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Antifeedant Effect of Commercial Chemicals and Plant Extracts Against Schistocerca americana (Orthoptera

Permanent Link: http://ufdc.ufl.edu/UFE0024748/00001

Material Information

Title: Antifeedant Effect of Commercial Chemicals and Plant Extracts Against Schistocerca americana (Orthoptera Acrididae) and Diaprepes abbreviatus (Coleoptera: Curculionidae)
Physical Description: 1 online resource (64 p.)
Language: english
Creator: Sandoval Mojica, Andres
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: antifeedants, ardisia, azadirachtin, ceratiola, diaprepes, illicium, rotenone, ryanodine, sabadilla, schistocerca
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: ANTIFEEDANT EFFECT OF COMMERCIAL CHEMICALS AND PLANT EXTRACTS AGAINST Schistocerca americana (ORTHOPTERA: ACRIDIDAE) AND Diaprepes abbreviatus (COLEOPTERA: CURCULIONIDAE) By Andres Felipe Sandoval Mojica August 2009 Chair: John Capinera Major: Entomology and Nematology I investigated the deterrent effect of seven botanical and three inorganic agricultural products against nymphs of the American bird grasshopper, Schistocerca americana, and adults of the sugarcane rootstock weevil, Diaprepes abbreviatus. Methanol and methylene chloride extracts of the Florida rosemary, Ceratiola ericoides, yellow star anise, Illicium parviflorum, and scratchthroat, Ardisia crenata, were also tested as potential feeding deterrents. Antifeedant activity was assayed using a leaf disk bioassay, in choice and no-choice tests. The residual activity of the agricultural products that showed a significant antifeedant activity in leaf disk bioassays was assayed by applying them in a no-choice test to foliage of Citrus paradisi plants exposed to three time intervals of sunlight. Sabadilla, azadirachtin and ryanodine effectively deterred S. americana whereas rotenone, sabadilla and ryanodine reduced the feeding activity of D. abbreviatus in choice and no-choice leaf disk bioassays. Rapid loss of effectiveness was observed under field conditions. Sabadilla was the only compound that maintained its antifeedant properties in the field, but only against S. americana. Methanol and methylene chloride extracts of C. ericoides deterred D. abbreviatus but only methylene chloride extract dissuaded S. americana. Methanol extract of A. crenata functioned as a feeding deterrent against both S. americana and D. abbreviatus, whose was also deterred by methylene chloride extract of A. crenata. Extracts of I. parvifolium only dissuaded the insects in choice bioassays. Based on their deterrency, some of the agricultural botanical products and plant extracts have potential for use as substitute crop protectants against these two species.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Andres Sandoval Mojica.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Capinera, John L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024748:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024748/00001

Material Information

Title: Antifeedant Effect of Commercial Chemicals and Plant Extracts Against Schistocerca americana (Orthoptera Acrididae) and Diaprepes abbreviatus (Coleoptera: Curculionidae)
Physical Description: 1 online resource (64 p.)
Language: english
Creator: Sandoval Mojica, Andres
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: antifeedants, ardisia, azadirachtin, ceratiola, diaprepes, illicium, rotenone, ryanodine, sabadilla, schistocerca
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: ANTIFEEDANT EFFECT OF COMMERCIAL CHEMICALS AND PLANT EXTRACTS AGAINST Schistocerca americana (ORTHOPTERA: ACRIDIDAE) AND Diaprepes abbreviatus (COLEOPTERA: CURCULIONIDAE) By Andres Felipe Sandoval Mojica August 2009 Chair: John Capinera Major: Entomology and Nematology I investigated the deterrent effect of seven botanical and three inorganic agricultural products against nymphs of the American bird grasshopper, Schistocerca americana, and adults of the sugarcane rootstock weevil, Diaprepes abbreviatus. Methanol and methylene chloride extracts of the Florida rosemary, Ceratiola ericoides, yellow star anise, Illicium parviflorum, and scratchthroat, Ardisia crenata, were also tested as potential feeding deterrents. Antifeedant activity was assayed using a leaf disk bioassay, in choice and no-choice tests. The residual activity of the agricultural products that showed a significant antifeedant activity in leaf disk bioassays was assayed by applying them in a no-choice test to foliage of Citrus paradisi plants exposed to three time intervals of sunlight. Sabadilla, azadirachtin and ryanodine effectively deterred S. americana whereas rotenone, sabadilla and ryanodine reduced the feeding activity of D. abbreviatus in choice and no-choice leaf disk bioassays. Rapid loss of effectiveness was observed under field conditions. Sabadilla was the only compound that maintained its antifeedant properties in the field, but only against S. americana. Methanol and methylene chloride extracts of C. ericoides deterred D. abbreviatus but only methylene chloride extract dissuaded S. americana. Methanol extract of A. crenata functioned as a feeding deterrent against both S. americana and D. abbreviatus, whose was also deterred by methylene chloride extract of A. crenata. Extracts of I. parvifolium only dissuaded the insects in choice bioassays. Based on their deterrency, some of the agricultural botanical products and plant extracts have potential for use as substitute crop protectants against these two species.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Andres Sandoval Mojica.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Capinera, John L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024748:00001


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5dabc1f9e528941179a261b3407b961fad357dfb







ANTIFEEDANT EFFECT OF COMMERCIAL CHEMICALS AND PLANT EXTRACTS
AGAINST Schistocerca americana (ORTHOPTERA: ACRIDIDAE) AND Diaprepes
abbreviatus (COLEOPTERA: CURCULIONIDAE)



















By

ANDRES FELIPE SANDOVAL MOJICA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2009


































2009 Andres Felipe Sandoval Mojica


































To my family, the source of my strength









ACKNOWLEDGMENTS

I thank my committee members Dr. John Capinera, Dr. Michael Scharf, and Dr. Heather

McAuslane for their advice, support and commitment to this study.









TABLE OF CONTENTS



A C K N O W L E D G M E N T S ............................................................................................... ............... 4

L IST O F T A B L E S ... .. ..................... .... ...................................................................... .............. ....... 7

L IST O F F IG U R E S ................................................................. 8

A B S T R A C T ..................................................................................................... 10

CHAPTER

1 IN T R O D U C T IO N ......................................................................................... 12

C conceptual Fram ew ork ...................................................... 12
Pest Insects...................... .................... ................ ........ 16
Com m ercial Chem icals Tested.................................... ............... 17
Plant Species Used To Obtain The Extracts ........................................................... ....20
O objectives .......... ............................................ 22

2 M ATERIALS AND M ETHODS ................................................. 23

In sect M material ............... .............................................................................................................. 2 3
C hem icals T ested ..................................................... 23
Plant E extracts T ested ...................................................... 24
B eh av ioral B io assay ................................................................................................. 2 4
Field Trial ......................... .................... 26

3 RESULTS ........................... .................... 29

Schistocerca am ericana ................... ............................................................... 29
Behavioral Bioassays: Commercial Formulations........................ ...........29
F field T ria l ................... .........................................................................................3 0
Behavioral Bioassays: Plant Extracts ................................ ................. 30
D iap rep es a bbrevia tus ............................................................................................................... 3 1
Behavioral Bioassays: Commercial Formulations ........................................... 31
Field Trial ............................ ....... ............................ ... 32
B behavioral B ioassays: Plant Extracts.................. ................... ........................................ 32

4 D ISCU SSION .......................... .......... ............... 46

Behavioral Bioassays: Commercial Formulations ......... .. ........... ......... ............... 46
F field T rial ........................... .... ..... ........ ............................................ ....... 52
Behavioral Bioassays: Plant Extracts......................................................................................... 52





5









R E F E R E N C E S ........................................ ................................................................................... 5 5

BIOGRAPHICAL SKETCH ......................................... 63









LIST OF TABLES

Table page

2-1 Chemicals evaluated for antifeedant activity against S. americana nymphs and D.
abbreviatus adults. .................................................... 28

2-2 Temperature (OC) profile of the days on which antifeedant's residual activity was
tested ......................................... ............ 28









LIST OF FIGURES


Figure p e

3-1 Total area consumed of untreated and treated leaf disks, by S. americana nymphs, in
choice bioassays ....... ........ .............................. 34

3-2 Total average area consumed of untreated and treated leaf disks, by S. americana
nymphs, in no-choice bioassays. .......................................................................... 35

3-3 Total leaf area eaten (cm2) by S. americana nymphs when exposed to the most
effective feeding deterrents in multiple-choice situations ............................................. 36

3-4 Consumption of azadirachtin treated and control citrus disks after three time intervals
of sunlight exposure on three trials .................................................. .............................. 36

3-5 Consumption ofryanodine treated and control citrus disks after three time intervals
of sunlight exposure on three trials .................................................. .............................. 37

3-6 Consumption of sabadilla treated and control citrus disks after three time intervals of
sunlight exposure on three trials..................................................... ............................ 37

3-7 Total area consumed of untreated and plant extracts-treated leaf disks, by S.
americana nymphs, in choice bioassays........................... ....................38

3-8 Total average area consumed of untreated and plant extracts-treated leaf disks, by S.
americana nymphs, in no-choice bioassays. .................. ....................................... 39

3-9 Total area consumed of untreated and treated leaf disks, by D. abbreviatus, in choice
bioassays. .......... ............................... ..................... 40

3-10 Total average area consumed of untreated and treated leaf disks, by D. abbreviatus
adults, in no-choice bioassays. ............................................... ........................................ 4 1

3-11 Total leaf area eaten by D. abbreviatus adults when exposed to the most effective
feeding deterrents in multiple-choice situations...................................... ...................... 42

3-12 Consumption ofrotenone treated and control citrus disks after three time intervals of
sunlight exposure on three trials..................................................... ............................ 42

3-13 Consumption ofryanodine treated and control citrus disks after three time intervals
of sunlight exposure on three trials. .......................................................................... ..... 43

3-14 Consumption of sabadilla treated and control citrus disks after three time intervals of
sunlight exposure on three trials..................................................... ............................ 43









3-15 Total area consumed of untreated and plant extracts-treated leaf disks, by D.
abbreviatus adults, in choice bioassays. ..................................................... ................44

3-16 Total average area consumed of untreated and plant extracts-treated leaf disks, by D.
abbreviatus adults, in no-choice bioassays ............................... 45









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ANTIFEEDANT EFFECT OF COMMERCIAL CHEMICALS AND PLANT EXTRACTS
AGAINST Schistocerca americana (ORTHOPTERA: ACRIDIDAE) AND Diaprepes
abbreviatus (COLEOPTERA: CURCULIONIDAE)

By

Andres Felipe Sandoval Mojica

August 2009

Chair: John Capinera
Major: Entomology and Nematology

I investigated the deterrent effect of seven botanical and three inorganic agricultural

products against nymphs of the American bird grasshopper, Schistocerca americana, and adults

of the sugarcane rootstock weevil, Diaprepes abbreviatus. Methanol and methylene chloride

extracts of the Florida rosemary, Ceratiola ericoides, yellow star anise, Illicium parviflorum, and

scratchthroat, Ardisia crenata, were also tested as potential feeding deterrents. Antifeedant

activity was assayed using a leaf disk bioassay, in choice and no-choice tests. The residual

activity of the agricultural products that showed a significant antifeedant activity in leaf disk

bioassays was assayed by applying them in a no-choice test to foliage of Citrus paradisi plants

exposed to three time intervals of sunlight. Sabadilla, azadirachtin and ryanodine effectively

deterred S. americana whereas rotenone, sabadilla and ryanodine reduced the feeding activity of

D. abbreviatus in choice and no-choice leaf disk bioassays. Rapid loss of effectiveness was

observed under field conditions. Sabadilla was the only compound that maintained its antifeedant

properties in the field, but only against S. americana. Methanol and methylene chloride extracts

of C. ericoides deterred D. abbreviatus but only methylene chloride extract dissuaded S.

americana. Methanol extract ofA. crenata functioned as a feeding deterrent against both S.









americana and D. abbreviatus, whose was also deterred by methylene chloride extract of A.

crenata. Extracts of parvifolium only dissuaded the insects in choice bioassays. Based on their

deterrency, some of the agricultural botanical products and plant extracts have potential for use

as substitute crop protectants against these two species.









CHAPTER 1
INTRODUCTION

Conceptual Framework

Feeding deterrents, or antifeedants, are chemical compounds that prevent or suspend the

feeding behavior of an insect when they are detected (Schoonhoven 1982). They can be found

amongst the major classes of secondary metabolites: alkaloids, terpenoids and phenolics (Koul

2008), but other organic and inorganic compounds can inhibit also food uptake by insects (Glen

et al. 1999, Wei et al. 2000). Insects are able to detect antifeedants through contact

chemoreceptors, characterized by the presence of a small number of bipolar sensory neurons

(three to 10) within short hairs, spines or bristles (sensilla trichoidea or sensilla chaetica), pegs

(sensilla basiconica), or cones (sensilla styloconica), with a single terminal pore. Sensory

(gustatory) neurons can be phagostimulatory or deterrent cells. Activity of these neurons in

response to appropriate stimuli would enhance or reduce feeding, respectively (Chapman 2003,

Koul 2008). Feeding deterrents may be perceived by insects due to several chemoreception

mechanisms (Schoonhoven 1982, Chapman et al. 1991):

1. Stimulation of specialized deterrent receptors

2. Distortion of the normal function of neurons that perceive phagostimulatory compounds.

3. Excitation of broad spectrum receptors.

4. Changes in complex and subtle sensory codes as a consequence of stimulation activity in
some nerve cells and inhibition in others.

5. Production of a highly abnormal impulse pattern, often at high frequencies, and/or bursting
activities.

Some antifeedants influence insect feeding activity through a combination of these modes

of action (Schoonhoven 1982, Chapman 2003, Koul 2008).









Reduction or complete inhibition of feeding, using organic derivatives, crude plant extracts

and/or pure allelochemicals as antifeedants, has been demonstrated in several orders such as

Lepidoptera, Coleoptera, Hemiptera and Orthoptera.

Regarding Lepidoptera, Akhtar and Isman (2004) observed antifeedant effects of crude

extracts of Melia volkensii (Meliaceae) on third instar larvae of the cabbage looper, Trichoplusia

ni Hubner (Noctuidae), and the armyworm, Pseudaletia unipuncta Haworth (Noctuidae).

Methanol, acetone and chloroform extracts ofJusticia adhatoda L. (Acanthaceae), Ageratum

conyzoides L. (Asteraceae) and Plumbago zeylanica L. (Plumbaginaceae), respectively, were

strongly deterrent to feeding of Spilarctia obliqua Walker (Noctuidae) at a dose of 10 mg/ml

(Prajapati et al. 2003). Ulrichs and collaborators (2008) detected antifeedant and toxic effects of

Porteresia coarctata Takeoka (Poaceae) hexane extracts on Spodoptera litura F. (Noctuidae)

third instar larvae due to the presence ofterpenoids in the plant. Meliaceae and Labiatae also

produce terpenes that modify the feeding behavior of insects: a mixture of limonoids, obtained

from seeds of Trichilia havanensis Jacq. (Meliaceae), and the diterpene scutecyprol A isolated

from Scutellaria valdiviana (Clos) Epling (Labiatae), reduced the feeding activity of Spodoptera

exigua (Hiibner) (Noctuidae) in choice and no choice bioassays (Caballero et al. 2008).

Azadirachtin, a triterpenoid obtained from the neem tree, Azadirachta indica A. Juss

(Meliaceae), reduced food consumption ofHeliothis virescens (F.) (Noctuidae) and Lymantria

dispar L. (Limantriidae) larvae (Yoshida and Toscano 1994, Kostic et al. 2008).

Concerning Coleoptera, extracts ofHumulus lupulus L. (Cannabaceae) and Xanthium

strumarium L. (Asteraceae) at a concentration of 20 g kg1, inhibited the feeding behavior of

Colorado potato beetle larvae, Leptinotarsa decemlineata Say (Gokce et al. 2006), which was

also deterred by ichangensin, a terpenoid (limonoid aglycone) present in the family Rutaceae









(Murray et al. 1999). Terpenes isolated from Lansium domesticum Corr. Serr. (Meliaceeae), as

well as crude extracts of the plant, showed antifeedant activity against the rice weevil Sitophilus

oryzae L. (Curculionidae) (Omar et al. 2007). Methanol extracts ofMomordica charantia L.

(Cucurbitaceae) strongly deterred cucurbitaceous feeding beetle species of the genus

Aulacophora and Epilachna (Abe and Matsuda 2000). Villani and Gould (1985), after screening

extracts from 78 plant species, discovered that Asclepias tuberosa L. (Asclepiadaceae) and

Hedera helix L. (Araliaceae) provided high levels of feeding deterrency against the corn

wireworm, Melanotus communis (Gyllenhal) (Elateridae).

Among the Hemiptera, the milkweed bug, Oncopeltusfasciatus Dallas (Lygaeidae), was

deterred from feeding by extracts of dried roots oflnula helenium L. (Asteraceae) when applied

to sunflower kernels (Alexenizer and Dorn 2007). Reuter and colleagues (1993), using an

electronic feeding monitor, observed a reduction in the time spent in ingestion by the green

peach aphid, Myzuspersicae (Sulzer) (Aphididae), when it was feeding on lettuce plants treated

with azadirachtin and an acrylic copolymer.

Within the Orthoptera, the graminivorous pest Locusta migratoria L. (Acrididae) is

deterred by crude methanol extracts of Cryptomeriajaponica (L. f) D. Don (Taxodiaceae)

(Kashiwagi et al. 2007), and also by azadirachtin, nicotine, coumarin and a mixture of phenolic

compounds obtained from Sorghum bicolor (L.) Moench (Poaceae) (Adams and Bernays 1978).

