Responses of the Catalpa Sphinx, Ceratomia Catalpae, and Its Primary Parasitoid, Cotesia Congregata, to Varying Levels of Iridoid Glycosides in Catalpa

Virginia Commonwealth University VCU Scholars Compass

Theses and Dissertations Graduate School

2015

RESPONSES OF THE CATALPA SPHINX, CERATOMIA CATALPAE, AND ITS PRIMARY PARASITOID, COTESIA CONGREGATA, TO VARYING LEVELS OF IRIDOID GLYCOSIDES IN CATALPA

Jessica L. Bray Virginia Commonwealth University

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RESPONSES OF THE CATALPA SPHINX, CERATOMIA CATALPAE, AND ITS

PRIMARY PARASITOID, COTESIA CONGREGATA, TO VARYING LEVELS

OF IRIDOID GLYCOSIDES IN CATALPA

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University.

JESSICA L. BRAY B.S., Longwood University, 2012

Major Professor: KAREN M. KESTER, PH.D. Associate Professor, Department of Biology

Virginia Commonwealth University Richmond, Virginia April, 2015

Acknowledgments

I thank my advisor, Dr. Karen Kester, for her guidance and mentorship on this project. I also thank my fellow graduate students, Justin Bredlau and Megan Ayers, for their help and moral support throughout graduate school. During my time at VCU, they have become very close friends. I also thank my fellow labmate, Kanchan Joshi, who took care of the hyperparasitoid colonies while I was away at conferences and the many Bug lab interns, especially LeAynne

Miller and Kathleen Lau, who helped rear caterpillars to maintain the wasp colonies. Thanks, too, to VCU independent study student Andrew Wood for collecting the 2011 oviposition data,

Mr. and Mrs. Buster Tyson for allowing me to collect catalpa leaves and catalpa sphinx caterpillars on their property, and the “Bowers laboratory” for analyzing leaf samples. Finally, I thank Dr. M. Deane Bowers, as well as the rest of my committee members, Dr. Michael Fine, Dr.

Salvatore Agosta, and Dr. Edward Boone, for their advice and feedback on my project.

Table of Contents

Page

Acknowledgements ...... i

Table of Contents ...... ii

List of Tables ...... iii

List of Figures ...... iii

Abstract………...... iv

Introduction ...... 1

Materials and Methods ...... 6

Results ...... 13

Discussion ...... 16

Acknowledgements ...... 19

Literature Cited ...... 20

Tables and Figures ...... 25

Vita ...... 35

List of Tables Page

Table 1: Species characteristics of catalpa trees of interest ...... 25

Table 2: Results of a two- way ANOVA comparing searching times of wild

and colony wasps ...... 26

List of Figures Page

Figure 1: Feeding preference assay design ...... 27

Figure 2: Percent dry weight total iridoid glycosides, catalpol and catalposide in catalpa

trees of interest ...... 28

Figure 3: Comparison of percent defoliation to percent dry weight total iridoid

glycosides, catalpol and catalposide ...... 30

Figure 4: Comparison of moth oviposition preference to high and low

iridoid glycoside trees ...... 32

Figure 5: Comparison of caterpillar feeding preferences to high and low

iridoid glycoside leaf discs ...... 33

Figure 6: Comparison of wasp searching responses to high and low

iridoid glycoside leaf discs ...... 34

iii

Abstract

RESPONSES OF THE CATALPA SPHINX, CERATOMIA CATALPAE, AND ITS

PRIMARY PARASITOID, COTESIA CONGREGATA, TO VARYING LEVELS OF

IRIDOID GLYCOSIDES IN CATALPA

By Jessica L. Bray

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology at Virginia Commonwealth University.

Virginia Commonwealth University, 2015

Director: Karen M. Kester, Ph.D. Associate Professor, Department of Biology

The catalpa sphinx, Ceratomia catalpae, is a specialist on Catalpa trees, which produce

iridoid glycosides (IGs). Whereas some trees are defoliated every year, others escape

herbivory. Caterpillar populations are either heavily parasitized by the braconid wasp,

Cotesia congregata, or remain unparasitized. We hypothesized that these patterns could

be explained by variable IG concentrations among trees and insect responses to these

chemicals. IG concentrations varied among trees. Percent defoliation was positively

related to IG concentration. In comparisons of insect responses to relatively high or low

IG concentrations, moths preferred to oviposit on trees with high IG concentrations.

Caterpillars did not display a feeding preference nor did wasps differ in searching responses to leaf discs with high or low IG concentrations. Results indicate that observed patterns of herbivory can be explained by moth oviposition preferences for trees with high IG concentrations.

INTRODUCTION

Plant secondary metabolites function as feeding and oviposition stimulants or deterrents for many phytophagous insects. Insect species vary greatly in their degree of host specialization and in their ovipositional and feeding responses to plants. Whereas generalists may accept a variety of plants as hosts, specialists typically require specific cues from secondary metabolites in order to accept a plant as a host (Bernays & Chapman 1994). For example, Pieris brassicae L.

(Pieridae) butterflies respond to glucosinolates produced by crucifers as oviposition stimulants

(van Loon et al. 1992, Chew & Renwick, 1995). Some specialists sequester secondary metabolites as a defense against their natural enemies (Dyer & Gentry 1999, Dicke 2000).

