2008). Larvae were held in the same dish throughout the duration of the experiment. All dishes were examined daily to record mortality and larval molts. Replicates were terminated either when predators pupated, or when none remained alive. The fresh weight of all surviving predators was measured on an analytical balance within 24 h of pupation. III.5. Data analysis Categorical variables such as plant survival, biological control outcome, and predator victory in IGP were analyzed pairwise using the Chi Square, Goodness of Fit test with one degree of freedom. Scalar variables such as fresh weight at pupation and larval developmental time were analyzed by one way ANOVA. These scalar variables were further analyzed with a two- way ANOVA with diet and treatment as factors (SPSS, 1998). Since there were no significant differences ( = 0.05) in any mean values between the two runs of experiment 1, results for the pooled data set are reported. 36

45 IV. Results IV.1. Temperature The mean temperature inside the conetainers during these experiments was 23.2 ± 0.6 C (mean minimum = 19.4 ± 0.2 C, mean maximum = 25.1 ± 0.9 C). The mean temperature inside the growth chamber during the Petri dish experiment was 21.6 ± 0.3 C (mean minimum = 19.5 ± 0.3 C, mean maximum = 22.6 C ± 0.3 C). IV.2. Pooling of Data Since there were no significant differences in outcomes for treatment one (both predators present) between the two replications of the conetainer experiment (plant survival: 86.8% vs 79.4%, Chi square = 0.93, ns; IGP victories for C. carnea: 66.2% vs 55.9%, Chi Square = 1.61, ns; biological control of aphids: 69.1% vs 70.6%, Chi square = 0.13, ns) the data were pooled for further analysis. Similarly, there was no significant effect of diet on IGP outcomes for C. carnea in Petri dishes (89.8% vs 88.0 %, Chi square = 0.15; ns) so diet treatments were pooled for analysis. IV.3. Plant Survival in Conetainers With the exception of one replicate in which an aphid colony failed to establish, all plants in the no predator treatment (n = 30), died as a result of aphid feeding. The survival rate of sorghum plants in replicates with two different predators was 86.8% and 79.4% in the first and second run, respectively (Chi Square = 0.93, ns). Plants in the treatment with a single C. maculata larva survived in 26 of 28 valid replicates (92.9%), whereas those with a single C. 37

46 carnea larva survived in 13 of 15 replicates (86.7%; Chi square = 0.87, ns). When two C. maculata larvae were present, plants survival was significantly higher (in 52 of 53 replicates), compared for the treatment with two C. carnea larvae (in 39 of 48 replicates) (Chi square = 9.89, p<0.05). IV.4. Biological Control of Aphids in Conetainers The mean number of aphids per replicate at the end of the experiment varied significantly among treatments (F4,241 = 2.524, P = 0.042; Table 2). The treatment with two C. maculata larvae finished the experiment with fewer aphids per replicate than treatments with two C. carnea with no other differences among treatments significant. Treatment Mean ± SE Significance 1 C. maculata and 1 C. carnea 54.5 ± 8.4 ab 1 C. maculata 54.3 ± 12.8 ab 1 C. carnea 57.2 ± 20.1 ab 2 C. maculata 18.9 ± 5.9 b 2 C. carnea 61.7 ± 13.5 a Table 2: Mean number of aphids per treatment. Means followed by different letters are significantly different at = 0.05 (Tukey s HSD) Control of the aphid population, defined as prevention of exponential growth by the colony, occurred significantly more often in the treatment with two C. maculata larvae than in the treatment with two C. carnea larvae (Chi square = 22.46, P < 0.001). Aphids were also controlled significantly more often by a mixed pair of predators than by two C. carnea (Chi square = 20.3, P < 0.001), whereas two C. maculata larvae controlled S. graminum more often than did one (Chi square = 6.20, P < 0.02). However, one C. carnea larva was just as likely to provide S. graminum control as were two (Chi square = 2.06, ns). 38