Gramine, 3-(dimethylaminomethyl)-indole, the principal alkaloid in barley, Hordeum vulgare L.

(Poaceae), inhibits as well the feeding behavior ofL. migratoria (Ishikawa and Kanke 2000).

Rathinasabapathi et al. (2007) observed that arsenic (As) present in the Chinese brake fern,

Pteris vittata L. (Pteridaceae), reduced frond tissue consumption by nymphs of the American

bird grasshopper, Schistocerca americana Drury (Acrididae).









Feeding deterrents can protect plants from insect herbivory by making the hosts less

palatable or even toxic to the arthropods. Therefore, antifeedants are a useful element for

integrated pest management programs that involve behavioral manipulation of insect pests, like

push-pull strategies where a protected plant resource is made unattractive or inappropriate to the

pests (push) while drawing them into an attractive source (pull) from where the pests are

subsequently removed (Jain and Tripathi 1993, Cook et al. 2007). Furthermore, antifeedants are

usually safer alternative crop protectants than conventional synthetic pesticides due to their low

toxicity, specificity, and protection to non-target organisms (insects, birds or mammals) that are

indispensable for interactions like natural biological control and pollination (Isman 2006).

Moreover, antifeedants are advantageous due to their effectiveness at small concentrations and

quick degradation. This can result in lower non-target hazard and reduced pollution problems

relative to conventional pesticides. Typical problems associated with conventional insecticides

include ground water contamination, the development of pesticide resistant pest populations, and

acute and chronic intoxication of applicators, farm workers and consumers (Isman 2006, Koul

2008, Rosell et al. 2008).

One of the weak points of investigations into the occurrence of antifeedant substances is

that in most cases only one test insect species is used. In most papers the structure of the

compounds) found to be active in laboratory tests on one or a few insect species is reported, but

no further attempts are made to find out against what other species the substance is active, or

how stable the compound is under natural conditions (Jermy 1990).

The purpose of the present study was to test the antifeedant effect of the botanical

derivatives azadirachtin, neem oil, sabadilla, rotenone, ryanodine, capsaicin and garlic juice, as

well as inorganic substances, such as diatomaceous earth, elemental sulfur and kaolin clay. Tests









were conducted under laboratory and field conditions, using behavioral bioassays, against

nymphs of the American bird grasshopper, Schistocerca americana (Drury) (Orthoptera:

Acrididae), and adults of the West Indian sugarcane rootstock borer weevil, Diaprepes

abbreviatus L. (Coleoptera: Curculionidae), two generalist herbivores. In addition, crude extracts

of the Florida rosemary, Ceratiola ericoides Michx (Empetraceae), yellow star anise, Illicium

parviJlorum Michaux ex Ventenat (Illiciaceae), and scratchthroat, Ardisia crenata Sims

(Myrsinaceae), were tested as potential antifeedants for both insect species.

Pest Insects

The American bird grasshopper, Schistocerca americana (Drury), is one of the most

destructive grasshopper species in the southeastern United States. It is found throughout the

eastern United States west to the Rocky Mountains. This grasshopper's range also includes the

Bahamas and Mexico (Thomas 1991). It occasionally reaches outbreak status, at least locally,

and can cause injury to corn, oats, rye, peanuts, sugarcane, tobacco, cotton, vegetables, flowers,

ornamental shrubs and citrus. The American grasshopper caused, during 1991, significant loss to

citrus and ornamental plants in west-central Florida and limited damage to field crops and

ornamentals in north-central Florida (Capinera 1993a). Young citrus trees are damaged by the

grasshopper's gnawing on the leaves and most of the feeding damage is caused by the third,

fourth and fifth instars, which have a much larger appetite than the adults. Although a highly

polyphagous acridid, S. americana does exhibit a clear preference (e.g., smooth crabgrass,

bahiagrass, hyssop spurge, soybean, corn, pecan) and non-preference (e. g., cucumber, nandina,

pepper) for certain host species (Capinera 1993a, Smith and Capinera 2005). The American

grasshopper has two generations per year and overwinters in the adult stage; therefore, it is

present throughout the year. Females lay their eggs in the soil, about 2 or 3 cm below the surface,

and are able to deposit up to three egg clusters (each cluster pod usually consist of 60 to 80









eggs). The groups of eggs are held together by proteinaceous foam which serves to cement the

surrounding soil particles together to form a more or less discrete capsule, or "pod". Nymphs

hatch from the eggs after three or four weeks and undergo between five and six molts before

reaching adulthood (Thomas 1991, Capinera 1993b).

The West Indian sugarcane rootstock borer weevil, Diaprepes abbreviatus L., is native to

the Lesser Antilles of the Caribbean region and has become an important long-term pest of citrus

and ornamental crops in Florida (Hall 1995). Since its introduction to Florida in 1964, D.

abbreviatus has spread into approximately 23,000 ha of commercial citrus in 17 counties and 94

commercial citrus and ornamental plant nurseries (Woodruff 1968, Hall 1995). It is a highly

polyphagous species; Simpson et al. (1996) listed numerous host plants associated with this pest,

including 270 species in 157 genera from about 59 families. Adult weevils feed on plant foliage,

leaving characteristic semicircular notches along the edges of young leaves, and oviposit on

preferred host plants. Females deposit clusters of eggs between leaves that are glued together by

secretion of a sticky substance that cements the leaf surfaces. Each cluster contains around 50

eggs, and female weevils are able to deposit as many as 5,000 to 29,000 eggs during their

lifespan (three to four months). As the eggs hatch, after seven to 10 days, the larvae fall to the

soil surface where they burrow into the soil to feed on the roots of the plant where they remain

for 8-12 months, until adult emergence (Woodruff 1968, Hall 1995, Mannion et al. 2003).

Commercial Chemicals Tested

Neem oil and azadirachtin are biopesticides obtained from the Indian neem tree,

Azadirachta indica A. Juss (Meliaceae). Neem oil is acquired by cold-pressing the seeds of the

neem tree and is useful in the control of soft-bodied insects (Bruce et al. 2004), mites and

phytopathogens due to the presence of disulfides that contribute to its bioactivity. Azadirachtin, a

limonoid or tetranortriterpenoid, is found in the extracts of the seed residues after removal of the









oil (Isman 2006). Several effects, both physiological and behavioral, have been reported on

insects (Schmutterer 1990): disruption of growth, development and reproduction by blocking the

synthesis and release of molting hormones, and repellency and oviposition deterrence.

Azadirachtin and neem seed extracts also seem to have a potent antifeedant effect on a large

number of pest insects, including orthopterans (Aerts and Mordue 1997, Capinera and Froeba

2007), hemipterans (Kumar and Poehling 2007), coleopterans (Baumler and Potter 2007), and

lepidopterans (Aerts and Mordue 1997, Yoshida and Toscano 1994, Liang et al. 2003, Showler et

al. 2004, Charlston et al. 2005).

Sabadilla is a botanical insecticide obtained from the seeds ofSchoenocaulon officinale

Grey (Liliaceae). The major active ingredients are cevadine and veratradine, which are esters of a

steroidal alkaloid named veracevine (Rosell et al. 2008). These alkaloids bind the activation gate

of the sodium channels in the nervous system of insects, resulting in persistent neuroexcitation.

Although sabadilla has a high mammalian toxicity, commercial preparations normally contain

less than 1% active ingredient, providing a margin of safety (Isman 2006). Sabadilla is used

mostly for control ofthrips populations in citrus and avocado (Hare and Morse 1997, Humeres

and Morse 2006) but it has potential as an antifungal agent (Oros and Ujvary 1999) and as a

feeding deterrent as well (Yoshida and Toscano 1994).

Rotenone is one of several isoflavonoid compounds produced in the roots of the tropical

legumes Derris, Lonchocarpus and Tephrosia (Leguminosae). Rotenone is a mitochondrial

poison that blocks the electron transport chain and prevents energy production. It has been used

as an insecticide, acaricide and piscicide and is commonly sold as a dust containing 1% to 5%

active ingredients for home and garden use, though liquid formulations used in organic

agriculture can contain between 8% and 15% (Isman 2006, Rosell et al. 2008). Rotenone can act









as a feeding deterrent against stored product insect pests (Nawrot et al. 1989) and polyphagous

noctuid species (Wheeler et al. 2001).

Ryanodine is an alkaloid obtained by grinding the wood of the Caribbean shrub Ryania

speciosa Vahl (Flacourtiaceae) (Isman 2006). This botanical pesticide is active on the muscular

system, specifically neuro-muscular calcium channels. Ryanodine acts as an agonist by

enhancing calcium output of the sarcosome tubule network that surrounds muscle fibers,

resulting in continual calcium availability and a continual state of muscle contraction (Nauen

2006). Ryanodine and related ryanoids deter feeding by lepidopteran and coleopteran pests

(Yoshida and Toscano 1994, Gonzalez-Coloma et al. 1999).

Capsaicin, extracted from the hot cayenne pepper, Capsicum annuum L. (Solanacaeae), is a

derivative of vanillyl amine (8-methyl-N-vanillyl-6-noneamide), a compound that produces the

"hotness" in some species of the plant genus Capsicum. Capsaicin is currently registered by the

US Environmental Protection Agency (EPA) for use as an insect repellent and toxicant as well as

a vertebrate repellent for dogs, birds, voles (Microtus spp), deer (Odocoileus spp), rabbits

(Sylvilagus spp.) and squirrels (Sciurus spp.) (EPA 1996). Capsaicin has been tested as a leaf

protector against scarab pests, including the rose chafer, Macrodactylus subspinosus (F.) (Isaacs

et al. 2004) and the Japanese beetle, Popillia japonica Newman (Baumler and Potter 2007) but

without consistent results.

Garlic extracts derived from Allium sativum L. (Liliaceae) have shown insecticidal activity

to dipteran pests (Prowse et al. 2006) as well as antifeedant effects on coleopteran stored-

products pests (Chiam et al. 1999) and moths (Gurusubramanian and Krishna 1996).

Diatomaceous earth is composed of the fossilized skeletons of various species of marine

and fresh water phytoplankton, predominantly diatoms and other siliceous algae which existed









during the Eocene and Miocene periods. The diatomaceous earth active ingredient is amorphous

silicon dioxide (silica), which can damage the insect's epicuticular lipids by hydrocarbon

absorption and abrasion, making the cuticle permeable and therefore causing death due to water

loss and desiccation (Korunic 1997). Diatomaceous earth has proved to be useful in the control

of stored-products insects such as Cryptolestesferrugineus Stephens (Laemophloeidae) (Fields

and Korunic 2000), Rhyzopertha dominica F. (Bostrichidae) (Ferizli and Beris 2005) and the

confused flour beetle, Tribolium confusum Jacquelin du Val (Tenebrionidae) (Dowdy and Fields

2002, Vayias et al. 2006).

Sulfur is a non-systemic contact and protectant fungicide with secondary acaricidal

activity. It is used primarily to control powdery mildews, certain rusts, leaf blights and fruit rots.

Spider mites, psyllids and thrips are also vulnerable to sulfur. This chemical is known to be of

low toxicity and poses little if any risk to human and animal health (Lamberth 2004).

Kaolin is a soft, white, clay mineral that can be mixed with water and sprayed on plants in

order to form a protective particle film. Kaolin is a nonabrasive aluminosilicate

(Al4Si4010(OH)8), that can reduce feeding and oviposition of arthropods by entangling

mouthparts and restraining mobility over treated plant surfaces (Glenn et al. 1999). Furthermore,

kaolin may interfere with insect's contact chemoreceptors, causing plants to be unrecognizable

as a host (Puterka et al. 2000). Spraying kaolin on crops has been effective against pests

belonging to different orders including Hemiptera (Glenn et al. 1999, Puterka et al. 2000, Cottrell

et al. 2002, Daniel et al. 2005), Coleoptera (Lapointe 2000) and Lepidoptera (Knight et al. 2000).

Plant Species Used To Obtain The Extracts

The Florida rosemary, Ceratiola ericoides Michx, is an indigenous plant restricted to the

Florida scrub community, growing on excessively to well-drained sandy soils. This plant is a

dioecious perennial evergreen shrub which can grow as tall as 2 m in height. Ceratiola ericoides









has a whorled branching pattern, with leaves strongly revolute (needle-like) of 8-12 mm

(Wunderlin and Hansen 2002). The absence of herbaceous growth around C. ericoides plants

demonstrates the allelopathic effects that this species exerts on others. Examples include

suppressing germination ofEryngium cuneifolium Small (Apiaceae) and Hypericum cumulicola

(Small) P. Adams (Hypericaceae) by leaf and litter leachates (Hunter and Menges 2002), and

affecting radicle growth and germination of sandhill grasses such as little bluestem,

Schizachyrium scoparium (Michx.) Nash (Poaceae) and green sprangletop, Leptochloa dubia

(Kunth) Ness (Poaceae), (Fischer et al. 1994). Several chemicals have been isolated from the

Florida rosemary including the dihydrochalcone flavonoid ceratiolin (Tanrisever et al. 1987, Tak

et al. 1993) which seems to be the precursor of the photochemically activated hydrocinnamic

acid, a germination and growth inhibitor of grasses and pines (Fischer et al. 1994).

Illiciumparviflorum Michaux ex Ventenat, commonly known as swamp star anise, or

yellow star anise, is an indigenous species of moist forests and swamps of central Florida. It is a

broadleaf evergreen large shrub or small tree with highly aromatic anise-scented foliage that can

grow up to 15 feet in height (Osorio 2001). Illiciumparviflorum is used in landscapes because of

its considerable drought tolerance, capacity to grow under a broad range of light conditions, and

resistance to pests. Sharma and Rich (2005) assessed reproduction of three root-knot nematode

species (Meloidogyne arenaria, M. incognita and M. javanica) on five native plants and three

non-native plants to the southeastern USA and observed very few or no galls on roots ofl.

parvifolium. Secondary metabolites such as sesquiterpene lactones (Schmidt 1999) have been

isolated from leaves of parviflorum, as well as safrole (68.14 0.88%), linalool (13.18

1.01%) and methyl eugenol (11.89 0.87%), the main components of the essential oil of yellow

anise tree (Tucker and Maciarello 1999).









Ardisia crenata Sims, or scratchthroat, is an evergreen small shrub (0.5-1 m in height)

native to Japan to north India that grows in multi-stemmed clumps. It has alternate leaves (dark

green above, waxy, glabrous with crenate margins) of 21 mm (Wunderlin and Hansen 2002).

Ardisia crenata was introduced to the USA for ornamental purposes and has become established

in much of northern and central Florida. In some areas, it is a serious pest, displacing native

species in the understory of hardwood forests by creating dense local shade (Kitajima et al.

2006). Several studies have revealed the presence, in the genus Ardisia, of phytochemicals such

as triterpenoid saponins, isocoumarins, quinones and alkylphenols (Kobayashi and de Mejia

2004, Liu et al 2007) that exhibit a wide range of bioactivities such as uterus contraction,

inhibition of cyclic adenosine monophosphate phosphodiesterase, cytotoxicity, anti-HIV and

anti-cancer among others (Kobayashi and de Mejia 2004). The presence of these secondary

metabolites may cause A. crenata to be an unsuitable host for arthropod feeders. For example,

Neal and colleagues (1998) observed a reduction in the number of eggs laid by the twospotted

spider mite, Tetranychus urticae Koch, as well as a higher nymphal mortality of the whiteflies

Bemisia argentifolii Bellows & Perring and Trialeurodes vaporariorum (Westwood) when

developing on A. crenata leaves, compared with other three host plants of the same genus.

Objectives

* To test the antifeedant properties of azadirachtin, neem oil, sabadilla, rotenone, ryanodine,
capsaicin, garlic juice, diatomaceous earth, elemental sulfur and kaolin clay against fifth
instar nymphs of S. americana and adults ofD. abbreviatus using behavioral bioassays.

* To test the residual activity under field conditions of the commercial formulations that are
the most deterrent to the feeding of the two species of insects in the laboratory.

* To obtain crude extracts from C. ericoides, I. parviflorum, and A. crenata plants, and to test
their antifeedant properties against nymphs of S. americana and adults ofD. abbreviatus
using behavioral bioassays.









CHAPTER 2
MATERIALS AND METHODS

Insect Material

The American grasshoppers used in this study were from a laboratory colony that has been

maintained for approximately 10 years in the Entomology and Nematology Department at the

University of Florida. The insects had free access to water and a dry diet consisting of whole

wheat flour (one part), soy flour (one part) and wheat bran (two parts), supplemented with

romaine lettuce, Lactuca sativa var. longifolia Lam, (Asteraceae). The nymphs and adults were

maintained at 270C, although they had access to 90 W light bulbs, approximately 10 cm away

from the cages, so they could attain a warmer temperature if desired. They also were provided

with a photoperiod of 16:8 (L:D) and a relative humidity of about 58%.

Adult sugarcane rootstock borer weevils were from a rearing facility of the Florida

Department of Agriculture & Consumer Services, Division of Plant Industry (DPI), at

Gainesville, Florida, where they had been maintained for 5 years. The insects were maintained in

plastic cages of 30 x 30 x 30 cm, 60 weevils per cage, with free access to water and a diet

consisting of romaine lettuce (Lactuca sativa var. longifolia Lam) and store-bought carrot roots

[Daucus carota L. (Apiaceae)]. The adult weevils were maintained at 280C and a relative

humidity of about 58%.