Levels of secondary metabolites can vary within individuals and both within and among populations of hostplants, and the absence or low levels of these plant cues may cause an insect to overlook an acceptable hostplant (van der Meijden 1996). This variation in plant chemistry can be affected by genetics (Berenbaum et al. 1986, Mihaliak et al. 1989) as well as environmental factors (Mihaliak et al. 1989). For example, in wild parsnip Pastinaca sativa L.

(Apiaceae) variation in plant defenses to its primary herbivore, Depressaria pastinacella

(Duponchel) (Elachistidae) is partially genetically controlled (Berenbaum et al. 1986). Likewise, iridoid glycoside concentrations in Plantago lanceolata L. (Plantaginaceae) vary within and among populations (Bowers & Stamp 1992, Bowers et al. 1992), partially determined by genotypic differences (Bowers & Stamp 1992, 1993, Adler et al. 1995, Reudler Talsma et al.

2008).

Iridoid glycosides (IGs) are a group of secondary metabolites common in over fifty different plant families (Bobbitt & Segebarth 1969, El- Naggar & Beal 1980, Bowers & Puttick

1986). Some lepidopterans are specialists on plants that produce iridoid glycosides (Bowers &

Puttick 1986, Optiz & Müller 2009). Specialist butterflies that are attracted to plants containing these compounds often prefer to oviposit on plants with higher concentrations of iridoid glycosides. For example, the Glanville fritillary, Melitaea cinxia L. (Nymphalidae), and the buckeye butterfly, Junonia coenia Hübner (Nymphalidae), both prefer to oviposit on P. lanceolata with high concentrations of iridoid glycosides (Nieminen et al. 2003, Pereyra &

Bowers 1988). Larvae of these species sequester iridoid glycosides as a defense against their natural enemies and therefore, sequestration may provide offspring with a higher chance of survival (Bowers & Stamp 1992).

The relative unpalatability of an insect often depends on the chemistry of its hostplant, particularly for specialist caterpillars that sequester secondary metabolites (Bowers 1980,

Belofsky et al. 1989, Bowers & Farley 1990, Bowers & Collinge 1992). Many of the lepidopteran larvae that sequester iridoid glycosides sequester catalpol or aucubin over other iridoids present in the hostplant (Optiz and Müller 2009). Catalpol is considered to be a more important defensive compound than many other iridoid glycosides, which may explain why it is sequestered over other iridoids (Klockers et al. 1993, Bowers 1991, Belofsky et al. 1989). For example, larvae of the butterfly Euphydryas anicia (Doubleday) (Nymphalidae) sequester catalpol instead of aucubin even though the concentrations of these compounds in the plant are similar (Stermitz et al. 1986). Gardner & Stermitz (1988) demonstrated that E. anicia can hydrolyze a catalpol ester into catalpol. The same may be true for other catalpol sequestering larvae such as the catalpa sphinx, although this has yet to be investigated.

Variation in the concentrations of secondary metabolites sequestered by specialist caterpillars is related to variation in rates of parasitism (Nieminen et al. 2003), depending on the parasitoids’ tolerance. For example, Cotesia melitaearum (Wilkinson) (Braconidae), a primary parasitoid of M. cinxia, prefers to parasitize larvae that sequester lower concentrations of the iridoid glycoside catalpol (Nieminen et al. 2003). To survive in a chemically toxic environment, endoparasitoids of specialists must adapt to living inside caterpillars that sequester secondary plant chemicals. Broad surveys demonstrate that many chemically defended insects have high rates of parasitism, possibly due to parasitoid specialization on particular hosts (Cornell &

Hawkins 1995, Gentry & Dyer 2002). Herbivores that sequester plant chemicals may be unpalatable to predators, which in turn, may protect the parasitoids from harm during development (Dyer & Gentry 1999, Gentry & Dyer 2002, Lampert et al. 2010). In addition, it is well established that parasitoids locate their respective hosts using volatile and contact cues from their specialist hosts’ hostplants (Vinson 1976, Vet & Dicke 1992); thus, intraspecific variation in plant chemistry may affect the ability of parasitoids to locate their hosts.

Deciduous catalpa trees (Catalpa spp.) (Bignoniaceae) are in found North America and

East Asia (Baerg 1935, Forest Service 1948). The southern catalpa (Catalpa bignonioides

Walter) is native to the southeast and midwest United States and the northern catalpa (Catalpa speciosa Warder) is native only to the midwest (Petrides & Wehr 1998). Both are widely planted as ornamentals outside of their native ranges and are known to hybridize naturally if in close proximity (Douglass 1906, Stone 1907, Scott 1911, Forest Service 1948). Both species occur in

Virginia. Catalpa trees produce iridoid glycosides, primarily catalpol and its ester catalposide

(von Poser et al. 2000, Castillo & Rossini 2010, Lampert et al. 2010). Average iridoid glycoside concentrations vary from 3-10% of the dry leaf weight, with catalpol and catalposide making up

the majority of this percentage (Lampert et al. 2010). These compounds are considered a deterrent to most insects, excluding the catalpa sphinx caterpillar, for which they appear to serve as feeding stimulants (Nayar & Fraenkel 1963, Lampert et al. 2010).