47 IV.5. Starvation and Larval Survival Whenever the aphid population in a conetainer was suppressed before the predators had completed development, the predators died of starvation due to exhaustion of prey. This occurred in 1/28 replicates (3.6%) with solitary C. maculata, in 3/15 replicates (20.0%) with solitary C. carnea, in 14/53 replicates (26.4%) for pairs of C. maculata, and in 12/48 replicates (25.0%) for pairs of C. carnea. For survivors in the IGP treatment, starvation occurred for 2/38 (7.9%) of C. maculata and 21/64 (31.3%) of C. carnea (Chi square = 13.08, P < 0.001). Even though Ephestia eggs were continuously available in the Petri dish experiment, not all larvae survived even when isolated. When solitary C. maculata developed on a diet of greenbug, only 8/20 (40%) survived, compared to 17/20 (85%) on a diet of Ephestia eggs (Chi square =16.87, P <0.001 ). The survival of solitary C. carnea in Petri dishes was significantly lower on greenbug 15/20 (75%) than on Ephestia eggs 19/20 (95%) (Chi square = 4.27, P < 0.05). IV.6. IGP Outcome Larvae of C. carnea were superior to C. maculata larvae in IGP interactions that occurred on plants in conetainers, winning 64/102 contests (62.7%, Chi square = 28.35, ns, Figure 8a). A comparison of the developmental stages of winners and losers by species reveals that C. maculata is particularly vulnerable to C. carnea in the first and third instars (Figure 7). Excluding one case of mutual elimination that occurred on the Ephestia egg diet, larvae of C. carnea won 88/99 contests (88.9%) that occurred in Petri dishes. Thus, C. maculata were almost three times more successful in IGP interactions on plants in conetainers as they were in interactions in Petri dishes (Figure 8b). Again, comparison of the developmental stages of winners and losers by species reveals the vulnerability of C. maculata to C. carnea while in the first instar (Figure 9). 39

48 Figure 7: Percentage of larvae in the conetainers that were subjected to IGP vs the percentage of larvae that engaged successfully in IGP per larval instar. Figure 8: The percentage of IGP winners per species. a = IGP winners in the conetainers; b = IGP winners in the Petri dishes. Figure 9: Percentage of larvae in the conetainers that were subjected to IGP vs the percentage of larvae that successfully engaged in IGP per larval instar. 40

49 IV.7. Cannibalism When two C. maculata larvae developed in the same conetainer, cannibalism occurred in 35/53 replications (66%), whereas it occurred in 44/48 replications (91.7%) with pairs of C. carnea larvae, suggesting that C. carnea was more cannibalistic (Chi square = 14.06, P < 0.001). In Petri dishes, paired C. maculata cannibalized in 24/30 replications (80%) on the Ephestia egg diet and 29/29 (100%) on the aphid diet (with two cases of mutual elimination), whereas C. carnea cannibalized in 100% of replicates with paired larvae, regardless of diet (n = 30 and 29, respectively). Figure 10 and 11 provide data on which developmental stages were subjected to cannibalism or successfully engaged in cannibalism wen fed on Ephestia eggs or S. graminum respectively. Figure 10: The percentages of cases in the Petri dishes in which a larval instar was subjected to cannibalism or successfully engaged in cannibalism wen fed on Ephestia Figure 11: The percentages of cases in the Petri dishes in which a larval instar was subjected to cannibalism or successfully engaged in cannibalism wen fed on S. graminum 41

50 IV.8. Development In the conetainer experiments, pupating C. carnea that fed only on aphids (n = 21) did not differ from those that vanquished and consumed C. maculata larvae in the IGP treatment (n = 35) in either larval developmental time (16.0 ± 0.8 d vs ± 0.6 d; F1,54 = 0.112, P = 0.740) or fresh weight at pupation (9.0 ± 0.5 mg vs. 8.9 ± 0.3 mg; F1,54 = 0.059, P = 0.809). Similarly, C. carnea larvae that cannibalized their counterpart in the paired treatment and survived to pupate (n = 24) required a mean of 17.5 ± 0.7 d to complete development and weighed a mean of 8.4 ± 0.4 mg at pupation, values that did not significantly differ from non- cannibals (F1,43 = 1.869, P = and F1,43 = 0.964, P = 0.332, respectively). Pupae of C. maculata in the conetainer experiment that fed only on aphids (n = 54) did not differ from those that had vanquished and consumed C. carnea larvae (n = 35) in either larval developmental time (16.3 ± 0.4 d vs ± 0.6 d; F1,87 = 0.000, P = 0.998) or fresh weight at pupation (12.0 ± 0.4 mg in both cases; F1,87 = 0.000, P = 0.995). However, C. maculata larvae that cannibalized their counterpart in the paired treatment and survived to pupate (n = 24) required a mean of 19.2 ± 0.8 d to complete development and weighed a mean of 10.6 ± 0.6 mg at pupation, values significantly greater and lower, respectively, compared to non- cannibals (F1,77 = , P < and F1,77 = 4.039, P = 0.048, respectively). A two- way ANOVA of C. carnea developmental time in the petri dish experiment was significant overall (F5,162 = 73.31, P < 0.001) and revealed a significant effect of both treatment (F2,162 = 10.57, P < 0.001) and diet (F1,162 = , P < ) and a significant treatment*diet interaction (F2,162 = 6.47, P = 0.002). Similarly, the two- way ANOVA of C. carnea pupal weight was significant (F5,162 = 16.15, P < 0.001) with significant effects of both treatment (F2,162 = 7.72, P = 0.001) and diet (F1,162 = 61.04, P < 0.001) and a significant treatment*diet interaction term (F2,162 = 8.36, P < 0.001). Disregarding treatments, larvae of C. carnea that survived to pupate on the Ephestia egg diet developed faster than those on the S. graminum diet (F1,166 = , P < ) and weighed more (F1,166 = 38.64, P < ). There was no effect of treatment on C. carnea developmental time on the S. graminum diet (F2,74 = 0.355, P = 0.702; Figure 12), but cannibals weighed significantly more than solitary individuals (F2,74 = 3.84, P < 0.01), with 42