Chemicals Tested

Ten bioinsecticides and putative antifeedants/repellents were evaluated for deterrence to S.

americana, and D. abbreviatus (Table 1). They included seven botanical derivatives

azadirachtinn, neem oil, sabadilla, rotenone, ryanodine, capsaicin and garlicjuice) and three

inorganic materials (diatomaceous earth, elemental sulfur and kaolin clay). Products were

applied at label rates, using the highest concentration commercially recommended.









Plant Extracts Tested

The plant extracts were prepared based on the procedures described by Gokce and

collaborators (2005). Samples of Florida rosemary, C. ericoides, were collected in the Ordaway-

Swisher Biological Station (Putnam County, Florida), during fall of 2007 and 2008. Samples of

yellow star anise tree, I parviflorum and scratchthroat, and A. crenata, were obtained in the city

of Gainesville (Alachua County, Florida) during fall of 2008. Leaf samples were dried at room

temperature in the dark for 3 weeks and afterward ground in a Wiley mill (Model 3383-L10,

Thomas Scientific, Swedesboro, NJ) using a mesh size of 40. The ground leaf material was

stored in plastic containers at -800C. Ten-gram samples of dried plants were placed into 125 ml

Erlenmeyer flasks and treated with 100 ml of methanol (Fisher Scientific, Fair Lawn, NJ) or

methylene chloride (Fisher Scientific, Fair Lawn, NJ). Flasks were covered with aluminum foil,

placed on an orbital shaker (Model 361, Fisher Scientific, Pittsburgh, PA), and shaken (120

oscillations/min) for 24 h in the dark at 240C. The suspension was filtered first through two

layers of cheese cloth (XL-400, Cotton TM, Worcester, MA) followed by vacuum filtration. The

filtrate was transferred into a 250 ml evaporating flask and dried at 400C in a rotary evaporator

(RE 111 Bichi, Switzerland), in order to evaporate the excess methanol or methylene chloride.

The resulting residues were weighed and mixed with acetone to yield a 20% (w/w) plant

suspension.

Behavioral Bioassay

Antifeedant activity of test substances was assayed using a leaf disk bioassay, in both

choice and no-choice formats. The bioassays were conducted in round transparent plastic

containers of 18 cm diameter and 8 cm height. A moist paper towel (Prime Source) was placed at

the bottom of each container in order to maintain high humidity and to keep the foliage fresh.

The leaf disks were pinned on a cork base of 1.3 cm height.









Leafs were removed from healthy romaine lettuce plants purchased from a grocery store,

and leaf disks were cut using a no. 15 cork borer (2 cm diameter, 3.14 cm2 area). Foliage disks

were cut immediately before application of treatment solutions, minimizing changes in leaf

quality. After leaf disks were cut, they were immediately dipped into one of the treatments

solutions for 10 sec. The disks were left to dry under a fume hood for 15 min at room

temperature. Control disks were dipped into water and were left to dry at room temperature. In

the case of the plant extracts, the control disks were dipped into acetone. A single fifth instar

nymph of S. americana, 2-3 days after molting from fourth instar, was added to each container

and allowed to feed for 24 h at 270C. Nymphal instars were determined according to the methods

of Capinera (1993b.). For D. abbreviatus, one 7-10 day-old adult was placed into each container

and allowed to feed, also for 24 h at 270C. The test insects were starved for 12 h prior to the

experiments. For every chemical substance evaluated, in both choice and no-choice situations,

twenty replicate containers were tested on three different days (n=20, N=60). On each day a

different set of insects was assayed and a fresh preparation of each compound was tested.

In choice tests, one treated and one control disk were placed in each container. The

distance between the two disks was 13 cm. In order to ensure that bioassays were not hunger-

biased, experiments were stopped when grasshoppers or weevils had consumed 50% of either

disk. The bioassays were checked periodically. If the insects had not consumed 50% of either

disk after 24 h, the experiment was terminated. In no-choice tests, either two treated leaf disks or

two untreated (control) disks were placed in each container. The average of the leaf area

consumed in both disks was used for data analysis. No-choice tests were only stopped after 24 h.

Control and treatment evaluations were done simultaneously. Only the biological pesticides that

demonstrated a significant antifeedant activity in the choice tests were evaluated using no-choice









tests. The remaining area of the disks (treated and controls) was measured using a leaf area meter

(LI-3000A, LI-COR, Lincoln, Nebraska, USA).

In order to determine the most effective insect antifeedant, multiple-choice bioassays that

included three treatments in the same container were carried out for 24 h. The treatments chosen

for comparison showed a significant reduction in the consumed leaf area in the control vs.

treatment no-choice assays.

The consumed area of the leaf disks (treated and controls) in both the choice and the no-

choice tests were used for the data analysis. This was obtained by subtracting the remaining area,

calculated with the leaf area meter, from the total area of each leaf disk (3.14 cm2). Shapiro-

Wilk normality tests and Levene tests of homogeneity of variances were employed to determine

the type of distribution for the data obtained in every experiment. For choice tests, paired t-tests

or sign tests, depending on data distribution, were used to test for significant differences in

consumption levels of treatment and control disks (Horton 1995). In no-choice bioassays, t-tests

for independent samples parametricc data) or Mann-Whitney Utest (nonparametric) were used to

evaluate the dissimilarities in area consumed in the control and treatment disks. Parametric one-

way ANOVAs or Kruskal-Wallis nonparametric one-way ANOVAs were used to compare the

leaf area consumed (control and treatment) between the three replicates (n=20, N=60) utilized to

assess every treatment in choice and no-choice bioassays. A Friedman ANOVA was used to

analyze the results of the multiple-choice bioassays, separating means with Mann-Whitney U

test. P values <0.05 were considered to be statistically significant. STATISTICA 6.0 (Stat Soft,

Inc., Tulsa, OK) software was used for the data analysis.

Field Trial

To test the residual activity under field conditions of the chemicals that showed a

significant antifeedant activity against S. americana and D. abbreviatus, applications of the most









effective treatments were made to 5-year-old, randomly selected, Citrus paradisi MacFad

(Rutaceae) potted plants. For each treatment, applications were made at 7 a.m, 11 a.m and 3 p.m.

At every time interval, treatments were applied by a number 4 flat brush to the adaxial surface of

five leaves (per treatment), on the same plant. The painted leaves were collected at 7 p.m. on the

day of application in order to obtain 4 h (3 p.m), 8 h (11 a.m) and 12 h (7 a.m) of sunlight

exposure. Also, five unpainted leaves were collected at 7 p.m. as controls. The selected leaves

were undamaged and apparently of the same age, based on size, coloration and turgidity. From

the painted leaves for each treatment, 10 leaf disks (2.25 cm, diameter, 3.97 cm2 area) were cut

and provided in pairs to individual fifth instar nymphs of S. americana or pairs of adults of D.

abbreviatus in a no-choice format (five replicates per time application, per treatment). Control

leaves were handled in the same way. After 24 h the average of the leaf area consumed in both

disks was used for data analysis. The field experiment was repeated twice for a total of three

trials, for both insect species, using a different citrus plant each time. Applications were made on

November 17, 23 and 27 of 2008 for the S. americana trials, and on February 17, 21 and 23 of

2009 for the D. abbreviatus trials. There was no precipitation on any of the application dates.

The temperature profile on the six dates is presented in the Table 3-2.

Factorial ANOVAs were used in order to analyze the individual and interactive effects of

the hours of sunlight exposure and the three trials made per treatment, on leaf consumption.

Means were separated with Tukey's test (P =0.05). STATISTICA 6.0 (Stat Soft, Inc., Tulsa, OK)

software was used for the data analysis.









Table 2-1. Chemicals evaluated for antifeedant activity against S. americana nymphs and D.
abbreviatus adults.


Active ingredient
(ai)


Neem oil


Azadirachtin


Sabadilla alkaloids


Rotenone


Ryanodine


Capsaicin and other
capsaicinoids

Garlic juice and oil


Diatomaceous earth


Elemental sulfur


Kaolin clay


Trade name


Pure Neem
Oil

Azatrol EC


Sabadilla
Pest Control

Rotenone 5


Ryan 50


Hot Pepper
Wax

Garlic Guard


Mother Earth
D

Liquid Sulfur


Surround WP


AI (% in
product)


Application rate
(ml or g/liter)


8 ml


5.6 ml


0.10


30 g


48 g


18g


0.00018 31.3 ml


50 ml


100


19.5 ml


95.0


60 g


Product Source


Dyna-Gro, San Pablo, CA


PBI/Gordon, Kansas City,
MO

Necessary Organics, New
Castle, VA


Bonide Products,
Yorkville, NY

Dunhill Chemical,
Rosemead, CA

Hot Pepper Wax,
Greenville, PA


Super-Natural Gardner,
Exeter, NH

Whitmire Micro-Gen, St.
Louis, Mo

Bonide Products,
Oriskany, NY

Extremely Green
Gardening, Abington, MA


Table 2-2. Temperature (OC) profile
tested.


of the days on which antifeedant's residual activity was


Date Max C Min C Average C
11/17/08 18.33 0.00 9.44
11/23/08 20.00 0.00 10.00
11/27/08 21.67 1.11 11.67
02/17/09 19.44 0.56 10.00
02/21/09 19.44 -3.33 8.33
02/23/09 16.67 0.56 9.44









CHAPTER 3
RESULTS

Schistocerca americana

Behavioral Bioassays: Commercial Formulations

In choice tests (Figure 4-1), American grasshoppers consumed significantly more untreated

(control) leaf disk material when presented simultaneously with leaf disks treated with either

azadirachtin (t = 12.6; df= 59; P < 0.001), sabadilla (z = 7.62; P < 0.001), ryanodine (t= 6.89; df

= 59; P < 0.001), neem oil (t = 9.50; df= 59; P < 0.001), capsaicin (t = 2.50; df= 59; P= 0.02),

or kaolin clay (t = 2.63; df = 59; P = 0.01). There was not a significant difference in the leaf area

consumed between the untreated and the disks treated with elemental sulfur (z = 7.62; P = 0.52),

diatomaceous earth (t= 1.33; df= 59; P = 0.19), garlic juice (z = 7.62; P = 0.79), or rotenone (t

=0.86; df= 59; P= 0.39).

In no-choice tests (Figure 4-2), azadirachtin (U= 388.00; Z= -7.41; P < 0.001), sabadilla

(U= 0.00; Z= -9.45; P < 0.001) and ryanodine (U= 546.50; Z = -6.58; P < 0.001) were

confirmed to be statistically significant antifeedants against S. americana. Sabadilla showed a

strikingly potent inhibition effect (Figure 4-2). Neem oil did not reduce the feeding behavior of

the American grasshoppers significantly (U= 1707.50; Z= 0.49; P = 0.63). Capsaicin and kaolin

clay were discarded as potential S. americana feeding deterrents after the first trial because

nymphs completely consumed the leaf disks treated with these chemicals.

The multiple-choice bioassays demonstrated that sabadilla was the most effective feeding

deterrent for S. americana fifth instar nymphs. It exerted a strong feeding inhibition effect that

was statistically superior to the one produced by ryanodine and azadirachtin (X2F = 51.11; df= 2;

P < 0.001). Although the average leaf consumption of azadirachtin treated leaf disks was lower









than in the ryanodine treated disks (Figure 4-3), there was not a statistically significantly

difference between these treatments (U=1566.00; Z= 1.23; P = 0.22).

Field Trial

Persistence of the potential antifeedants under field conditions was variable. There was no

effect of sunlight exposure intervals on consumption of foliage treated with azadirachtin (F =

0.06; df = 2; P = 0.95) or ryanodine (F=3.06; df = 2; P = 0.053). Azadirachtin (Figure 4-4) did

not significantly reduce herbivory by S. americana nymphs during any of the exposure periods in

each of the three trials. Ryanodine (Figure 4-5) only exerted significant leaf protection after 8 h

of exposure to sunlight, during the second evaluation. Sabadilla (Figure 4-6) was the only

treatment that reduced significantly the feeding by S. americana at 4 h, 8 h and 12 h of sunlight

exposure, and did so in all the three trials. Consumption of leaf material in foliage treated with

sabadilla did not differ statistically among the hours of exposure (F= 2.24; df = 2; P = 0.11).

There were no differences among replicates for consumption of foliage treated with azadirachtin

(F= 1.33; df = 2; P = 0.27), ryanodine (F=1.15; df = 2; P= 0.32) or sabadilla (F=3.00; df= 2;

P = 0.06), as well as the interaction of this factor with the hours of exposure azadirachtinn: F=

0.33; df= 4; P = 0.86; ryanodine: F=1.83; df= 4; P = 0.13; sabadilla: F= 0.68; df= 4; P=

0.61).

Behavioral Bioassays: Plant Extracts

The plant extracts obtained from C. ericoides, I. parviflorum and A. crenata significantly

reduced the consumption of treated leaf disks, compared to untreated (control) disks in choice

tests (Figure 4-7). Both methanol (C. ericoides: z = 2.34; P = 0.02; I. parviflorum: t = 11.31; df=

59; P < 0.001; A. crenata: t = 5.70; df= 59; P < 0.001) and methylene chloride extracts (C.

ericoides: z =5.29; P < 0.001; 1. parviflorum: t = 14.40; df = 59; P < 0.001; A. crenata: z = 4.26;

P < 0.001) significantly protected the treatment disks in this type ofbioassay (Figure 4-7).









In no-choice bioassays, only the C. ericoides methylene chloride extract (U=1294.50; Z=

-2.65; P= 0.008) and the A. crenata methanol extract (t= -2.1; df = 118; P = 0.04) functioned as

antifeedants that significantly reduced herbivory by nymphs of S. americana (Figure 4-8). Leaf

disks treated with I. parviflorum extracts were consumed in higher proportion than the control

disks (methanol: t= 3.28; df= 118; P < 0.001; methylene chloride: t= 2.21; df= 118; P 0.03).

Treatment of leaf disks with C. ericoides methanol extract (t = -0.48; df= 118; P = 0.63) and A.

crenata methylene chloride extract (t = -0.58; df= 118; P = 0.56) did not statistically modify the

feeding behavior of the grasshoppers (Figure 4-8).

Diaprepes abbreviatus

Behavioral Bioassays: Commercial Formulations

Feeding by the sugarcane rootstock borer weevils in choice tests was reduced in leaf disks

treated with azadirachtin (t= 4.26; df = 59; P< 0.001), neem oil (t= 2.25; df = 59; P = 0.03),

sabadilla (z = 2.47; P = 0.01), rotenone (z = 6.17; P < 0.001) and ryanodine (z = 2.76; P = 0.01),

as compared with untreated disks (Figure 4-9). Elemental sulfur (z = 1.04; P = 0.30),

diatomaceous earth (t = -1.05; df = 59; P = 0.29), garlic juice (t= 0.07, df = 59; P = 0.95),

kaolin clay (t= -1.08; df= 59; P = 0.86) and capsaicin (t= 1.82; df= 59; P = 0.07) did not

significantly modify the feeding behavior of the weevils (Figure 4-9).

Ryanodine (t= -6.02; df= 118; P< 0.001), rotenone (U= 1403; Z = -2.08; P = 0.04) and

sabadilla (U=627; Z = -2.08; P < 0.001) were the treatments that caused a significant antifeedant

effect over D. abbreviatus adults in no-choice bioassays (Figure 4-10). There was not a

significant difference between the area consumed of azadirachtin-treated leaf disks and untreated

ones (t = 1.87; df= 118; P = 0.06). Neem oil-treated disks were eaten in a higher proportion than

control disks (U=1319.50; Z= 2.52; P = 0.01).









The multiple-choice bioassays (Figure 4-11) did not expose a significant difference

amongst ryanodine, rotenone and sabadilla in terms of leaf area consumed by the weevils (X2F

=1.78; df = 2; P= 0.41).

Field Trial

The hours of sunlight exposure did not affect significantly the herbivory of citrus leaves

treated with rotenone (F= 0.28; df= 2; P = 0.75), ryanodine (F= 1.21; df= 2; P = 0.30) or

sabadilla (F = 0.70; df = 2; P = 0.5). At each time interval (4 h, 8 h and 12 h) these chemicals

were ineffective as feeding deterrents. Rotenone (Figure 4-12), ryanodine (Figure 4-13) and

sabadilla (Figure 4-14) failed to reduce leaf consumption, compared to untreated leaves, during

the field evaluations. Replication did not have an effect on the leaf area consumed in foliage

treated with rotenone (F= 2.62; df= 2; P = 0.08), ryanodine (F= 2.09; df= 2; P = 0.13) or

sabadilla (F = 2.52; df = 2; P = 0.09), as well as the interaction of this factor with the hours of

sunlight exposure (rotenone : F= 1.55; df= 4; P =0.20; ryanodine: F= 0.68; df= 4; P = 0.61;

sabadilla: F = 0.62; df = 4; P = 0.65).

Behavioral Bioassays: Plant Extracts

The plant extracts reduced feeding by sugarcane rootstock borer weevils. Regardless of the

solvent used, weevils consumed significantly more of the untreated lettuce leaf disks than disks

treated with C. ericoides (methanol: z = 7.62; P < 0.001; methylene chloride: z = 7.29; P <

0.001), I. parviflorum (methanol: t =4.88; df= 59; P < 0.001; methylene chloride: z =3.88; P <

0.001) andA. crenata (methanol: z = 7.10; P < 0.001; methylene chloride: z = 5.56; P< 0.001)

extracts, in choice tests (Figure 4-15).