The catalpa sphinx, Ceratomia catalpae (Boisduval) (Lepidoptera: Sphingidae), is a specialist on trees in the genus Catalpa (Baerg 1935, Nayar & Fraenkel 1963, Bowers 2003). It is native to the Eastern and Midwestern United States and in many areas, is an important pest and the only major defoliator of catalpa trees (Hodges, 1971). Catalpa sphinx moths oviposit clusters of up to several hundred eggs. Moths do not feed and survive only for 5-6 days in the laboratory

(Bray pers. obs.). Caterpillars are gregarious in the first few instars (Hodges, 1971) and become more solitary in their later instars (4th and 5th) (Bray pers. obs.). Larvae feed heavily on catalpa trees and in large numbers, are capable of defoliating entire trees. Catalpa sphinx larvae sequester catalpol, from their hostplant, in their hemolymph and are generally considered to be unpalatable to most predators (Bowers 2003, Lampert et al. 2010). Catalpol levels can be as high as 5 to 20% of the caterpillars’ dry weight (Lampert et al. 2010). In Virginia, they have 2 to 3 generations a year from late June to the beginning of October and overwinter as pupae.

Catalpa sphinx populations are often heavily parasitized by the gregarious endoparasitoid

Cotesia congregata (Say) (Hymenoptera: Braconidae), which is the only primary hymenopterous parasitoid of the catalpa sphinx in the mid-Atlantic region (Kester pers. obs.). Cotesia congregata is reported to attack several sphingid species (Krombein et al. 1979); however, recent evidence suggests that as a result of adaptations to different host-hostplant complexes, C. congregata that attacks the catalpa sphinx and C. congregata that attacks Manduca sexta L. on tobacco, are in the process of diverging and are likely incipient species (Bredlau & Kester, in press, Kester et al., in press). Cotesia congregata attacks 2nd and 3rd instar larvae of the catalpa

sphinx, depositing anywhere between a few to over a hundred eggs inside each host. Parasitoid larvae feed on nutrients in the host hemolymph for 2 to 3 weeks then egress by chewing through the host cuticle, spin silken cocoons that remain attached to the host, pupate, and 6-8 days later, emerge as adults (Kester & Barbosa 1991). Lampert et al. (2010) found that survivorship of C. congregata was not significantly affected by catalpol sequestration and suggested this was due to an evolved tolerance to catalpol in the host hemolymph. To date, the effects of iridoid glycoside concentration on parasitoid responses to plant cues that may be used in host location have not been studied. Because the catalpa sphinx is monophagous and C. congregata is its only primary hymenopterous parasitoid, this association provides an excellent model system for studying the effects of chemical variation in tritrophic interactions.

Observations of catalpa trees at several sites in Richmond, VA and a site in Cumberland

County, VA (designated the “Tyson site”) over the past 16 years indicate that certain catalpa trees are heavily infested by the catalpa sphinx while other trees have no or low levels of infestation (Kester pers. obs.). Further, parasitism by C. congregata also is highly variable; some catalpa sphinx populations are heavily parasitized while others are not (Kester pers. obs.). We hypothesized that iridoid glycoside concentrations varied among trees and that insect responses to relatively high or low concentrations of iridoid glycosides could explain the patterns of catalpa sphinx infestation and parasitism by C. congregata.

To investigate how variation in leaf iridoid glycosides could affect the behavior of catalpa sphinx and its primary parasitoid, we asked three questions: 1) Do adult females of the catalpa sphinx prefer to oviposit on catalpa trees with relatively high or low levels of iridoid glycosides? 2) Do caterpillars of the catalpa sphinx prefer to feed on leaves with relatively high

or low levels of iridoid glycosides? 3) Do C. congregata females display differential searching responses to catalpa leaves with relatively high or low levels of iridoid glycosides?

MATERIALS AND METHODS

Field sites The Tyson site in Cumberland County, VA (37.712, -78.163) consists of 23 trees planted

15 to 20 years ago in a grove from the same seed source (Tyson pers. comm.). Only 17 of the 23 trees on the Tyson property were used due to presence of the owner’s beehives at the base of six trees. The three Richmond, VA sites consist of one or two trees along roadsides in an urban environment. The three Richmond sites are the Monument Ave. tree (37.595, -77.531), the 30th street trees (corner & side) (37.520, -77.466) and the Lakeside tree (37.622, -77.462).

Catalpa observations

Because the northern (C. speciosa) and southern (C. bignonioides) catalpa co-occur in

Virginia and can hybridize (e.g., Douglass 1906), we first determined the species status of each tree at each site (as described above) to evaluate whether differences in defoliation were related to catalpa species. Morphological characteristics used to determine species included: bark texture, trunk branching, flowering time, number of seed pods per cluster, seed pod width, seed width and location of seed hairs (Scott 1911, Petrides & Wehr 1998). Only selected traits were used because others described by Scott (1911) and Petrides & Wehr (1998) overlap, e.g., seed pod length (6-18 in for C. bignonioides, 7-20 in for C. speciosa), and others, such as leaf shape

(leaves vary in shape and size) and the presence of notched flower petals, were difficult to define. Seed pod and seed-related traits were not assessed for all trees at the at Tyson site due to

lack of fruit production over multiple years; as reported in Stephenson (1980, 1982), catalpa trees attacked by the catalpa sphinx caterpillar will abort fruits on heavily infested branches.