51 intraguild predators not significantly different from either cannibals or solitary individuals (Figure 13). However, on the Ephestia egg diet, treatment affected both developmental time (F2,88 = 28.69, P < 0.001) and fresh weight at pupation (F2,88 = 12.87, P < 0.001). Solitary larvae developed faster than cannibals that, in turn, developed faster than intraguild predators (Figure 12) and intraguild predators weighed less on average than either cannibals or solitary individuals, the latter groups being not significantly different (Figure 13). Figure 12: Mean (+SE) developmental times of C. carnea larvae that pupated either as solitary individuals or following acts of cannibalism or intraguild predation when reared on each of two diets. Columns bearing the same letters were not significantly different among treatments within diets (Tukey s HSD, α = 0.05) Figure 13: Mean (+SE) fresh pupal weights of C. carnea larvae that pupated either as solitary individuals, or following acts of cannibalism or intraguild predation when reared on each of two diets. Columns bearing the same letters were not significantly different among treatments within diets (Tukey s HSD, α = 0.05). 43

52 A two- way ANOVA of developmental time for C. maculata larvae in the Petri dish experiment was significant overall (F5,71 = 17.08, P < 0.001) and revealed significant effects of both treatment (F2,71 = 15.65, P < 0.001) and diet (F1,71 = 28.73, P < 0.001) but the treatment*diet interaction was not significant (F2,71 = 0.15, P = 0.860). The two- way ANOVA of C. maculata pupal weight was significant overall (F5,71 = 5.99, P < 0.001) with a significant effect of treatment (F2,71 = 9.56, P < 0.001) but not diet (F1,71 = 3.27, P = 0.075) and the treatment*diet interaction was not significant (F2,71 = 2.84, P < 0.065). Disregarding treatments, larvae fed Ephestia eggs developed faster (F1,63 = 39.49, P < 0.001) and weighed more at pupation (F1,63 = 4.56, P = 0.037) than larvae fed S. graminum. On the aphid diet, treatment affected both developmental time (F2,25 = 4.40, P = 0.023) and pupal weight (F2,25 = 4.40, P = 0.023). Solitary larvae developed faster than cannibals but intraguild predators were not different from either (Figure 14). Cannibal pupae weighed less than solitary pupae and intraguild predators were not significantly different from either (Figure 15). Treatment also affected developmental time (F2,34 = 14.53, P < 0.001) and pupation weight (F2,34 = 5.61, P = 0.008) when C. maculata larvae were fed Ephestia eggs. Solitary larvae developed faster than either intraguild predators or cannibals, with the latter two groups not significantly different (Figure 14). Similarly, pupae of solitary individuals were heavier than those of intraguild predators or cannibals, with the latter two groups not significantly different (Figure 15). 44

53 Figure 14: Mean (+SE) developmental times of C. maculata larvae that pupated either as solitary individuals, or following acts of cannibalism or intraguild predation when reared on each of two diets. Columns bearing the same letters were not significantly different among treatments within diets (Tukey s HSD, α = 0.05). Figure 15: Mean (+SE) fresh pupal weights of C. maculata larvae that pupated either as solitary individuals, or following acts of cannibalism or intraguild predation when reared on each of two diets. Columns bearing the same letters were not significantly different among treatments within diets (Tukey s HSD, α = 0.05). 45