In no-choice bioassays, both extracts of C. ericoides (methanol: U= 144.50; Z = -8.69; P <

0.001; methylene chloride: U=58.00; Z= -9.14; P < 0.001) andA. crenata (methanol: t= -34.54;

df= 118; P< 0.001; methylene chloride: U= 89.50; Z= -8.98; P< 0.001) were confirmed to be









feeding deterrents against the sugarcane rootstock borer weevils (Figure 4-16). Leaf disks treated

with I. parviflorum extracts were consumed in the same proportion as the control disks

(methanol: t=-1.14; df= 118; P = 0.25; methylene chloride: t=-1.45; df= 118; P = 0.15).















Azadirachtm
P<0001










E
Control
Treatment

Sabadilla
P <0001
0+
5



, --- ,----c



















Control
Treatment

Ryanodine
P<0001
o




























Control
Treatment
P<0001






























Control Treatment
P00
Control



















Elemental sulenfur
P=052
P=052


Control Treatment


* Mean
SSE
SSD


* Mean
SE
SSD














* Mean
l SE
SSD















B Mean
SE
SD














Mean
SE
SD


Neem oil
P<0001




0
5



S0
'5 ---- ----- ----- ----- ---- [T



Control
Treatment

Rotenone
P =039












Control
Treatment

Capsaicm
P =002












Control
Treatment

Diatomaceous earth
P=019
|0


Control
Treatment

Kaohn clay
P 001


Control


Treatment


* Mean
SE
" SD














Mean
S-SE
I SD














Mean
S-SE
I SD














E Mean
SE
SSD














M Mean
--SE
SD


Figure 3-1. Total area consumed of untreated and treated leaf disks, by S. americana nymphs, in

choice bioassays.

















35

30

I 25

I 20

15
l
co


S10oo


<


Azadirachtin
P 0.001



















Control
Treatment

Ryanodine
P 0.001



















Control
Treatment


Sabadilla
P 0.001
35
35 ---i --------I------- ------ i--

30

"25

S20

15
cz
10

S 05
00


-0 5 --- ------------------ ---
Control
Treatment

Neem oil
P =0.63
35


0

02


0
CZ 15

10
r0
0 5



Control
Treatment


Figure 3-2. Total average area consumed of untreated and treated leaf disks, by S. americana

nymphs, in no-choice bioassays.


* Mean
- SE
I SD






















* Mean
- SE
" SD


) 25
C 30




0

15
I "





0
<


* Mean
- SE
T SD






















* Mean
- SE
" SD











P< 0.001





0,n

rn ---


o Mean
S-- SE
+SD


Figure 3-3. Total leaf area eaten (cm2) by S. americana nymphs when exposed to the most
effective feeding deterrents in multiple-choice situations.


55


45
o
40
-e


30
O
(2
20

( 15
2,


05 '
4 8 12

Hours of exposure
Treatment


4 8 12

Hours of exposure
Control


- Evaluation
1
--a-- Evaluation
2
----- Evaluation


Figure 3-4. Consumption of azadirachtin treated and control citrus disks after three time
intervals of sunlight exposure on three trials. Disks were provided to S. americana
nymphs in no-choice situations. Vertical bars denote 0.95 confidence intervals.


a













50

S 45

S40
-3
35

I 30
3
d 25

20


215
10

05


4 8 12

Hours of exposure
Control


-- Evaluation
1
-o- Evaluation
2
--- Evaluation
3


Figure 3-5. Consumption of ryanodine treated and control citrus disks after three time intervals
of sunlight exposure on three trials. Disks were provided to S. americana nymphs in
no-choice situations. Vertical bars denote 0.95 confidence intervals.


4 8 12

Hours of exposure
Treatment


4 8 12

Hours of exposure
Control


-- Evaluation
1
-o- Evaluation
2
...... Evaluation
3


Figure 3-6. Consumption of sabadilla treated and control citrus disks after three time intervals of
sunlight exposure on three trials. Disks were provided to S. americana nymphs in no-
choice situations. Vertical bars denote 0.95 confidence intervals.


55


4 8 12

Hours of exposure
Treatment












C. encodes (CHOH)
P 002









++


Control
Treatment

I. parvflorum (CH30H)
P <0 001












Control
Treatment


* Mean
SSE
I SD


SMean
SSE
I SD


A. crenata (CH3OH)
P< 0.001


Control
Treatment


Mean
I SE
I SD


C. erzcoides (CH2C12)
P< 0.001












Control
Treatment

L. parvflorum (CH2C12)
P <0 001












Control
Treatment
A crenata (CH2C2)
P<0001


Control


Treatment


Figure 3-7. Total area consumed of untreated and plant extracts-treated leaf disks, by S.
americana nymphs, in choice bioassays.


Mean
SSE
I SD














* Mean
SSE
I SD















J Mean
SE
SD















C. ericoides (CH3OH)
P =0.63


Control
Treatment


I. parviflorum (CH3 OH)
P< 0.001


14
Control
Treatment

A. crenata (CH3OH)
P 0.04
34
32
30
28
26
24
22
20
18
16
14
12
10
08
Control
Treatment


C. ericoides (CH2C12)
P = 0.008


* Mean
- SE
T SD



















* Mean
O SE
I SD



















* Mean
- SE
" SD


Control
Treatment


I. parviflorum (CH2C12)
P 0.03


Control
Treatment

A. crenata (CH2C12)
P 0.56
34
32
30
28
26
24
22
20
18
16
14
12
10
Control
Treatment


Figure 3-8. Total average area consumed of untreated and plant extracts-treated leaf disks, by S.

americana nymphs, in no-choice bioassays.


* Mean
- SE
T SD



















* Mean
- SE
I SD



















* Mean
SSE
SD



















Azadirachtm

P<0001
26
22
20P
18
1 6
14
12
10
08
06
04
02
00
:00 2 +
04l
Control
Treatment

Sabadilla
P=001

2 o


1 6


















24 | ---- i ---- i ---- i ---- i ----
12


18
0 6
14
0 2








08
0 2

















:04 ----------=------
Control Treatment


Ryanohne











P=09501
24
22

18
16
14
12
O 0
08
06
04
02
00
-02 2
-04
Control
Treatment

Garlicjuice
P=095
26


Control Treatment


Elemental sulfur
P =030
20
18
16
14
12

08
06
04
02
oo
42
Control
TreaHtment


* Mean
SSE

SSD



















Mean
SE
SSD


















SMean
\SE
SSD



















m Mean
-SE
SD


* Mean
SSE
SSD


Neem oil
P =003
26
24
22
20
18
16
14
12
10
08
06
04
02
00
-02
-04
Control
Treatment

Rotenone
P<0001
24
24
20
18
16
14
12
10
08
06
04
02
00

-02
Control
Treatment


Capsalcm
P =007
26
24
22
20
1 8
1 6
14
1 2
1 0
08
06
04
02
00
-02
Control
Treatment

Diatomaceous earth
P =029
26
24
22
20
18
16
14
12
10
08
06
04
02 +
00
-02-
Control
Treatment


Kaohn clay
P =086

20
1 8
16
14



08
06
04
02

Control
Treatmennt


Figure 3-9. Total area consumed of untreated and treated leaf disks, by D. abbreviatus, in choice

bioassays.


* Mean
SSE
SSD


















* Mean
I SE
1 SD


















* Mean
- SE
1 SD


















E Mean
- SE
- SD


















* Mean
SSE
I SD


















3 35

S30


S-
2


0

C 1 5
0

S10
b0

05

00


Control
Treatment


Azadirachtin
P 0.06




















Control
Treatment

Rotenone
P 0.04











E-


7



cz











7
t
C
c.)


Zn









C.)
t

C





C.)

Zn


Ryanodme
P <0 001
35

30

25

20

15

10

05

00
Control
Treatment

Sabadilla
P< 0.001
36
34
32
30
28
26
24


20
18
16
14
1 2


Control
Treatment


Neem oil
P 0.01




















Control
Treatment


Figure 3-10. Total average area consumed of untreated and treated leaf disks, by D. abbreviatus

adults, in no-choice bioassays.


* Mean
'SE
I SD























SMean
-l SE
"I SD


Mean
I SE
I SD























Mean
- SE
" SD


* Mean
- SE
T SD












P = 0.41
18
16
14
12
10
08
06
04
02
00
-02 -
-Ga


SMean
I +SE
Z +SD


Figure 3-11. Total leaf area eaten by D. abbreviatus adults when exposed to the most effective
feeding deterrents in multiple-choice situations.
4.5

4.0

0.35
S 3.0 .






T -
1. 2.5

U 210 TT





13
010 .-.--- Evaluation

-0.5
-1.0 -- Evaluation

-1.5 1 E
4 8 12 4 8 12 ..."---" Evaluation


Hours of exposure
Treatment


Hours of exposure
Control


Figure 3-12. Consumption of rotenone treated and control citrus disks after three time intervals
of sunlight exposure on three trials. Disks were provided to D. abbreviatus adults in
no-choice situations. Vertical bars denote 0.95 confidence intervals.











30


00

-05


-1 0 i
4 8 12

Hours of exposure
Treatment


4 8 12

Hours of exposure
Control


--- Evaluation
1
---- Evaluation
2
------ Evaluation

3


Figure 3-13. Consumption of ryanodine treated and control citrus disks after three time intervals
of sunlight exposure on three trials. Disks were provided to D. abbreviatus adults in
no-choice situations. Vertical bars denote 0.95 confidence intervals.

35

30

(Na
-e
20

15





S-05 ---- Evaluation

2
o-10

4 12 4 12 .... -- Evaluation

-os -a- Evaluation
2
4 8 12 4 8 12 ---o-- Evaluation


Hours of exposure
Treatment


Hours of exposure
Control


Figure 3-14. Consumption of sabadilla treated and control citrus disks after three time intervals
of sunlight exposure on three trials. Disks were provided to D. abbreviatus adults in
no-choice situations. Vertical bars denote 0.95 confidence intervals.














C. encodes (CHOH)
P <0001















Control
Treatment

. parvflorum (CH3OH)
P <0 001


Control
Treatment


A. crenata (CH3OH)
P <0.001
30

25

20

15

10

05

00

-05
Control
Treatment


* Mean
-SE
I SD


* Mean
- SE
I SD


C. encodes (CH2C12)
P <0 001















Control
Treatment

L parvflorum (CH2C12)
P <0 001


Control
Treatment


A. crenata (CH2C12)
P <0 001


Mean
[ SE
IT SD


Control
Treatment


Figure 3-15. Total area consumed of untreated and plant extracts-treated leaf disks, by D.

abbreviatus adults, in choice bioassays.


* Mean
SSE
I SD


* Mean
SSE
I SD


SMean
- SE
I SD
















0
I)
0,-
0
I)
I)
cli

I)


Control
Treatment


C. encoides (CH,OH)
P<0001













Control
Treatment

I. parviflorum (CH3OH)
P 0.25





m







Control
Treatment

A. crenata (CH30H)
P <0.001

0o +..----







50


15


SMean
I SE
" SD


C. ericodes (CH2C12)
P< 0.001






5


5



Control
Treatment

parvflorum (CH2C12)
P 015





I ,


Control
Treatment


A. crenata (CH2C12)
P <0.001


+


Control
Treatment


Figure 3-16. Total average area consumed of untreated and plant extracts-treated leaf disks, by
D. abbreviatus adults, in no-choice bioassays.


Mean
SSE
I SD
















SMean
- SE
I SD


Mean
- SE
I SD
















SMean
SSE
I SD


Mean
I SE
I SD









CHAPTER 4
DISCUSSION

Behavioral Bioassays: Commercial Formulations

In this research, sabadilla was the most effective feeding deterrent against S. americana.

Alkaloids, the active ingredient in sabadilla, have previously been show to produce an

antifeedant effect on grasshoppers. Nicotine hydrogen tartrate, as well as gramine, were able to

reduce the feeding behavior of S. americana (White and Chapman 1990, Chapman et al. 1991,

Bernays 1991), L. migratoria (Ishikawa and Kanke 2000), Ageneotettix deorum Scudder and

Phoetaliotes nebrascensis Scudder (Mole and Joern 1994). The two former species were also

deterred by the alkaloid eserine (Mole and Joern 1994). Alkaloids seem to be perceived in the

American bird grasshopper by the stimulation of a specialized deterrent receptor. Prior

electrophysiological studies have shown that contact chemoreceptors on the tibia and tarsus of S.

americana are stimulated by alkaloids, and have demonstrated an association between the neuron

activity and the antifeedant response (White and Chapman 1990, Chapman et al. 1991).

Therefore, it is possible that the sabadilla alkaloids cevadine and veratradine were detected by

the alkaloid-sensitive neurons. Based on the rapid and persistent rejection of sabadilla-treated

leaf disks by S. americana nymphs observed during the bioassays, it is plausible that the sensory

coding that elicited the sabadilla-deterrent effect resulted from "labeled line" responses

(Schoonhoven 1982, van Loon 1996, Koul 2008). This means that each neuron in a contact

chemoreceptor conveys a specific message, in this case a deterrent signal, which can be

interpreted by the central nervous system without additional information from other neurons,

leading to immediate rejection of a food source without feeding.

Sabadilla also effectively reduced the feeding behavior ofD. abbreviatus. The feeding

inhibition that this chemical elicited on the weevils was not as potent as the deterrence observed









for the grasshoppers; thus, the sensory input that the sabadilla alkaloids generated in D.

abbreviatus and S. americana nervous system, is perhaps different. Instead of a labeled line

response, the reduction in feeding behavior by D. abbreviatus could be produced by an across-

fiber pattern (Schoonhoven 1982, van Loon 1996, Koul 2008), in which the combined input of

two or more receptors, with different stimulus thresholds, determines the acceptance or rejection

of a host. For example, alkaloids from the family Solanaceae do not have any specific disruptive

effects on several taste neurons nor inhibit the activity of the taste cell sensitive to

phagostimulants in the red turnip beetle, Entomoscelis americana Brown (Mitchell and Gregory

1979), suggesting an across-fiber pattern as sensory coding.

Although not as potent as sabadilla, azadirachtin caused a significant reduction in the

feeding behavior of S. americana. Previous studies had documented the antifeedant properties of

azadirachtin against different orders of insects, including Orthoptera (Mordue (Luntz) and

Blackwell 1993, Aerts and Mordue 1997, Capinera and Froeba 2007). Inhibition of feeding

behavior by this triterpenoid could be the result of stimulation of a deterrent receptor, or

blockage of the input from neurons that detect phagostimulatory compounds such as

carbohydrates, or both. Winstanley and Blaney (1978) studied the behavioral and sensory

response ofSchistocerca gregaria Forskal to a set of solutions including azadirachtin. They

proposed that the deterrent effect of azadirachtin on this grasshopper was caused by the

stimulation of a specialized deterrent cell. Pieris brassicae L. larvae also possess a deterrent

receptor, located in the medial sensilla styloconica on the maxilla, which is excited by

azadirachtin (Schoonhoven 1982). Charleston and colleagues (2005) observed feeding deterrent

activity by plants extracts from Melia azedarach L. (Meliaceae) and Azadirachta indica against

the diamondback moth, Plutella xylostella L. The authors suggested that triterpenoids present in









the plant extracts, including azadirachtin, disrupted the normal function of chemoreceptors

responsible for perceiving glucosinolates, a strong phagostimulant for this lepidopteran.

The antifeedant effect of azadirachtin varies among insect species. In this study, D.

abbreviatus was deterred by azadirachtin during the choice tests, consuming more control disks

than treated ones. But in the no-choice bioassays the terpenoid did not reduce herbivory of the

treated leaf disks. These results concurred with the observations of Showler and collaborators

(2004), who evaluated three commercial neem-based insecticides (Agroneem, Ecozin and

Neemix) as potential feeding and oviposition deterrents against gravid female boll weevils,

Anthonomus grandis grandis Boheman on cotton plants. They found that in choice assays, only

Ecozin deterred the weevils from feeding, whereas in no-choice tests, none of the products

reduced the consumption of cotton leaves after 24 h. It is probable that D. abbreviatus had

habituated to azadirachtin due to repeated exposure to the chemical during the 24 h time interval

of the experiments. Desensitization to azadirachtin has been reported previously. Fifth instar S.

litura larvae became desensitized to pure azadirachtin, in both choice and no-choice assays, after

being exposed to the terpenoid for 2 h (Bomford and Isman 1996). Similarly, Held and

colleagues (2001) observed adults of the Japanese beetle, P. japonica, to habituate to a

commercially formulated neem extract, applied to linden, Tilia cordata L., in a series of 4-h

choice or no-choice tests over four successive days.

Neem oil reduced feeding of treated leaf disks by S. americana and D. abbreviatus in

paired choice assays but was unable to deter both insects in no-choice tests. D. abbreviatus even

consumed significantly more neem oil-treated leaf disks than untreated ones. The increased

acceptability of neem oil by the insects may be due to desensitization. The biological activity of

neem oil is closely related to its azadirachtin content (Isman et al. 1990, Isman 2006). Neem









seeds normally contain 0.2% to 0.6% azadirachtin by weight, while azadirachtin formulations

contain 10% to 50% (Isman 2006). Desensitization occurs more frequently when an antifeedant

exerts weak inhibitory stimuli (Held et al. 2001), in this case lower concentrations of

azadirachtin. Although neem oil includes considerable quantities of other triterpenoids such as

salannin and nimbin (Schmutterer 1990, Isman 2006) that can act as antifeedants as well (Koul et

al. 2004), the feeding strategy of S. americana and D. abbreviatus may have counteracted their

effect. The capacity for habituation in insects may be greater in polyphagous than in

oligophagous or monophagous species (Jermy 1990, Held et al. 2002) because the taste

sensitivity of insect herbivores to deterrents is lower in generalists than in specialists (Bernays et

al. 2000).