Caterpillar defoliation levels were quantified for catalpa trees at each site from late July through October over three consecutive years. In 2014, percent defoliation of each tree was visually estimated more definitively every two weeks and these biweekly observations were used to determine the most consistent estimate of percent defoliation for each tree during the 2014 growing season. Percent defoliation and % dry weight of total iridoid glycosides were compared using linear regression in JMP v11 (SAS Institute). Percent defoliation and % dry weight of catalpol or catalposide were compared using separate linear regressions in JMP v11 (SAS

Institute).

Iridoid glycoside analyses

Leaves were collected from 21 catalpa trees at the Tyson site (n=17) and three Richmond sites (n=4). Fifteen to twenty leaves of roughly the same size were collected from two opposite sides of each tree to account for leaf age and variability within an individual tree (typically,

North/South or East/West). Leaves were collected in the middle of June before catalpa caterpillar infestation. Shortly after collection, leaves were dried, crushed, and sent to Dr. M Deane Bowers at the University of Colorado for gas chromatograph analyses of iridoid glycosides.

Gas chromatography was performed as described in Bowers and Stamp (1993) and

Bowers (2003). Briefly, portions of dried, powdered leaf samples (25 mg per sample) were extracted in 95% methanol overnight. Solid material was filtered out, and the methanol was evaporated. Phenyl-β-D-glucopyranoside (used as an internal standard) was added to each sample. Water and diethyl ether were added to separate out hydrophobic compounds, then the

water was evaporated and the ether layer removed. The leftover residue was suspended in methanol. Before evaporating the methanol, some of the residue was removed. The residue was derivitized using Tri-Syl Z™ (Pierce Chemical Company, Rockford, IL, USA) in pyridine before being injected into the gas chromatograph. All leaf samples were analyzed for both milligrams of catalpol and catalposide and % dry weight was calculated (milligrams catalpol or catalposide x

100/mg sample weight).

To investigate whether iridoid glycoside levels are related to tree age, the circumference

(cm) of each tree was measured 1 m above the ground. The relationship between tree age and % dry weight of total iridoid glycosides, catalpol, and catalposide were analyzed using separate linear regressions in JMP v11 (SAS Institute).

Plants & Insects Trees with particularly high or low levels of iridoid glycosides were chosen for seeds to grow seedlings for the behavioral experiments described. Catalpa seeds were planted in a pesticide free greenhouse in mid–May 2013. Seeds were collected from the Monument Ave. tree and 30th street corner tree in the City of Richmond (low levels of iridoid glycosides) and Tree A at the Tyson site (high levels of iridoid glycosides). Catalpa trees were fertilized every two months with Osmocote® outdoor/indoor plant food. Trees used in the oviposition assay described below were 1.5 years old.

First instar catalpa sphinx caterpillars were collected from mid-July to early October at the Tyson site to provide unparasitized caterpillars to rear out for oviposition work. Larvae were randomly assigned to a maintenance diet of leaves collected from Tree O at the Tyson site or one time from the 30th St. side tree (high levels of iridoid glycosides) or the Monument tree (low levels of iridoid glycosides). Third and fourth instar caterpillars were collected from early

August to late October to rear out C. congregata, which heavily parasitizes the second generation of the catalpa sphinx caterpillar. Unparasitized larvae were reared through pupation and also used in oviposition preference studies. Wild wasps were raised in the laboratory through

Manduca sexta L. (Sphingidae) for one generation to provide an adequate number of wasps for experimental assays.

Moth Oviposition Assay

Moth oviposition preference for trees with high or low iridoid glycoside levels was determined using a choice assay. Moth pupae were kept in netted wire mesh collapsible cages

(30.5 cm x 30.5 cm x 30.5 cm) inside a growth chamber set at 28º C (16:8 light-dark cycle) to mimic the mid-summer in Virginia when these moths are out in the field. As the moths emerged, they were placed in another netted wire mesh cage with moths of the same age and given 24 hours to mate. Catalpa trees (1.5 years old), one with high iridoid glycosides and the other with low iridoid glycosides were placed in a netted PVC pipe enclosure (1.83 m x 1.83 m x 0.91 m) inside the greenhouse.

Male and female moths were placed into the experimental enclosure to further encourage mating; the total number of moths varied from 10 (5 males to 5 females) to 20 (10 males to 10 females). Females were identified by their thicker abdomens, thinner antennae and the lack of certain male specific sensory receptors found on the antennae (Messenger 1997). Equal numbers of males and females were placed on the base of each tree. Multiple moths were used to mimic the natural environment in which moths likely emerge around the same time at the base of the same catalpa tree. Preliminary experiments using only one male and one female or small numbers of females did not result in successful mating or any oviposition.

Females were allowed 12 hours overnight (8 pm to 8 am) for oviposition. The following morning, all moths were removed from the enclosure and returned to the mesh collapsible cage.

The location of the high and low iridoid glycoside trees was reversed between each trial. The number of egg masses and total number of eggs oviposited on each tree or in each corresponding area of the enclosure were counted. Moths were 2-3 days old when used in the oviposition assays. Oviposition assays were a blind assay because in 2011, seedlings were chosen based on infestation level of the tree that was their seed source. The total number of eggs oviposited and number of egg masses on high and low iridoid glycoside trees in both the 2011 and 2014 oviposition trials were compared using separate paired t-tests in JMP v11 (SAS Institute).

Caterpillar Feeding Assay

A leaf disc choice assay developed by Jermy et al. (1968) and modified by deBoer &

Hanson (1984) was used to determine whether catalpa sphinx caterpillars preferred to feed on leaves with high or low iridoid glycoside levels. To control for potential confounding effects of previous feeding experience, first and second instar larvae collected from the field were placed in plastic shoebox containers and randomly assigned to a consistent diet of high or low iridoid glycoside leaves. Leaves fed to experimental caterpillars were collected from Tree O at the

Tyson site which was found to have a high level of iridoid glycosides in the iridoid glycoside analyses or the Monument Ave. tree that had a low level of iridoid glycosides and was easily accessible (see “Plants & Insects”). Leaves were stored in the refrigerator to ensure freshness.

Caterpillars were raised on each leaf diet until they reached the 3rd or 4th instar. As determined in preliminary feeding assays, newly molted 3rd or 4th instar larvae were starved for approximately

4 hours to allow time for their mandibles to harden and become hungry before being placed in the feeding arena.

Caterpillars were offered two high and two low iridoid glycoside leaf discs. Leaf discs

(12.7 mm in diameter) were cut, using a cork borer, from leaves from high or low iridoid glycoside trees grown in the greenhouse and arranged in an alternating pattern inside a closed foam bottomed petri dish (9 cm in diameter) (Figure 1). Foam was used as the substrate at the bottom of the petri dish because it retained moisture better than filter paper and it did not impede caterpillar movement or feeding in preliminary assays. At the start of the experiment, a single caterpillar was placed in the center of the arena and allowed approximately two minutes to adjust. Caterpillars were observed continuously over 2 hours; after 2 hours leaf discs began to dry out. Once a 3rd instar caterpillar had eaten 25% of the total leaf area the overall choice was recorded. Fourth instar caterpillars were assayed the same as the third instars except an overall choice was not recorded until 50% of the leaf area was eaten. This was a blind assay in that the exact levels of iridoid glycosides present in each leaf disc were not known at the time of the assay. Because parasitization has been reported to induce behavioral changes (Fritz 1982), caterpillars were reared out individually to determine parasitization status and whether parasitization status would affect feeding preferences. Feeding choices of parasitized and unparasitized caterpillars were compared using a Fisher’s Exact test performed in JMP v11 (SAS

Institute). If parasitization status was found to affect feeding preferences then data for parasitized and unparasitized caterpillars would be analyzed separately and if not data would be combined.

Feeding choices for 3rd and 4th instar caterpillars were compared using separate Fisher’s Exact tests in JMP v11 (SAS Institute). Caterpillars that fed on less than 15% of the total leaf area were excluded from the analyses, because an overall choice could not be visually discerned.

Wasp Searching Assay

Searching time of presumably mated females of C. congregata on catalpa leaves with high or low iridoid glycosides was quantified using a no choice searching assay (Kester &

Barbosa 1991). Searching times of both wild wasps from the Tyson site (Parent and F1 generation) and colony wasps (F9 generation) collected from the Tyson site the previous year and reared through Manduca sexta feeding on artificial diet in the laboratory. Caterpillars with parasitoid cocoons were isolated into individual plastic cups (4 x 7.5 cm, 4 oz.) to await parasitoid emergence. When the parasitoids were close to emergence, a kimwipe was torn and placed in their environment for four hours, to provide a substrate after emergence. Normally, wasps emerge on a plant surface and have their hosts’ plant as a substrate after emergence. Then, all wasps from multiple broods were placed in a plastic container (11 x 18 cm) with honey agar and water, and left undisturbed for 48 hours. After 48 hours, female wasps were isolated and individually transferred to 1-dram glass vials. Each female was given 2 to 5 minutes to adjust to the vial before being assayed. While the wasps adjusted, leaf discs were cut (12.7 mm in diameter) from one side of the midvein of a high or low iridoid glycoside leaf using a cork borer and placed on a moist piece of filter paper to ensure freshness prior to the assay. For wild wasp assays, leaf discs were also cut from the opposite side of the midvein, so that we could perform iridoid glycoside level comparison to the leaf disc used in the assay. The leaf discs not used in the wild searching assay were dried and frozen in a glassine envelope for iridoid glycoside analysis.