Rotenone was ineffective as a feeding deterrent of S. americana in this research. There is

evidence that some species of grasshoppers are able to detect flavonoids (Bernays and Chapman

2000). Chapman and co-workers (1991) detected that salicin, a phenolic glycoside that stimulates

a deterrent neuron in S. americana, had a phagostimulatory effect on this grasshopper at low

concentrations. But it seemed that the activity of the deterrent cells at higher concentrations of

salicin was sufficient to override the phagostimulatory effects of the sucrose-sensitive cells,

producing an antifeedant effect (Chapman et al. 1991). Therefore, the inefficiency of rotenone in

reducing herbivory by S. americana may be a consequence of the imbalance between the weak

deterrent stimuli generated by the isoflavonoid and the strong phagostimulatory effect of the

carbohydrates present in the lettuce leaf disks.

Contrary to the effect observed with the grasshoppers, rotenone applied to lettuce leaf disks

effectively deterred D. abbreviatus adults in both choice and no-choice bioassys. This result

supports previous studies that tested rotenone against curculionids and other coleopterans.









Rotenone showed a strong antifeedent effect against adults and larvae of the wheat weevil,

Sitophilus granarius L., adults of the confused flour beetle, Tribolium confusum Jacquelin du

Val, and larvae of the khapra beetle, Trogoderma granarium Everts (Nawrot et al. 1989). The

grass grub, Costelytra zealandica (White) was also deterred by rotenone in an artificial diet

(Lane et al. 1985).

Ryanodine was the only chemical tested other than sabadilla that significantly reduced the

feeding behavior of S. americana and D. abbreviatus in both choice and no-choice tests. The

antifeedant effect of ryanodine on insects has been proven previously. Ryanodine reduced the

food consumption of the tobacco budworm, Heliothis virescens (F.), and the tobacco cutworm,

Spodoptera litura F., under laboratory conditions (Yoshida and Toscano 1994, Gonzalez-Coloma

et al. 1996). Larvae of the African cotton leafworm, Spodoptera littoralis Boisduval, were more

sensitive to the deterrent action of ryanodine than adults of the Colorado potato beetle, L.

decemlineata (Gonzalez-Coloma et al. 1999). The observed antifeedant action of the alkaloid

ryanodine potentially implicates the involvement of a common ligand-gated ion channel that

mediates the taste response to these compounds in both the grasshoppers and weevils nerve cells.

A possible candidate could be a ryanodine receptor. Ryanodine receptors are ryanodine-sensitive

intracellular Ca2+ release channels (Nauen 2006). Ca2+is an intracellular messenger which

intercedes in many cellular and physiological activities such as neurotransmitter release,

hormone secretion, gene expression and muscle contraction (Nauen 2006).

In this study, capsaicin did not exert a relevant antifeedent effect against S. americana or

D. abbreviatus. The ineffectiveness of capsaicin as an insect feeding deterrent has been

previously documented. Capsaicin did not decrease herbivory of grape leaves by the rose chafer,

Macrodactylus subspinosus F. (Isaacs et al. 2004). Potato plots treated with a capsaicin extract









did not decrease the incidence of the Colorado potato beetle, Leptinotarsa decemlineata Say

(Moreau et al. 2006). Baumler and Potter (2007) also reported the inefficacy of capsaicin in

reducing defoliation of linden (Tilia cordata L.) by the Japanese beetle.

Kaolin clay, an aluminosilicate material, did not effectively deter any of the insects tested

in this study. Schistocerca americana consumed less kaolin-treated leaf disks than untreated ones

in choice bioassays, but in a no-choice scenario the presence of the aluminosilicate did not

prevent the entire consumption of the treated disks. Against D. abbreviatus, kaolin clay did not

even exert a significant protection for treated leaf disks, in choice tests. Other studies have

observed successful suppression of arthropod pests on fruit plants coated with kaolin (Glenn et

al. 1999, Puterka et al. 2000). According to these studies, a plant coated with a hydrophobic

particle film barrier, such as kaolin clay, can repel arthropods not only by making the plant

visually or tactilely unrecognizable as a potential host but also by restraining mobility and

snaring the arthropod's mouthparts. But kaolin clay as a plant protector is maybe only effective

against small arthropods with piercing-sucking mouthparts, such as the ones tested in these

studies (pear psylla, Cacopsyllapyricola Foerster, spirea aphid, Aphis spirecola Patch, potato

leaf hopper, Empoascafabae Harris, twospotted spider mite, Tetramychus urticae Koch and pear

rust mite, Epitrimeruspyri Nalepa). For large insects with chewing mouthparts, such as D.

abbreviatus or S. americana, kaolin clay does not represent a physical barrier. Our results also

contrast with those of Lapointe (2000), who observed a reduction in feeding by D. abbreviatus

adults on citrus plants treated with kaolin clay under laboratory conditions. Lapointe suggested

that the antifeedant effect was caused by interference with tactile recognition of citrus plants as

hosts, using the research of Puterka and colleagues (2000) as a reference. This mode of action is

probably correct against specialist arthropods, like the pear psylla and the pear rust mite, which









depend on very specific visual, olfactory, gustatory and mechano-sensory stimuli in order to

select a host plant, but this may not apply to generalist insects that rely more, at short distances,

on tasting most plants and eating those that lack strong feeding deterrent compounds in order to

select a host plant (Schoonhoven et al. 2005). The contrasting results suggest a necessity for

further studies of the antifeedant effect of kaolin clay on D. abbreviatus.

Field Trial

Residual activity of the tested antifeedants under field conditions proved to be quite brief.

All the chemicals tested did not significantly reduce herbivory by S. americana or D. abbreviatus

after 4 h of exposure to sunlight. The only exception was sabadilla, which showed an antifeedant

effect against nymphs of S. americana after 4 h, 8 h and 12 h of exposure. Photodegradation of

botanical pesticides under sunlight has been reported previously (Liang et al. 2003, Showler et

al. 2004, Capinera and Froeba 2007) and represents one of several problems affecting plant-

based insecticides under field conditions.

Behavioral Bioassays: Plant Extracts

In this study, both methanol and methylene chloride extracts from the Florida rosemary, C.

ericoides, deterred D. abbreviatus adults; whereas only the methylene chloride extracts from this

plant species exerted an antifeedant effect on S. americana nymphs. Chemical analysis of C.

ericoides has previously demonstrated the presence of several classes of flavonoids including,

dihydrochalcones, flavones, catechins and epicatechins (Tanrisever et al. 1987). Flavonoids may

affect the feeding behavior of insects by stimulation of a specialized deterrent receptor

(Simmonds 2001, Koul 2008). Flavonoids are compounds (Ding 1998) that are only soluble in a

polar solvent such as methanol. Therefore, these phenolic compounds may be the basis of the

antifeedant effect of C. ericoides methanol extracts on D. abbreviatus. The feeding inhibition

that the C. ericoides methylene chloride extracts observed against S. americana and D.









abbreviatus suggests the presence of a non-polar chemical that interacts with the chemosensory

system of the two insect species. A possible candidate would be a class ofterpenoid, due to the

relative non-polar character of these plant chemicals (Fischer et al. 1994) and their potential as

feeding deterrents (Koul 2008).

Methanol extracts from A. crenata functioned as a feeding deterrent against both S.

americana nymphs and D. abbreviatus adults. The latter insect species was also deterred by

methylene chloride extracts from A. crenata. Triterpenoid saponins have been isolated from A.

crenata plants (Liu et al. 2007). These non-polar terpenoids are able to modify the feeding

behavior of insects. Larvae of the diamondback moth, Plutella xylostella L., were deterred by a

triterpenoid saponin extracted using chloroform from wintercress, Barbarea vulgaris W. T.

Aiton (Shinoda et al. 2002). Terpenoids can inhibit insect feeding by distortion of the normal

function of phagostimulant receptors, excitation of deterrent receptors and/or stimulation of

broad spectrum receptors, among others (Koul 2008). Isocoumarins are another type of

phytochemical that has been identified in members of the genus Ardisia (Kobayashi and de

Mejia 2004). Isocoumarins are phenolic compounds that can produce a feeding deterrent effect

by antagonizing y-aminobutyric acid (GABA) (Ozoe et al. 2004). GABA and related

aminobutyric acids have been shown to stimulate feeding and induce taste cell responses among

herbivorous insects of four orders including Orthoptera and Coleoptera (Mullin et al. 1994).

GABA-gated chloride channels in the peripheral nervous system of insects participate in

chemoreception. In excitable cells, binding by a ligand (GABA, in this case) changes channel

conformation, which leads to an opening of the ion pore and an inhibitory inward C1- movement,

due to high external C1-. But, according to Mullin et al. (1994), opening of these channels under

low C1- concentrations of plant tissues, in the absence of a mechanism to maintain high









extracellular C1- in the sensillar fluid (which is a mucopolysaccharide material surrounding the

tip of the dendrites of the contact chemoreceptors), could result in an outward C1- movement

leading to depolarization of the sensory neuron. Thus, if a phytochemical (i.e. isocoumarins)

antagonizes GABA at the binding site of a ligand-gated chloride channel in a gustatory cell,

feeding deterrence will be induced.

In summary, of the ten chemicals tested only sabadilla, azadirachtin and ryanodine deterred

S. americana under laboratory conditions. Sabadilla was the only compound that maintained its

remarkable antifeedant properties against the grasshoppers after 12 h of exposure to sunlight.

Sabadilla's deterrent effect and relative durability under field conditions makes it a potential tool

for integrated management of the American bird grasshopper. Against D. abbreviatus,

ryanodine, rotenone and sabadilla acted as feeding deterrents, but only in the laboratory

bioassays. The stability of these chemicals in the field must be improved if effective protection

against the sugarcane rootstock borer weevil is desired. The effectiveness of the extracts obtained

from C. ericoides and A. crenata in reducing herbivory of the two insect species tested is an

indication that many plants contain phytochemicals that could potentially be developed as

antifeedants, and examination of these compounds is a logical next step for this research.









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BIOGRAPHICAL SKETCH

Andres Felipe Sandoval Mojica was born on 1981 in Bogota, Colombia. The youngest of

four children, he grew up in Tunja, Boyaca, Colombia graduating from Colegio de Boyaca in

1997. He obtained his undergraduate degree in biology from the Pontificia Universidad

Javeriana, where he developed an interest in entomology after taking the course "Biology of

Arthropods". Due to an excellent academic performance in this subject, he became the teaching

assistant for the same course in the year 2001. His undergraduate thesis identified the pattern of

altitudinal variation in richness, abundance, diversity and composition of the Orthoptera

community in an altitudinal gradient between 2,000 m and 3,000 m. in an Andean cloud forest.

This research contributed to understand the controversial relationship that exists between altitude

and species richness. It also provided information about factors affecting insect distribution in

environmental gradients. Due to the significance of this work, he received financial support, as a

scholarship, from the Colombian Society ofEntomology (SOCOLEN). The research was

nominated as the best student paper at the XXXII meeting of the same institution in 2005 and the

results published in the 32nd volume ofRevista Colombiana de Entomologia in 2006. In 2005, he

was hired by Fundaci6n OMACHA (OMACHA Foundation, an organization committed to the

sustainable development, research and conservation of the Colombian natural resources, with

emphasis in aquatic ecosystems), where he studied the entomological fauna that exist in the

Bojonawi NaturalReserve, in the Colombian Orinoquia. He proposed and developed a research

subject that compared the structure and composition of dung beetles, ants and butterflies

communities in three land units that are present at the reserve: savannah, galleria forest and palm

trees. This study, which was presented at the XXXIII meeting of the Colombian Society of

Entomology, was nominated as the best study presented by a professional in 2006. A new report

ofPhanaeus haroldi Kirsch (Coleoptera: Scarabaeidae) for the Department of Vichada









(Colombia) was also obtained. During 2006, he worked as a volunteer at the Colombian

Corporation ofAgricultural Research (CORPOICA).





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1 ANTIFEEDANT EFFECT OF COMMERCIAL CHEMICALS AND PLANT EXTRACTS AGAINST Schistocerca americana (ORTHOPTERA: ACRIDIDAE) AND Diaprepes abbreviatus (COLEOPTERA: CURCULIONIDAE) By ANDRES FELIPE SANDOVAL MOJICA A THESIS PRESENTE D TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Andres Felipe Sandoval Mojica

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3 To my family, the source of my strength

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4 AC KNOWLEDGMENTS I thank my committee members Dr. John Capinera, Dr. Michael Scharf, and Dr. Heather McAuslane for their advice, support and commitment to this study.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 7 LIST OF FIGURES .............................................................................................................................. 8 ABSTRACT ........................................................................................................................................ 10 CHAPTER 1 INTRODUCTION ....................................................................................................................... 12 Conceptual Framework ............................................................................................................... 12 Pest Insects ................................................................................................................................... 16 Commercial Chemicals Tested ................................................................................................... 17 Plant Species Used To Obtain The Extracts .............................................................................. 20 Objectives .................................................................................................................................... 22 2 MATERIALS AND METHODS ............................................................................................... 23 Insect Material ............................................................................................................................. 23 Chemicals Tested ........................................................................................................................ 23 Plant Extracts Tested ................................................................................................................... 24 Behavioral Bioassay .................................................................................................................... 24 Field Trial .................................................................................................................................... 26 3 RESULTS .................................................................................................................................... 29 Schistocerca americana .............................................................................................................. 29 Behavioral Bioassays: Commercial Formulations ............................................................. 29 Field Trial ............................................................................................................................. 30 Behavioral Bioassays: Plant Extracts ................................................................................. 30 Diaprepes abbreviatus ................................................................................................................ 31 Behavioral Bioassays: Commercial Formulations ............................................................. 31 Field Trial ............................................................................................................................. 32 Behavioral Bioassays: Plant Extracts ................................................................................. 32 4 DISCUSSION .............................................................................................................................. 46 Behavioral Bioassays: Commercial Formulations .................................................................... 46 Field Trial .................................................................................................................................... 52 Behavioral Bioassays: Plant Extracts ......................................................................................... 52

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6 REFERENCES ................................................................................................................................... 55 BIOGRAPHICAL SKETCH ............................................................................................................. 63

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7 LIST OF TABLES Table page 2 1 Chemicals evaluated for antifeedant activity against S americana nymphs and D abbreviatus adults. ................................................................................................................. 28 2 2 Temperature (C) profile of the days on which antifeedants residual activity was tested ....................................................................................................................................... 28

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8 LIST OF FIGURES Figure page 3 1 Total area consumed of untreated and treated leaf disks, by S. americana nymphs, in choice bioassays ..................................................................................................................... 34 3 2 Total average area consumed of untreated and treated leaf disks, by S. americana nymphs, in no-choice bioassays. ........................................................................................... 35 3 3 Total leaf area eaten (cm2) by S. americana nymphs when exposed to the most effective feeding deterrents in multiple -choice situatio ns. .................................................. 36 3 4 Consumption of azadirachtin treated and control citrus disks after three time intervals of sunlight exposure on three trials ....................................................................................... 36 3 5 Consumption of ryanodine treated and control citrus disks after three time intervals of sunlight exposure on three trials ....................................................................................... 37 3 6 Consumption of sabadilla treated and control citrus di sks after three time intervals of sunlight exposure on three trials ............................................................................................ 37 3 7 Total area consumed of untreated and plant extracts -treated leaf disks, by S. americana nymphs, in choice bioassays. .............................................................................. 38 3 8 Total average area consumed of untreated and plant extracts -treated leaf disks, by S. americana nymphs, in no -choice bioassays. ........................................................................ 39 3 9 Total area consumed of untreated and treated leaf disks, by D. abbreviatus in choice bioassays. ................................................................................................................................ 40 3 10 Total average area consumed of untreated and treated leaf disks, by D. abbreviatus adults, in no-choice bioassays. .............................................................................................. 41 3 11 Total leaf area eaten by D. abbreviatus adults when exposed to the most effective feeding deterrents in multiple -choice situations ................................................................... 42 3 12 Consumption of rotenone treated and control citrus disks after three time intervals of sunlight exposure on three trials ............................................................................................ 42 3 13 Consumption of r yanodine treated and control citrus disks after three time intervals of sunlight exposure on three trials. ...................................................................................... 43 3 14 Consumption of sabadilla treated and control citrus disks after three time inte rvals of sunlight exposure on three trials ............................................................................................ 43

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9 3 15 Total area consumed of untreated and plant extracts -treated leaf disks, by D. abbreviatus adults, in choice bioassays. ............................................................................... 44 3 16 Total average area consumed of untreated and plant extracts -treated leaf disks, by D. abbreviatus adults, in no choice bioassays. .......................................................................... 45

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10 Abstract of Thesis Presente d to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ANTIFEEDANT EFFECT OF COMMERCIAL CHEMICALS AND PLANT EXTRACTS AGAINST Schistocerca americana (ORTHOPTERA: ACRIDIDAE) AND D iaprepes abbreviatus (COLEOPTERA: CURCULIONIDAE) By Andres Felipe Sandoval Mojica August 2009 Chair: John Capinera Major: Entomology and Nematology I investigated the deterrent effect of seven botanical and three inorganic agricultural products against nymphs of the American bird grasshopper, Schistocerca americana, and adults of the sugarcane rootstock weevil, Diaprepes abbreviatus Methanol and methylene chloride extracts of the Florida rosemary, Ceratiola ericoides yellow star anise, Illicium parviflorum and scratchthroat, Ardisia crenata were also tested as potential feeding deterrents. Antifeedant activity was assayed using a leaf disk bioassay, in choice and no -choice tests. The residual activity of the agricultural products that showed a signif icant antifeedant activity in leaf disk bioassays was assayed by applying them in a no choice test to foliage of Citrus paradisi plants exposed to three time intervals of sunlight. Sabadilla, azadirachtin and ryanodine effectively deterred S. americana whe reas rotenone, sabadilla and ryanodine reduced the feeding activity of D. abbreviatus in choice and no -choice leaf disk bioassays. Rapid loss of effectiveness was observed under field conditions. Sabadilla was the only compound that maintained its antifeed ant properties in the field, but only against S. americana. Methanol and methylene chloride extracts of C. ericoides deterred D. abbreviatus but only methylene chloride extract dissuaded S. americana. Methanol extract of A. crenata functioned as a feeding deterrent against both S.