Each female was given either a high iridoid glycoside leaf disc or a low iridoid glycoside leaf disc and allowed to search for 3 minutes. Searching was defined by antennal palpation on the leaf disc and measured with a digital stopwatch. Each female was only used once. Assays

were conducted on a rolling basis over multiple days from 10 am to 3 pm, when wasps are most active (Kester pers. obs.). Also, because searching times may be affected by environmental variables, all assays were performed at 26 ± 4°C, 60 ± 6% relative humidity, and at an atmospheric pressure above 29.1 (inHg). Searching data were transformed by (square root

(searching time + 0.5)) to meet assumptions for parametric analysis. Searching times on high and low iridoid glycoside leaf discs between colony and wild wasps were compared using a two-way

ANOVA and a Tukey HSD post hoc test in JMP v11 (SAS Institute). Searching times on high and low iridoid glycoside leaf discs of colony wasps and wild wasps were compared using separate two-sample independent t-tests in JMP v11 (SAS Institute).

RESULTS

Catalpa observations

Surveys of the morphological characteristics of catalpa trees at the Richmond sites did not display characteristics allowing us to identify them as distinctly Catalpa bignonioides or

Catalpa speciosa and may be hybrid varieties of the two species (Table 1). The Tyson site trees have traits described for the Southern catalpa (C. bignonioides) (Table 1). The majority of the trees located at the Richmond, VA sites have traits described for both northern (C. speciosa) and southern (C. bignonioides) catalpa (Table 1).

Trees with low iridoid glycoside levels in the City of Richmond (Monument Ave. and the

30th street corner) have not experienced herbivory by the catalpa sphinx for at least the past decade (Kester pers. obs.). At the Tyson site in Cumberland County, trees with high levels of iridoid glycosides tended to have caterpillars first and had more caterpillars over the season

(Bray pers. obs.). Trees with high levels of iridoid glycosides were more likely to be completely

defoliated each season (Bray pers. obs.). Trees with low iridoid glycoside levels at the Tyson site received fewer caterpillars than the high iridoid glycoside trees. Trees in the City of Richmond with high iridoid glycoside levels (30th street along roadside and Lakeside), have experienced infestation over the past decade, but for unknown reasons have not had caterpillars in the past two years.

Iridoid glycoside analyses

Total iridoid glycoside levels varied both within and among sites (Figure 2). Catalposide levels tended to be higher than catalpol levels in the majority of the trees (Figure 2). There was no relationship between % dry weight of catalpol and catalposide (r2=2.11e-5, P=0.9842). Tree age was not related to % dry weight of total iridoid glycosides in leaf samples (r2=0.07,

P=0.2397). Tree age was also not related to % dry weight catalpol (r2=0.002, P=0.8462) or % dry weight catalposide in the leaf samples (r2=0.14, P=0.1017).

Within the Tyson site there was a relationship between percent defoliation and % dry weight of total iridoid glycosides (r2=0.39, P=0.0071) (Figure 3). Percent defoliation and % dry weight catalposide were also significantly related (r2=0.39, P=0.0067) (Figure 3). There was no significant relationship between percent defoliation and % dry weight catalpol (r2=0.08,

P=0.2655) (Figure 3).

Moth Oviposition Assay

Moths significantly preferred to oviposit on high iridoid glycoside trees in 2011 but apparently not in 2014 (Figure 4). In 2011, both the number of total eggs oviposited on high iridoid glycoside trees across 6 trials (t=-4.19, df=41, P=0.0001) and the total number of egg

masses oviposited on high iridoid glycoside trees across six trials (t= -5.18, df=10, P=0.0004) was significant (Figure 4) In 2014, moths oviposited more eggs on high (235 eggs) than on low

(44 eggs) iridoid glycoside trees, but these differences were not significant (t=1.08, df =5,

P=0.3261) (Figure 4). The total number of eggs oviposited in 2014 (279 eggs) was much lower than the total number of eggs oviposited in 2011 (1762 eggs).

Caterpillar Feeding Assay

Parasitization status of third instar caterpillars did not affect feeding preferences (Fisher’s

Exact, n=19 vs. 22, P=0.9248), therefore, data from both parasitized and unparasitized caterpillars were combined for the 3rd instar feeding preference assays performed. Regardless of pre-assay treatment, neither 3rd (Fisher’s Exact, n=20 vs. 30, P=0.2361) nor 4th (Fisher’s Exact, n=15 vs. 30, P=0.9146) instar caterpillars displayed a preference for high or low iridoid glycoside leaf discs (Figure 5). Both 3rd and 4th instar caterpillars often moved around the feeding arena exploring and tasting the different leaf discs before deciding on which to feed

(Bray pers. obs.). Third instars were more likely to move from disc to disc without finishing a full disc before moving to another, whereas, fourth instars completed eating a disc before moving to another (Bray pers. obs.).

Wasp Searching Assay

Wild wasps (Parent/F1 generation) and colony wasps (F9 generation) differed in their searching responses to catalpa leaf discs (F=10.99, df=3, P < 0.001, Table 2), but did not differ in responses to high and low iridoid glycoside level leaf discs within each wasp source (Figure 6).

Colony and wild wasps differed in their searching times to high iridoid glycoside level leaf discs

(P < 0.001), but not to low iridoid glycoside level leaf discs (P=0.3062). Neither colony wasps

(t= -1.26, df=65, P=0.2086) nor wild wasps (t=1.85, df=42, P=0.0702) displayed a difference in searching responses to high and low iridoid glycoside leaf discs (Figure 6).