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11 americana and D. abbreviatus whose was also deterred by methylene chloride extract of A. crenata Extracts of I. parvifolium only dissuaded the insects in choice bioassays. Based on their deterrency, some of the agricultural bota nical products and plant extracts have potential for use as substitute crop protectants against these two species

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12 CHAPTER 1 INTRODUCTION Conceptual Framework Feeding deterrents, or antifeedants, are chemical compounds that prevent or suspend the feedin g behavior of an insect when they are detected (Schoonhoven 1982). They can be found amongst the major classes of secondary metabolites: alkaloids, terpenoids and phenolics (Koul 2008), but other organic and inorganic compounds can inhibit also food uptake by insects (Glen et al. 1999, Wei et al. 2000). Insects are able to detect antifeedants through contact chemoreceptors, characterized by the presence of a small number of bipolar sensory neurons (three to 10) within sh ort hairs, spines or bristles ( sensil la trichoidea or sensilla chaetica ), pegs (sensilla basiconica), or cones ( sensilla styloconica ), with a single terminal pore. Sensory (gustatory) neurons can be phagostimulatory or deterrent cells. Activity of these neurons in response to appropriate stim uli would enhance or reduce feeding, respectively (Chapman 2003, Koul 2008). Feeding deterrents may be perceived by insects due to several chemoreception mechanisms (Schoonhoven 1982, Chapman et al. 1991): 1 Stimulation of specialized deterrent receptors 2 Dis tortion of the normal function of neurons that perceive phagostimulatory compounds. 3 Excitation of broad spectrum receptors. 4 Changes in complex and subtle sensory codes as a consequence of stimulation activity in some nerve cells and inhibition in others. 5 Production of a highly abnormal impulse pattern, often at high frequencies, and/or bursting activities. Some antifeedants influence insect feeding activity through a combination of these modes of action (Schoonhoven 1982, Chapman 2003, Koul 2008).

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13 Reducti on or complete inhibition of feeding, using organic derivatives, crude plant extracts and/or pure allelochemicals as antifeedants, has been demonstrated in several orders such as Lepidoptera, Coleoptera, Hemiptera and Orthoptera. Regarding Lepidoptera, Ak htar and Isman (2004) observed antifeedant effects of crude extracts of Melia volkensii (Meliaceae) on third instar larvae of the cabbage looper, Trichoplusia ni Hbner (Noctuidae), and the armyworm, Pseudaletia unipuncta Haworth (Noctuidae). Methanol, ace tone and chloroform extracts of Justicia adhatoda L. (Acanthaceae), Ageratum conyzoides L. (Asteraceae) and Plumbago zeylanica L. (Plumbaginaceae), respectively, were strongly deterrent to feeding of Spilarctia obliqua Walker (Noctuidae) at a dose of 10 m g/ml (Prajapati et al. 2003). Ulrichs and collaborators (2008) detected antifeedant and toxic effects of Porteresia coarctata Takeoka (Poaceae) hexane extracts on Spodoptera litura F. (Noctuidae) third instar larvae due to the presence of terpenoids in the plant. Meliaceae and Labiatae also produce terpenes that modify the feeding behavior of insects: a mixture of limonoids, obtained from seeds of Trichilia havanensis Jacq. (Meliaceae), and the diterpene scutecyprol A isolated from Scutellaria valdiviana (Clos) Epling (Labiatae), reduced the feeding activity of Spodoptera exigua (Hbner) (Noctuidae) in choice and no choice bioassays (Caballero et al. 2008). Azadirachtin, a triterpenoid obtained from the neem tree, Azadirachta indica A. Juss (Meliaceae), redu ced food consumption of Heliothis virescens (F.) (Noctuidae) and Lymantria dispar L. (Limantriidae) larvae (Yoshida and Toscano 1994, Kostic et al. 2008). Concerning Coleoptera, extracts of Humulus lupulus L. (Cannabaceae) and Xanthium strumarium L. (Aster aceae) at a concentration of 20 g kg1, inhibited the feeding behavior of Colorado potato beetle larvae, Leptinotarsa decemlineata Say ( Gke et al. 2006), which was also deterred by ichangensin, a terpenoid (limonoid aglycone) present in the family Rutace ae

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14 (Murray et al. 1999). Terpenes isolated from Lansium domesticum Corr. Serr. (Meliaceeae), as well as crude extracts of the plant, showed antifeedant activity against the rice weevil Sitophilus oryzae L. (Curculionidae) (Omar et al. 2007). Methanol extra cts of Momordica charantia L. (Cucurbitaceae) strongly deterred cucurbitaceous feeding beetle species of the genus Aulacophora and Epilachna (Abe and Matsuda 2000). Villani and Gould (1985), after screening extracts from 78 plant species, discovered that A sclepias tuberosa L. (Asclepiadaceae) and Hedera helix L. (Araliaceae) provided high levels of feeding deterrency against the corn wireworm, Melanotus communis (Gyllenhal) (Elateridae). Among the Hemiptera, the milkweed bug, Oncopeltus fasciatus Dallas (Ly gaeidae), was deterred from feeding by extracts of dried roots of Inula helenium L. (Asteraceae) when applied to sunflower kernels (Alexenizer and Dorn 2007). Reuter and colleagues (1993), using an electronic feeding monitor, observed a reduction in the ti me spent in ingestion by the green peach aphid, Myzus persicae (Sulzer) (Aphididae), when it was feeding on lettuce plants treated with azadirachtin and an acrylic copolymer. Within the Orthoptera, the graminivorous pest Locusta migratoria L. (Acrididae) i s deterred by crude methanol extracts of Cryptomeria japonica (L. f.) D. Don (Taxodiaceae) (Kashiwagi et al. 2007), and also by azadirachtin, nicotine, coumarin and a mixture of phenolic compounds obtained from Sorghum bicolor (L.) Moench (Poaceae) (Adams and Bernays 1978). Gramine, 3 (dimethylaminomethyl) indole, the principal alkaloid in barley, Hordeum vulgare L. (Poaceae), inhibits as well the feeding behavior of L. migratoria (Ishikawa and Kanke 2000). Rathinasabapathi et al. (2007) observed that arsenic (As) present in the Chinese brake fern, Pteris vittata L. (Pteridaceae), reduced frond tissue consumption by nymphs of the American bird grasshopper, Schistocerca americana Drury (Acrididae).

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15 Feeding deterrents can protect plants from insect herbivory by making the hosts less palatable or even toxic to the arthropods. Therefore, antifeedants are a useful element for integrated pest management programs that involve behavioral manipulation of insect pests, like push -pull strategies where a protected plant resource is made unattractive or inappropriate to the pests (push) while drawing them into an attractive source (pull) from where the pests are subsequently removed (Jain and Tripathi 1993, Cook et al. 2007). Furthermore, antifeedants are usually safer alt ernative crop protectants than conventional synthetic pesticides due to their low toxicity, specificity, and protection to non target organisms (insects, birds or mammals) that are indispensable for interactions like natural biological control and pollinat ion (Isman 2006). Moreover, antifeedants are advantageous due to their effectiveness at small concentrations and quick degradation. This can result in lower non-target hazard and reduced pollution problems relative to conventional pesticides. Typical probl ems associated with conventional insecticides include ground water contamination, the development of pesticide resistant pest populations, and acute and chronic intoxication of applicators, farm workers and consumers (Isman 2006, Koul 2008, Rosell et al. 2008). One of the weak points of investigations into the occurrence of antifeedant substances is that in most cases only one test insect species is used. In most papers the structure of the compound(s) found to be active in laboratory tests on one or a fe w insect species is reported, but no further attempts are made to find out against what other species the substance is active, or how stable the compound is under natural conditions (Jermy 1990). The purpose of the present study was to test the antifeedan t effect of the botanical derivatives azadirachtin, neem oil, sabadilla, rotenone, ryanodine, capsaicin and garlic juice, as well as inorganic substances, such as diatomaceous earth, elemental sulfur and kaolin clay. Tests

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16 were conducted under laboratory a nd field conditions, using behavioral bioassays, against nymphs of the American bird grasshopper, Schistocerca americana (Drury) (Orthoptera: Acrididae), and adults of the West Indian sugarcane rootstock borer weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), two generalist herbivores. In addition, crude extracts of the Florida rosemary, Ceratiola ericoides Michx (Empetraceae), yellow star anise, Illicium parviflorum Michaux ex Ventenat (Illiciaceae), and scratchthroat, Ardisia crenata Sims (Myr sinaceae) were tested as potential antifeedants for both insect species. Pest Insects The American bird grasshopper, S chistocerca americana (Drury), is one of the most destructive grasshopper species in the southeastern United States. It is found througho ut the eastern United States west to the Rocky Mountains. T his grasshopper's range also includes the Bahamas and Mexico (Thomas 1991). It occasionally reaches outbreak status, at least locally, and can cause injury to corn, oats, rye, peanuts, sugarcane, t obacco, cotton, vegetables, flowers, ornamental shrubs and citrus. The American grasshopper caused, during 1991, significant loss to citrus and ornamental plants in west -central Florida and limited damage to field crops and ornamentals in north -central Flo rida (Capinera 1993a). Young citrus trees are damaged by the grasshoppers gnawing on the leaves and most of the feeding damage is caused by the third, fourth and fifth instars, which have a much larger appetite than the adults. Although a highly polyphago us acridid, S. americana does exhibit a clear preference (e.g., smooth crabgrass, bahiagrass, hyssop spurge, soybean, corn, pecan) and non -preference (e. g., cucumber, nandina, pepper) for certain host species (Capinera 1993a, Smith and Capinera 2005). The American grasshopper has two generations per year and overwinters in the adult stage; therefore, it is present throughout the year. Females lay their eggs in the soil, about 2 or 3 cm below the surface, and are able to deposit up to three egg clusters (ea ch cluster pod usually consist of 60 to 80

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17 eggs). The groups of eggs are held together by proteinaceous foam which serves to cement the surrounding soil particles together to form a more or less discrete capsule, or pod. Nymphs hatch from the eggs after three or four weeks and undergo between five and six molts before reaching adulthood (Thomas 1991, Capinera 1993b). The West Indian sugarcane rootstock borer weevil, Diaprepes abbreviatus L., is native to the Lesser Antilles of the Caribbean region and has become an important long-term pest of citrus and ornamental crops in Florida (Hall 1995). Since its introduction to Florida in 1964, D. abbreviatus has spread into approximately 23,000 ha of commercial citrus in 17 counties and 94 commercial citrus and or namental plant nurseries (Woodruff 1968, Hall 1995). It is a highly polyphagous species; Simpson et al. (1996) listed numerous host plants associated with this pest, including 270 species in 157 genera from about 59 families. Adult weevils feed on plant fo liage, leaving characteristic semicircular notches along the edges of young leaves, and oviposit on preferred host plants. Females deposit clusters of eggs between leaves that are glued together by secretion of a sticky substance that cements the leaf surf aces. Each cluster contains around 50 eggs, and female weevils are able to deposit as many as 5,000 to 29,000 eggs during their lifespan (three to four months). As the eggs hatch, after seven to 10 days, the larvae fall to the soil surface where they burro w into the soil to feed on the roots of the plant where they remain for 8 12 months, until adult emergence (Woodruff 1968, Hall 1995, Mannion et al. 2003). Commercial Chemicals Tested Neem oil and azadirachtin are biopesticides obtained from the Indian nee m tree, Azadirachta indica A. Juss (Meliaceae). Neem oil is acquired by cold -pressing the seeds of the neem tree and is useful in the control of soft bodied insects (Bruce et al. 2004), mites and phytopathogens due to the presence of disulfides that contri bute to its bioactivity. Azadirachtin, a limonoid or tetranortriterpenoid, is found in the extracts of the seed residues after removal of the

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18 oil (Isman 2006). Several effects, both physiological and behavioral, have been reported on insects (Schmutterer 1 990): disruption of growth, development and reproduction by blocking the synthesis and release of molting hormones, and repellency and oviposition deterrence. Azadirachtin and neem seed extracts also seem to have a potent antifeedant effect on a large numb er of pest insects, including orthopterans (Aerts and Mordue 1997, Capinera and Froeba 2007), hemipterans (Kumar and Poehling 2007), coleopterans (Baumler and Potter 2007), and lepidopterans (Aerts and Mordue 1997, Yoshida and Toscano 1994, Liang et al. 2003, Showler et al. 2004, Charlston et al. 2005). Sabadilla is a botanical insecticide obtained from the seeds of Schoenocaulon officinale Grey (Liliaceae). The major active ingredients are cevadine and veratradine, which are esters of a steroidal alkaloid named veracevine (Rosell et al. 2008). These alkaloids bind the activation gate of the sodium channels in the nervous system of insects, resulting in persistent neuroexcitation. Although sabadilla has a high mammalian toxicity, commercial preparations nor mally contain less than 1% active ingredient, providing a margin of safety (Isman 2006). Sabadilla is used mostly for control of thrips populations in citrus and avocado (Hare and Morse 1997, Humeres and Morse 2006) but it has potential as an antifungal a gent (Oros and Ujvary 1999) and as a feeding deterrent as well (Yoshida and Toscano 1994). Rotenone is one of several isoflavonoid compounds produced in the roots of the tropical legumes Derris, Lonchocarpus and Tephrosia (Leguminosae). Rotenone is a mitoc hondrial poison that blocks the electron transport chain and prevents energy production. It has been used as an insecticide, acaricide and piscicide and is commonly sold as a dust containing 1% to 5% active ingredients for home and garden use, though liqui d formulations used in organic agriculture can contain between 8% and 15% (Isman 2006, Rosell et al. 2008). Rotenone can act

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19 as a feeding deterrent against stored product insect pests (Nawrot et al. 1989) and polyphagous noctuid species (Wheeler et al. 2001). Ryanodine is an alkaloid obtained by grinding the wood of the Caribbean shrub Ryania speciosa Vahl (Flacourtiaceae) (Isman 2006). This botanical pesticide is active on the muscular system, specifically neuro -muscular calcium channels. Ryanodine acts a s an agonist by enhancing calcium output of the sarcosome tubule network that surrounds muscle fibers, resulting in continual calcium availability and a continual state of muscle contraction (Nauen 2006). Ryanodine and related ryanoids deter feeding by lepidopteran and coleopteran pests (Yoshida and Toscano 1994, Gonzalez -Coloma et al. 1999). Capsaicin, extracted from the hot cayenne pepper, Capsicum annuum L. (Solanacaeae), is a derivative of vanillyl amine (8 -methyl N -vanillyl 6 -noneamide), a compound tha t produces the hotness in some species of the plant genus Capsicum Capsaicin is currently registered by the US Environmental Protection Agency (EPA) for use as an insect repellent and toxicant as well as a vertebrate repellent for dogs, birds, voles ( Mi crotus spp), deer (Odocoileus spp), rabbits (Sylvilagus spp.) and squirrels ( Sciurus spp.) (EPA 1996). Capsaicin has been tested as a leaf protector against scarab pests, including the rose chafer, Macrodactylus subspinosus (F.) (Isaacs et al. 2004) and th e Japanese beetle, Popillia japonica Newman (Baumler and Potter 2007) but without consistent results. Garlic extracts derived from Allium sativum L. (Liliaceae) have shown insecticidal activity to dipteran pests (Prowse et al. 2006) as well as antifeedant effects on coleopteran stored products pests (Chiam et al. 1999) and moths (Gurusubramanian and Krishna 1996). Diatomaceous earth is composed of the fossilized skeletons of various species of marine and fresh water phytoplankton, predominantly diatoms and other siliceous algae which existed

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20 during the Eocene and Miocene periods. The diatomaceous earth active ingredient is amorphous silicon dioxide (silica), which can damage the insects epicuticular lipids by hydrocarbon absorption and abrasion, making the cuticle permeable and therefore causing death due to water loss and desiccation (Korunic 1997). Diatomaceous earth has proved to be useful in the control of stored products insects such as Cryptolestes ferrugineus Stephens (Laemophloeidae) (Fields and Kor unic 2000), Rhyzopertha dominica F. (Bostrichidae) (Ferizli and Beris 2005) and the confused flour beetle, Tribolium confusum Jacquelin du Val (Tenebrionidae) (Dowdy and Fields 2002, Vayias et al. 2006). Sulfur is a non-systemic contact and protectant fung icide with secondary acaricidal activity. It is used primarily to control powdery mildews, certain rusts, leaf blights and fruit rots. Spider mites, psyllids and thrips are also vulnerable to sulfur. This chemical is known to be of low toxicity and poses l ittle if any risk to human and animal health (Lamberth 2004). Kaolin is a soft, white, clay mineral that can be mixed with water and sprayed on plants in order to form a protective particle film. Kaolin is a nonabrasive aluminosilicate (Al4Si4O10(OH)8), th at can reduce feeding and oviposition of arthropods by entangling mouthparts and restraining mobility over treated plant surfaces (Glenn et al. 1999). Furthermore, k aolin may interfere with insects contact chemoreceptors, causing plants to be unrecognizable as a host (Puterka et al. 2000). Spraying kaolin on crops has been effective against pests belonging to different orders including Hemiptera (Glenn et al. 1999, Puterka et al. 2000, Cottrell et al. 2002, Daniel et al. 2005), Coleoptera (Lapointe 2000) a nd Lepidoptera (Knight et al. 2000). Plant S pecies Used To Obtain The Extracts The Florida rosemary, Ceratiola ericoides Michx is an indigenous plant restricted to the Florida scrub community, growing on excessively to well -drained sandy soils. This plant is a dioecious perennial evergreen shrub which can grow as tall as 2 m in height. Ceratiola ericoides