Pressure, relative humidity and temperature were not related to searching times of colony wasps (Pressure, r2=0.006, P=0.5114; RH, r2=0.005, P=0.5525; Temp., r2=0.003, P=0.6150). For wild wasps, both relative humidity and temperature were not related to searching times (RH, r2=0.079, P=0.0631; Temp., r2=0.032, P=0.2417), however, pressure was positively related to searching times (r2=0.286, P=0.0002).

DISCUSSION

This study is the first to investigate the relationship between defoliation and levels of iridoid glycosides in Catalpa trees. Long term field observations indicate that certain catalpa trees are heavily infested and defoliated by the catalpa sphinx caterpillar, Ceratomia catalpae, while other trees escape herbivory. Likewise, some populations of catalpa sphinx larvae are heavily parasitized by the braconid wasp, Cotesia congregata, while other populations escape parasitism. Results of this study demonstrate that the observed patterns of herbivory are best explained by moth ovipositional preferences for trees with relatively high levels of iridoid glycosides. In contrast, variability in parasitism among populations cannot be explained by levels of iridoid glycosides.

Percent dry weight of the total and major iridoid glycosides of catalpa, catalpol and catalposide, varied both among sites and within the Tyson site (Figure 2). Trees that suffered heavy defoliation by catalpa sphinx caterpillars had relatively high levels of iridoid glycosides and trees with no or low levels of defoliation had low levels of iridoid glycosides (Figure 3). Of

the two major iridoid glycosides, only % dry weight of catalposide was significantly related to percent defoliation (Figure 3). This indicates that levels of catalposide may be higher than those of catalpol in trees that are heavily infested and defoliated each year. Percent dry weight of total iridoid glycoside levels did not vary with respect to tree age (r2=0.07, P=0.2397, n=21 trees). An examination of morphological traits revealed that the 23 trees at the Tyson site (Cumberland

County, VA) that originated from a single seed source are clearly C. bignonioides (southern catalpa) and the four trees at three urban sites in Richmond, VA are likely hybrids of C. bignonioides and C. speciosa (northern catalpa) (Table 1).

Results from behavioral experiments indicate that observed variability in infestation levels among trees is likely due to moth oviposition preferences for trees with relatively high levels of iridoid glycosides (Figure 4). Moths displayed a strong preference for high iridoid glycoside trees in the 2011 greenhouse oviposition assay; however, this was not apparent in the

2014 assays. This discrepancy is likely due to a malfunctioning humidity control unit in 2014 which resulted in suboptimal levels of humidity and more unsuccessful trials. Moth mating and oviposition in Manduca sexta L. (Lepidoptera: Sphingidae) are reduced in low humidity environments (Baumhover et al. 1977), this is likely the same for the catalpa sphinx. Oviposition on high iridoid glycoside level trees likely gives the offspring feeding on those trees potential fitness benefits because they sequester one of these iridoid glycosides, catalpol, for defense.

Another potential explanation for moth oviposition preference for trees with high levels of iridoid glycosides is that there are developmental benefits for the offspring. Higher levels of iridoid glycosides increase larval weight and pupal size, and decrease development time in a few species (Harvey et al. 2005, Saastamoinen et al. 2007). Future studies of catalpa caterpillar

development while feeding on high or low iridoid glycoside level leaves may provide insight into fitness benefits for the caterpillar other than defense against natural enemies.

Despite potential benefits of feeding on high iridoid glycosides, mid-instar catalpa sphinx larvae did not prefer to feed on leaf discs with high (or low) levels of iridoid glycosides (Figure

5). Parasitized and unparasitized caterpillars did not differ in their feeding responses (Fisher’s

Exact, P=0.9248, n=19 parasitized vs. 22 unparasitized). The catalpa sphinx moth, which does not retain sequestered iridoid glycosides (Bowers & Puttick 1986), is palatable to birds (Bowers

& Farley 1990); however the palatability of larvae has not been tested. Studies with other iridoid glycoside-sequestering species demonstrate that some birds and ants reject feeding on larvae or adults that sequester these compounds (Bowers 1980, Belofsky et al. 1989, Bowers & Farley

1990, Dyer & Bowers 1996, Ness 2003). For example, ants reject J. coenia larvae with high levels of iridoid glycosides (Dyer & Bowers 1996).

Cotesia congregata females did not differ in their inherent searching responses to high or low iridoid glycoside leaf discs (Figure 6). This suggests that C. congregata may use plant chemicals other than iridoid glycosides to locate catalpa sphinx caterpillars. In any case, results do not offer insight into why some populations of catalpa sphinx caterpillars are heavily parasitized and others do not experience parasitism. What we do know is that populations of C. congregata originating from the catalpa sphinx are more highly inbred than those from M. sexta

(Kester et al., in press), suggesting that catalpa populations may be more localized and therefore more susceptible to local extinction. Future studies should consider the effects of prior catalpa exposure on orientation responses to the complete host-plant complex.