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21 has a whorled branching pattern, with leaves strongly revolute (needle like) of 8 12 mm (Wunderlin and Hansen 2002). The absence of herbaceous growth aro und C. ericoides plants demonstrates the allelopathic effects that this species exerts on others. Examples include suppressing germination of Eryngium cuneifolium Small (Apiaceae) and Hypericum cumulicola (Small) P. Adams (Hypericaceae) by leaf and litter leachates (Hunter and Menges 2002), and affecting radicle growth and germination of sandhill grasses such as little bluestem, Schizachyrium scoparium (Michx.) Nash (Poaceae) and green sprangletop, Leptochloa dubia (Kunth) Ness (Poaceae), (Fischer et al. 19 94). Several chemicals have been isolated from the Florida rosemary including the dihydrochalcone flavonoid ceratiolin (Tanrisever et al. 1987, Tak et al. 1993) which seems to be the precursor of the photochemically activated hydrocinnamic acid, a germinat ion and growth inhibitor of grasses and pines (Fischer et al. 1994). Illicium parviflorum Michaux ex Ventenat, commonly known as swamp star anise, or yellow star anise, is an indigenous species of moist forests and swamps of central Florida. It is a broadl eaf evergreen large shrub or small tree with highly aromatic anise -scented foliage that can grow up to 15 feet in height (Osorio 2001). Illicium parviflorum is used in landscapes because of its considerable drought tolerance, capacity to grow under a broad range of light conditions, and resistance to pests. Sharma and Rich (2005) assessed reproduction of three root knot nematode species ( Meloidogyne arenaria, M. incognita and M. javanica) on five native plants and three non -native plants to the southeastern USA and observed very few or no galls on roots of I. parvifolium Secondary metabolites such as sesquiterpene lactones (Schmidt 1999) have been isolated from leaves of I. parviflorum as well as safrole (68.14 0.88%), linalool (13.18 1.01%) and methyl eugenol (11.89 0.87%), the main components of the essential oil of yellow anise tree (Tucker and Maciarello 1999).

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22 Ardisia crenata Sims, or scratchthroat, is an evergreen small shrub (0.5 1 m in height) native to Japan to north India that grows in mult i -stemmed clumps. It has alternate leaves (dark green above, waxy, glabrous with crenate margins) of 21 mm ( Wunderlin and Hansen 2002). Ardisia crenata was introduced to the USA for ornamental purposes and has become established in much of northern and cen tral Florida. In some areas, it is a serious pest, displacing native species in the understory of hardwood forests by creating dense local shade (Kitajima et al. 2006). Several studies have revealed the presence, in the genus Ardisia of phytochemicals suc h as triterpenoid saponins, isocoumarins, quinones and alkylphenols (Kobayashi and de Mejia 2004, Liu et al 2007) that exhibit a wide range of bioactivities such as uterus contraction, inhibition of cyclic adenosine monophosphate phosphodiesterase, cytotox icity, antiHIV and anti -cancer among others (Kobayashi and de Mejia 2004). The presence of these secondary metabolites may cause A. crenata to be an unsuitable host for arthropod feeders. For example, Neal and colleagues (1998) observed a reduction in the number of eggs laid by the twospotted spider mite, Tetranychus urticae Koch, as well as a higher nymphal mortality of the whiteflies Bemisia argentifolii Bellows & Perring and Trialeurodes vaporariorum (Westwood) when developing on A crenata leaves, comp ared with other three host plants of the same genus. O bjectives To test the antifeedant properties of azadirachtin, neem oil, sabadilla, rotenone, ryanodine, capsaicin, garlic juice, diatomaceous earth, elemental sulfur and kaolin clay against fifth instar nymphs of S. americana and adults of D abbreviatus using behavioral bioassays. To test the residual activity under field conditions of the commercial formulations that are the most deterrent to the feeding of the two species of insects in the laboratory. To obtain crude extracts from C. ericoides I. parviflorum and A. crenata plants, and to test their antifeedant properties against nymphs of S. americana and adults of D. abbreviatus using behavioral bioassays.

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23 CHAPTER 2 MATERIALS AND METHOD S Insect M a terial The American grasshoppers used in this study were from a laboratory colony that has been maintained for approximately 10 years in the Entomology and Nematology Department at the University of Florida. The insects had free access to water and a dry d iet consisting of whole wheat flour (one part), soy flour (one part) and wheat bran (two parts), supplemented with romaine lettuce, Lactuca sativa var. longifolia Lam, (Asteraceae). The nymphs and adults were maintained at 27C, although they had access to 90 W light bulbs, approximately 10 cm away from the cages, so they could attain a warmer temperature if desired. They also were provided with a photoperiod of 16:8 (L:D) and a relative humidity of about 58%. Adult sugarcane rootstock borer weevils were fr om a rearing facility of the Florida Department of Agriculture & Consumer Services, Division of Plant Industry (DPI), at Gainesville, Florida, where they had been maintained for 5 years. The insects were maintained in plastic cages of 30 x 30 x 30 cm, 60 w eevils per cage, with free access to water and a diet consisting of romaine lettuce ( Lactuca sativa var. longifolia Lam) and store -bought carrot roots [Daucus carota L. (Apiaceae)]. The adult weevils were maintained at 28C and a relative humidity of about 58%. Chemicals Tested Ten bioinsecticides and putative antifeedants/repellents were evaluated for deterrence to S. americana, and D. abbreviatus (Table 1). They included seven botanical derivatives (azadirachtin, neem oil, sabadilla, rotenone, ryanodine, capsaicin and garlic juice) and three inorganic materials (diatomaceous earth, elemental sulfur and kaolin clay). Products were applied at label rates, using the highest concentration commercially recommended.

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24 Plant Extracts Tested The plant extracts were prepared based on the procedures described by Gke and collaborators (2005). Samples of Florida rosemary, C. ericoides were collected in the Ordaway Swisher Biological Station (Putnam County, Florida), during fall of 2007 and 2008. Samples of yellow sta r anise tree, I. parviflorum and scratchthroat, and A. crenata were obtained in the city of Gainesville (Alachua County, Florida) during fall of 2008. Leaf samples were dried at room temperature in the dark for 3 weeks and afterward ground in a Wiley mill ( Model 3383-L10 Thomas Scientific Swedesboro, NJ) using a mesh size of 40. The ground leaf material was stored in plastic containers at 80C. Ten gram samples of dried plants were placed into 125 ml Erlenmeyer flasks and treated with 100 ml of methanol (Fisher Scientific, Fair Lawn, NJ) or methylene chloride (Fisher Scientific, Fair Lawn, NJ). Flasks were covered with aluminum foil, placed on an orbital shaker (Model 361, Fisher Scientific, Pittsburgh, PA), and shaken (120 oscillations/min) for 24 h in the dark at 24C. The suspension was filtered first through two layers of cheese cloth (XL 400, Cotton TM, Worcester, MA) followed by vacuum filtration. The filtrate was transferred into a 250 ml evaporating flask and dried at 40C in a rotary evaporator (RE 111 Bchi Switzerland), in order to evaporate the excess methanol or methylene chloride. The resulting residues were weighed and mixed with acetone to yield a 20% (w/w) plant suspension. Behavioral Bioassay Antifeedant activity of test substances was assayed using a leaf disk bioassay, in both choice and no-choice formats. The bioassays were conducted in round transparent plastic containers of 18 cm diameter and 8 cm height. A moist paper towel (Prime Source) was placed at the bottom of each container i n order to maintain high humidity and to keep the foliage fresh. The leaf disks were pinned on a cork base of 1.3 cm height.

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25 Leafs were removed from healthy romaine lettuce plants purchased from a grocery store, and leaf disks were cut using a no. 15 cork borer (2 cm diameter, 3.14 cm2 area). Foliage disks were cut immediately before application of treatment solutions, minimizing changes in leaf quality. After leaf disks were cut, they were immediately dipped into one of the treatments solutions for 10 sec The disks were left to dry under a fume hood for 15 min at room temperature. Control disks were dipped into water and were left to dry at room temperature. In the case of the plant extracts, the control disks were dipped into acetone. A single fifth inst ar nymph of S. americana, 2 3 days after molting from fourth instar, was added to each container and allowed to feed for 24 h at 27C. Nymphal instars were determined according to the methods of Capinera (1993b.). For D. abbreviatus one 7 10 dayold adult was placed into each container and allowed to feed, also for 24 h at 27C. The test insects were starved for 12 h prior to the experiments. For every chemical substance evaluated, in both choice and no -choice situations, twenty replicate containers were t ested on three different days (n=20, N=60). On each day a different set of insects was assayed and a fresh preparation of each compound was tested. In choice tests, one treated and one control disk were placed in each container. The distance between the t wo disks was 13 cm. In order to ensure that bioassays were not hunger biased, experiments were stopped when grasshoppers or weevils had consumed 50% of either disk. The bioassays were checked periodically. If the insects had not consumed 50% of either disk after 24 h, the experiment was terminated. In no -choice tests, either two treated leaf disks or two untreated (control) disks were placed in each container. The average of the leaf area consumed in both disks was used for data analysis. No choice tests we re only stopped after 24 h. Control and treatment evaluations were done simultaneously. Only the biological pesticides that demonstrated a significant antifeedant activity in the choice tests were evaluated using no -choice

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26 tests. The remaining area of the disks (treated and controls) was measured using a leaf area meter (LI 3000A, LI -COR, Lincoln, Nebraska, USA). In order to determine the most effective insect antifeedant, multiple choice bioassays that included three treatments in the same container were c arried out for 24 h. The treatments chosen for comparison showed a significant reduction in the consumed leaf area in the control vs. treatment no choice assays. The consumed area of the leaf disks (treated and controls) in both the choice and the nochoi ce tests were used for the data analysis. This was obtained by subtracting the remaining area, calculated with the leaf area meter, from the total area of each leaf disk (3.14 cm2). Shapiro Wilk normality tests and Levene tests of homogeneity of variances were employed to determine the type of distribution for the data obtained in every experiment. For choice tests, paired t tests or sign tests, depending on data distribution, were used to test for significant differences in consumption levels of treatment and control disks (Horton 1995). In nochoice bioassays, t -tests for independent samples (parametric data) or Mann Whitney U test (nonparametric) were used to evaluate the dissimilarities in area consumed in the control and treatment disks. Parametric one way ANOVAs or Kruskal -Wallis nonparametric one -way ANOVAs were used to compare the leaf area consumed (control and treatment) between the three replicates (n=20, N=60) utilized to assess every treatment in choice and no -choice bioassays. A Friedman ANOVA w as used to analyze the results of the multiple-choice bioassays, separating means with Mann Whitney U test. P values <0.05 were considered to be statistically significant. STATISTICA 6.0 (Stat Soft, Inc., Tulsa, OK) software was used for the data analysis. Field Trial To test the residual activity under field conditions of the chemicals that showed a significant antifeedant activity against S. americana and D. abbreviatus applications of the most

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27 effective treatments were made to 5 year -old, randomly selec ted, Citrus paradisi MacFad (Rutaceae) potted plants. For each treatment, applications were made at 7 a.m, 11 a.m and 3 p.m. At every time interval, treatments were applied by a number 4 flat brush to the adaxial surface of five leaves (per treatment), on the same plant. The painted leaves were collected at 7 p.m. on the day of application in order to obtain 4 h (3 p.m), 8 h (11 a.m) and 12 h (7 a.m) of sunlight exposure. Also, five unpainted leaves were collected at 7 p.m. as controls. The selected leaves were undamaged and apparently of the same age, based on size, coloration and turgidity. From the painted leaves for each treatment, 10 leaf disks (2.25 cm, diameter, 3.97 cm2 area) were cut and provided in pairs to individual fifth instar nymphs of S. amer icana or pairs of adults of D. abbreviatus in a no -choice format (five replicates per time application, per treatment). Control leaves were handled in the same way. After 24 h the average of the leaf area consumed in both disks was used for data analysis. The field experiment was repeated twice for a total of three trials, for both insect species, using a different citrus plant each time. Applications were made on November 17, 23 and 27 of 2008 for the S. americana trials, and on February 17, 21 and 23 of 2 009 for the D. abbreviatus trials. There was no precipitation on any of the application dates. The temperature profile on the six dates is presented in the Table 3 2. Factorial ANOVAs were used in order to analyze the individual and interactive effects of the hours of sunlight exposure and the three trials made per treatment, on leaf consumption. Means were separated with Tukeys test ( P =0.05). STATISTICA 6.0 (Stat Soft, Inc., Tulsa, OK) software was used for the data analysis.

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28 Table 2 1. Chemicals ev aluated for antifeedant activity against S americana nymphs and D abbreviatus adults. Active ingredient (ai) Trade name AI (% in product) Application rate (ml or g/liter) Product Source Neem oil Pure Neem Oil 100 8 ml Dyna Gro, San Pablo, CA Azadirachtin Azatrol EC 1.2 5.6 ml PBI/Gordon, Kansas City, MO Sabadilla alkaloids Sabadilla Pest Control 8 30 g Necessary Organics, New Castle, VA Rotenone Rotenone 5 5 48 g Bonide Products, Yorkville, NY Ryanodine Ryan 50 0.10 18 g Dunhill Chemical, Rosemead, CA Capsaicin and other capsaicinoids Hot Pepper Wax 0.00018 31.3 ml Hot Pepper Wax, Greenville, PA Garlic juice and oil Garlic Guard 40 50 ml Super Natural Gardner, Exeter, NH Diatomaceous earth Mother Earth D 100 NA Whitmire Micro -Gen, St. Louis, Mo Elemental sulfur Liquid Sulfur 52 19.5 ml Bonide Products, Oriskany, NY Kaolin clay Surround WP 95.0 60 g Extremely Green Gardening, Abington, MA Table 2 2. T emperature (C) profile of the days o n which antifeedants residual activity was tested Date Max C Min C Average C 11/17/08 18.33 0.00 9.44 11/23/08 20.00 0.00 10.00 11/27/08 21.67 1.11 11.67 02/17/09 19.44 0.56 10.00 02/21/09 19.44 3.33 8.33 02/23/09 16.67 0.56 9.44

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29 CHAPTER 3 RES ULTS Schistocerca americana Behavioral Bioassays : Commercial Formulations In choice tests (Figure 4 1), American grasshoppers consumed significantly more untreated (control) leaf disk material when presented simultaneously with leaf disks treated with eith er azadirachtin ( t = 12.6; df = 59; P < 0.001), sabadilla ( z = 7.62; P < 0.001), ryanodine ( t = 6.89; df = 59; P < 0.001), neem oil ( t = 9.50; df = 59; P < 0.001), capsaicin ( t = 2.50; df = 59; P = 0.02), or kaolin clay ( t = 2.63; df = 59; P = 0.01). There was not a significant difference in the leaf area consumed between the untreated and the disks treated with elemental sulfur ( z = 7.62; P = 0.52), diatomaceous earth ( t = 1.33; df = 59; P = 0.19), garlic juice ( z = 7.62; P = 0.79), or rotenone ( t = 0.86; df = 59; P = 0.39). In no -choice tests (Figure 4 2), azadirachtin ( U = 388.00; Z = 7.41; P < 0.001), sabadilla (U = 0.00; Z = 9.45; P < 0.001) and ryanodine ( U = 546.50; Z = 6.58; P < 0.001) were confirmed to be statistically significant antifeedants a gainst S. americana. Sabadilla showed a strikingly potent inhibition effect (Figure 4 2). Neem oil did not reduce the feeding behavior of the American grasshoppers significantly ( U = 1707.50; Z = 0.49; P = 0.63). Capsaicin and kaolin clay were discarded as potential S americana feeding deterrents after the first trial because nymphs completely consumed the leaf disks treated with these chemicals. The multiple -choice bioassays demonstrated that sabadilla was the most effective feeding deterrent for S. amer icana fifth instar nymphs. It exerted a strong feeding inhibiti on effect that 2 F = 51.11; df = 2; P < 0.001). Although the average leaf consumption of azadirachtin treated leaf disks was lower

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30 than in the ryanodine treated disks (Figure 4 3 ), there was not a statistically significantly difference between these treatments ( U =1566.00; Z = 1.23; P = 0.22). Field Trial Persistence of the potential antifeedants under field conditions was variable. There was no effect of sunlight exposure interv als on consumption of foliage treated with azadirachtin ( F = 0.06; df = 2; P = 0.95) or ryanodine ( F =3.06; df = 2; P = 0.053). Azadirachtin (Figure 44) did not significantly reduce herbivory by S. americana nymphs during any of the exposure periods in ea ch of the three trials. Ryanodine (Figure 4 5) only exerted significant leaf protection after 8 h of exposure to sunlight, during the second evaluation. Sabadilla (Figure 46) was the only treatment that reduced significantly the feeding by S. americana at 4 h, 8 h and 12 h of sunlight exposure, and did so in all the three trials. Consumption of leaf material in foliage treated with sabadilla did not differ statistically among the hours of exposure ( F = 2.24; df = 2; P = 0.11). There were no differences amo ng replicates for consumption of foliage treated with azadirachtin (F = 1.33; df = 2; P = 0.27), ryanodine ( F =1.15; df = 2; P = 0.32) or sabadilla ( F =3.00; df = 2; P = 0.06), as well as the interaction of this factor with the hours of exposure (azadirach tin: F = 0.33; df = 4; P = 0.86; ryanodine: F =1.83; df = 4; P = 0.13; sabadilla: F = 0.68; df = 4; P = 0.61). Behavioral Bioassays: Plant Extracts The plant extracts obtained from C. ericoides I. parviflorum and A. crenata significantly reduced the cons umption of treated leaf disks, compared to untreated (control) disks in choice tests (Figure 4 7). Both methanol ( C. ericoides : z = 2.34; P = 0.02; I. parviflorum : t = 11.31; df = 59; P < 0.001; A. crenata : t = 5.70; df = 59; P < 0.001) and methylene chlo ride extracts ( C. ericoides : z =5.29; P < 0.001; I. parviflorum : t = 14.40; df = 59; P < 0.001; A. crenata : z = 4.26; P < 0.001) significantly protected the treatment disks in this type of bioassay (Figure 4 7).