Ongoing work analyzing the levels of iridoid glycosides in archived leaf discs used in this study will allow for more robust statistical analyses of caterpillar feeding preferences and wasp

searching responses to iridoid glycoside levels. Future studies examining moth oviposition preference for trees with and without catalpa sphinx damage, may aid in understanding why some trees are defoliated multiple times within a single season. Understanding chemical variation in hostplants within and among populations is essential for explaining observed patterns of herbivory and parasitism, especially for chemical sequestering species.

ACKNOWLEDGMENTS

I thank Dr. M. Deane Bowers and her laboratory for analyzing the leaf samples; many Bug lab interns that assisted with insect rearing. This research was funded by the Thomas F. Jeffress and

Kate Miller Jeffress Memorial Trust to KMK.

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Table 1: Species characteristics of four catalpa trees (Catalpa) located in Richmond, VA and a group of trees from the Tyson site in Cumberland County, VA (from the same seed source). The southern catalpa, Catalpa bignonioides, (S) and the northern catalpa, Catalpa speciosa, (N) characteristics are marked with and X for each tree. Traits as described in Douglass 1906, Scott 1911, and Petrides & Wehr 1998.

Table 2: Results of the two- way ANOVA examining the effect of wasp source (colony vs. wild) and plant assayed (high vs. low levels of iridoid glycosides in leaves) on wasp searching times. Source of variation df F P Plant 1 0.81 0.3697 Wasp source 1 24.39 < 0.001 Plant*Wasp source 1 5.52 0.0205 Error 107 Total 110

Figure 1: Feeding arena setup for the catalpa caterpillar (Ceratomia catalpae) feeding preference assay. Two high (H) and two low (L) iridoid glycosides leaf discs from catalpa (Catalpa) were placed in an alternating pattern in a closed foam bottomed petri dish. Each caterpillar was placed in the center of the arena at the start of the assay.

A) Total Iridoid glycosides

B) Catalpol

C) Catalposide

Figure 2: A) Percent dry weight of total iridoid glycosides in 21 catalpa trees of interest in Richmond, VA (n=4) and the Tyson site in Cumberland Co. VA (n=17). B) Percent dry weight of catalpol in 21 catalpa trees of interest in Richmond, VA (n=4) and the Tyson site in Cumberland Co. VA (n=17). C) Percent dry weight catalposide in the 21 catalpa trees of interest in the City of Richmond, VA (n=4) and the Tyson site in Cumberland Co. VA (n=17). For A, B, and C zeros indicate trees that have experienced no herbivory by catalpa sphinx larvae (Ceratomia catalpae) over the past decade. Asterisks indicate trees that are heavily infested and completely defoliated by catalpa sphinx larvae.

A) Total Iridoid glycosides

B) Catalpol

C) Catalposide

Figure 3: A) Percent dry weight of total iridoid glycosides of 17 catalpa trees at the Tyson site in Cumberland County, VA was significantly related to percentage of the tree defoliated by the catalpa sphinx caterpillar, Ceratomia catalpae (r2=0.39, P=0.0071). B) Percent dry weight catalpol of Tyson site trees was not significantly related to percent defoliation by the catalpa sphinx caterpillar (r2=0.08, P=0.2655). C) Percent dry weight catalposide of Tyson site trees was significantly related to percent defoliation by the catalpa sphinx caterpillar (r2=0.39, P=0.0067).

Figure 4: A) In 2011, catalpa sphinx moths (Ceratomia catalpae) oviposited more egg masses (P=0.0004) and more total eggs overall (6 trials; P=0.0001) on high iridoid glycoside (IG) trees but in B) 2014 the total eggs oviposited on high iridoid glycoside (IG) trees did not differ (3 trials; P=0.3261) from the total eggs oviposited on low iridoid glycoside trees. (n = the total number of egg masses oviposited on each tree over all trials).

Figure 5: Regardless of pre-assay feeding treatment, A) 3rd instars catalpa sphinx larvae (Ceratomia catalpae) did not display a preference for high or low iridoid glycoside (IG) leaf discs (n=20 vs. 30, P=0.2361), nor did B) 4th instars (n=15 vs. 30, P=0.9146).

Figure 6: Searching responses of A) colony wasps (Cotesia congregata) to high or low iridoid glycoside (IG) leaf discs (mean ± SE) did not differ (n=32 vs. 35, P=0.2086) nor did searching responses of B) wild wasps (C. congregata) (n=19 vs. 25, P=0.0702).

Vita

Jessica Lauren Bray was born in Mechanicsville, VA on August 6, 1990. She graduated from Atlee High School in Mechanicsville in 2008. In 2012, she graduated from Longwood

University with a B.S. in Biology. From 2010 to 2012 she worked on a research project with Dr.

Mary Lehman and Dr. Amanda Lentz-Ronning investigating the feeding and ovipositional preferences of Pieris rapae. She began her M.S. in Biology in 2012 at Virginia Commonwealth

University in the Insect Ecology & Behavior Lab. Jessica enjoys being outside hiking and looking for insects. Jessica has several pets, mostly various insects and spiders as well as a leopard gecko named Arya. Jessica will remain at VCU through the summer and fall of 2015 to continue research and teach Entomology Lab and Invertebrate Zoology Lab. Afterwards, she plans to pursue a career in teaching at the high school or community college level.