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31 In no -choice bioassays, only the C. ericoi des methylene chloride extract ( U =1294.50; Z = 2.65; P = 0.008) and the A. crenata methanol extract ( t = 2.1; df = 118; P = 0.04) functioned as antifeedants that significantly reduced herbivory by nymphs of S. americana (Figure 4 8). Leaf disks treated with I. parviflorum extracts were consumed in higher proportion than the control disks (methanol: t = 3.28; df = 118; P < 0.001; methylene chloride: t = 2.21; df = 118; P = 0.03). Treatment of leaf disks with C. ericoides methanol extract ( t = 0.48; df = 118; P = 0.63) and A. crenata methylene chloride extract ( t = 0.58; df = 118; P = 0.56) did not statistically modify the feeding behavior of the grasshoppers (Figure 4 8). Diaprepes abbreviatus Behavioral Bioassays: Commercial Formulations Feeding by the sugarcane rootstock borer weevils in choice tests was reduced in leaf disks treated with azadirachtin ( t = 4.26; df = 59; P < 0.001), neem oil ( t = 2.25; df = 59; P = 0.03), sabadilla ( z = 2.47; P = 0.01), rotenone ( z = 6.17; P < 0.001) and ryanodine ( z = 2.76; P = 0.01), as compared with untreated disks (Figure 4 9). Elemental sulfur ( z = 1.04; P = 0.30), diatomaceous earth ( t = 1.05; df = 59; P = 0.29), garlic juice ( t = 0.07, df = 59; P = 0.95), kaolin clay ( t = 1.08; df = 59; P = 0.86) and capsaicin (t = 1.82; df = 59; P = 0.07) did not significantly modify the feeding behavior of the weevils (Figure 4 9). Ryanodine ( t = 6.02; df = 118; P < 0.001), rotenone ( U = 1403; Z = 2.08; P = 0.04) and sabadilla ( U =627; Z = 2.08; P < 0.001) were the treatme nts that caused a significant antifeedant effect over D. abbreviatus adults in no -choice bioassays (Figure 4 10). There was not a significant difference between the area consumed of azadirachtin treated leaf disks and untreated ones ( t = 1.87; df = 118; P = 0.06). Neem oil treated disks were eaten in a higher proportion than control disks ( U =1319.50; Z = 2.52; P = 0.01).

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32 The multiple -choice bioassays (Figure 4 11) did not expose a significant difference amongst ryanodine, rotenone and sabadilla in terms o2 F =1.78; df = 2; P = 0.41). Field Trial The hours of sunlight exposure did not affect significantly the herbivory of citrus leaves treated with rotenone ( F = 0.28; df = 2; P = 0.75), ryanodine ( F = 1.21; df = 2; P = 0 .30) or sabadilla ( F = 0.70; df = 2; P = 0.5). At each time interval (4 h, 8 h and 12 h) these chemicals were ineffective as feeding deterrents. Rotenone (Figure 4 12), ryanodine (Figure 4 13) and sabadilla (Figure 4 14) failed to reduce leaf consumption, compared to untreated leaves, during the field evaluations. Replication did not have an effect on the leaf area consumed in foliage treated with rotenone ( F = 2.62; df = 2; P = 0.08), ryanodine ( F = 2.09; df = 2; P = 0.13) or sabadilla ( F = 2.52; df = 2; P = 0.09), as well as the interaction of this factor with the hours of sunlight exposure (rotenone : F = 1.55; df = 4; P =0.20; ryanodine: F = 0.68; df = 4; P = 0.61; sabadilla: F = 0.62; df = 4; P = 0.65). Behavioral Bioassays: Plant Extracts The plant extracts reduced feeding by sugarcane rootstock borer weevils. Regardless of the solvent used, weevils consumed significantly more of the untreated lettuce leaf disks than disks treated with C. ericoides (methanol: z = 7.62; P < 0.001; methylene chloride: z = 7.29; P < 0.001), I. parviflorum (methanol: t =4.88; df = 59; P < 0.001; methylene chloride: z =3.88; P < 0.001) and A. crenata (methanol: z = 7.10; P < 0.001; methylene chloride: z = 5.56; P < 0.001) extracts, in choice tests (Figure 4 15). In no -choice bioassays, both extracts of C. ericoides (methanol: U = 144.50; Z = 8.69; P < 0.001; methylene chloride: U =58.00; Z = 9.14; P < 0.001) and A. crenata (methanol: t = 34.54; df = 118; P < 0.001; methylene chloride: U = 89.50; Z = 8.98; P < 0.001) were confirmed to be

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33 feeding deterrents against the sugarcane rootstock borer weevils (Figure 4 16). Leaf disks treated with I. parviflorum extracts were consumed in the same proportion as the control disks (methanol: t = 1.14; df = 118; P = 0.25; methyle ne chloride: t = 1.45; df = 118; P = 0.15).

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34 Azadirachtin P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Neem oil P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Sabadilla P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Rotenone P = 0.39 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Ryanodine P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Capsaicin P = 0.02 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Garlic juice P = 0.79 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Diatomaceous earth P = 0.19 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Elemental sulfur P = 0.52 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Kaolin clay P = 0.01 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Figure 3 1. Total area consumed of untreated and treated leaf disks, by S. americana nymphs, in choice bioassays

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35 Azadirachtin P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) Sabadilla P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) Ryanodine P < 0.001 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) Neem oil P = 0.63 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) Figure 3 2 Total average area consumed of untreated and treated leaf disks, by S. americana nymphs, in no-choice bioassays.

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36 Mean SE SD Azadirachtin Sabadilla Ryanodine P < 0.001 0.0 0.5 1.0 1.5 2.0 2.5Area consumed (cm2) Figure 3 3 Total leaf area eaten (cm2) by S. americana nymphs when exposed to the most effective feeding deterrents in multiple -choice situations. Evaluation 1 Evaluation 2 Evaluation 3 Hours of exposure Treatment 4 8 12 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Mean area consumed (cm2) Hours of exposure Control 4 8 12 Figure 3 4. Consumption of azadirachtin treated and control citrus disks after three time intervals of sunlight exposure on three trials. Disks were provided to S. americana nymphs in no -choice situations. Vertical bars denote 0.95 confidence intervals

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37 Evaluation 1 Evaluation 2 Evaluation 3 Hours of exposure Treatment 4 8 12 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Mean area consumed (cm2) Hours of exposure Control 4 8 12 Figure 3 5. Consumption of ryanodine treated and control citrus disks after three time intervals of sunlight exposure on three trials. Disks were provided to S. americana nymphs in no -choice situations. Vertical bars denote 0.95 confidence intervals Evaluation 1 Evaluation 2 Evaluation 3 Hours of exposure Treatment 4 8 12 -2 -1 0 1 2 3 4 5 6Mean area consumed (cm2) Hours of exposure Control 4 8 12 Figure 3 6. Consumption of sabadilla treated and control citrus disks after three time int ervals of sunlight exposure on three trials. Disks were provided to S. americana nymphs in nochoice situations. Vertical bars denote 0.95 confidence intervals.

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38 C. ericoides (CH3OH) P = 0.02 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) C. ericoides (CH2Cl2) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) I. parviflorum (CH3OH) P < 0.001 Mean SE SD Control Treatment -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Area consumed (cm2) I. parviflorum (CH2Cl2) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Area consumed (cm2) A. crenata (CH3OH) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) A. crenata (CH2Cl2) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) Figure 3 7 Total area consumed of untreated and plant extracts -treated leaf disks, by S. americana nymphs, in choice bioassays.

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39 C. ericoides (CH3OH) P = 0.63 Mean SE SD Control Treatment 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Average Area consumed (cm2) C. ericoides (CH2Cl2) P = 0.008 Mean SE SD Control Treatment 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6Average Area consumed (cm2) I. parviflorum (CH3OH) P < 0.001 Mean SE SD Control Treatment 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Average Area consumed (cm2) I. parviflorum (CH2Cl2) P < 0.03 Mean SE SD Control Treatment 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6Average Area consumed (cm2) A. crenata (CH3OH) P = 0.04 Mean SE SD Control Treatment 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Average Area consumed (cm2) A. crenata (CH2Cl2) P = 0.56 Mean SE SD Control Treatment 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Average Area consumed (cm2) Figure 3 8 Total average area consumed of untreated and plant extracts treated leaf disks, by S. americana nymphs, in no -choice bioassays.

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40 Azadirachtin P < 0.001 Mean SE SD Control Treatment -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Area consumed (cm2) Neem oil P = 0.03 Mean SE SD Control Treatment -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Area consumed (cm2) Sabadilla P = 0.01 Mean SE SD Control Treatment -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2Area consumed (cm2) Rotenone P < 0.001 Mean SE SD Control Treatment -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Area consumed (cm2) Ryanodine P = 0.01 Mean SE SD Control Treatment -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Area consumed (cm2) Capsaicin P = 0.07 Mean SE SD Control Treatment -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Area consumed (cm2) Garlic juice P = 0.95 Mean SE SD Control Treatment 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Area consumed (cm2) Diatomaceous earth P = 0.29 Mean SE SD Control Treatment -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Area consumed (cm2) Elemental sulfur P = 0.30 Mean SE SD Control Treatment -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Area consuemed (cm2) Kaolin clay P = 0.86 Mean SE SD Control Treatment 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2Area consumed (cm2) Figure 3 9 Total area consumed of untreated and treated leaf disks, by D. abbreviatus in choice bioassays.

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41 Azadirachtin P = 0.06 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) Ryanodine P < 0.001 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) Rotenone P = 0.04 Mean SE SD Control Treatment 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Average Area consumed (cm2) Sabadilla P < 0.001 Mean SE SD Control Treatment 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6Average Area consumed (cm2) Neem oil P = 0.01 Mean SE SD Control Treatment 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6Average Area consumed (cm2) Figure 3 10. Total average area consumed of untreated and treated leaf disks, by D. abbreviatus adults, in no-choice bioassays.

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42 P = 0.41 Mean SE SD Rotenone Sabadilla Ryanodine -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8Area consumed (cm2) Figure 3 11. Total leaf area eaten by D. abbreviatus adults when exposed to the most effective feeding deterrents in multiple -choice situations Evaluation 1 Evaluation 2 Evaluation 3 Hours of exposure Treatment 4 8 12 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Mean area consumed (cm2) Hours of exposure Control 4 8 12 Figure 3 12. Consumption of rotenone treated and control citrus disks after three time intervals of sunlight exposure on three trials. Disks were provided to D. abbreviatus adults in no -choice situations. Vertical bars denote 0.95 confidence intervals.

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43 Evaluation 1 Evaluation 2 Evaluation 3 Hours of exposure Treatment 4 8 12 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0Mean area consumed (cm2) Hours of exposure Control 4 8 12 Figure 3 13. Consumption of ryanodine treated and control citrus disks after three time intervals of sunlight exposure on three trials. Disks were provided to D. abbreviatus adults in no -choice situations. Vertical bars denote 0.95 confidence intervals. Evaluation 1 Evaluation 2 Evaluation 3 Hours of exposure Treatment 4 8 12 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Mean area consumed (cm2) Hours of exposure Control 4 8 12 Figure 3 14. Consumption of sabadilla treated and control citrus disks after three time intervals of sunlight exposure on three trials. Disks were provided to D. abbreviatus adults in no -choice situations. Vertical bars denote 0.95 confidence intervals.

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44 C. ericoides (CH3OH) P < 0.001 Mean SE SD Control Treatment -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Area consumed (cm2) C. ericoides (CH2Cl2) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) I. parviflorum (CH3OH) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Area consumed (cm2) I. parviflorum (CH2Cl2) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0Area consumed (cm2) A. crenata (CH3OH) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0Area consumed (cm2) A. crenata (CH2Cl2) P < 0.001 Mean SE SD Control Treatment -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Area consumed (cm2) Figure 3 15. Total area consumed of untreated and plant extracts treated leaf disks, by D. abbreviatus adults, in choice bioassays.

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45 C. ericoides (CH3OH) P < 0.001 Mean SE SD Control Treatment -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) C. ericoides (CH2Cl2) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) I. parviflorum (CH3OH) P = 0.25 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) I. parviflorum (CH2Cl2) P = 0.15 Mean SE SD Control Treatment 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) A. crenata (CH3OH) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Average Area consumed (cm2) A. crenata (CH2Cl2) P < 0.001 Mean SE SD Control Treatment -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Average Area consumed (cm2) Figure 3 16. Total average area consum ed of untreated and plant extracts -treated leaf disks, by D. abbreviatus adults, in no -choice bioassays.

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46 CHAPTER 4 DISCUSSION Behavioral Bioassays: Commercial Formulations In this research, sabadilla was the most effective feeding deterrent against S. americana. Alkaloids, the active ingredient in sabadilla, have previously been show to produce an antifeedant effect on grasshoppers. Nicotine hydrogen tartrate, as well as gramine, were able to reduce the feeding behavior of S. americana (White and Chapman 1990, Chapman et al. 1991, Bernays 1991), L. migratoria (Ishikawa and Kanke 2000), Ageneotettix deorum Scudder and Phoetaliotes nebrascensis Scudder (Mole and Joern 1994). The two former species were also deterred by the alkaloid eserine (Mole and Jo ern 1994). Alkaloids seem to be perceived in the American bird grasshopper by the stimulation of a specialized deterrent receptor. Prior electrophysiological studies have shown that contact chemoreceptors on the tibia and tarsus of S. americana are stimula ted by alkaloids, and have demonstrated an association between the neuron activity and the antifeedant response (White and Chapman 1990, Chapman et al. 1991). Therefore, it is possible that the sabadilla alkaloids cevadine and veratradine were detected by the alkaloid -sensitive neurons. Based on the rapid and persistent rejection of sabadilla treated leaf disks by S. americana nymphs observed during the bioassays, it is plausible that the sensory coding that elicited the sabadilla -deterrent effect resulted from labeled line responses (Schoonhoven 1982, van Loon 1996, Koul 2008). This means that e ach neuron in a contact chemoreceptor conveys a specific message, in this case a deterrent signal, which can be interpreted by the central nervous system without a dditional information from other neurons leading to immediate rejection of a food source without feeding. Sabadilla also effectively reduced the feeding behavior of D. abbreviatus The feeding inhibition that this chemical elicited on the weevils was not as potent as the deterrence observed

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47 for the grasshoppers; thus, the sensory input that the sabadilla alkaloids generated in D. abbreviatus and S. americana nervous system, is perhaps different. Instead of a labeled line response, the reduction in feeding behavior by D. abbreviatus could be produced by an across fiber pattern (Schoonhoven 1982, van Loon 1996, Koul 2008), in which the combined input of two or more receptors, with different stimulus thresholds, determines the acceptance or rejection of a host For example, alkaloids from the family Solanaceae do not have any specific disruptive effects on several taste neurons nor inhibit the activity of the taste cell sensitive to phagostimulants in the red turnip beetle, Entomoscelis americana Brown (Mitchel l and Gregory 1979), suggesting an across -fiber pattern as sensory coding. Although not as potent as sabadilla, azadirachtin caused a significant reduction in the feeding behavior of S. americana. Previous studies had documented the antifeedant properties of azadirachtin against different orders of insects, including Orthoptera (Mordue (Luntz) and Blackwell 1993, Aerts and Mordue 1997, Capinera and Froeba 2007). Inhibition of feeding behavior by this triterpenoid could be the result of stimulation of a dete rrent receptor, or blockage of the input from neurons that detect phagostimulatory compounds such as carbohydrates, or both. Winstanley and Blaney (1978) studied the behavioral and sensory response of Schistocerca gregaria Forskal to a set of solutions inc luding azadirachtin. They proposed that the deterrent effect of azadirachtin on this grasshopper was caused by the stimulation of a specialized deterrent cell. Pieris brassicae L. larvae also possess a deterrent receptor, located in the medial sensilla sty loconica on the maxilla, which is excited by azadirachtin (Schoonhoven 1982). Charleston and colleagues (2005) observed feeding deterrent activity by plants extracts from Melia azedarach L. (Meliaceae) and Azadirachta indica against the diamondback moth, P lutella xylostella L. The authors suggested that triterpenoids present in

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48 the plant extracts, including azadirachtin, disrupted the normal function of chemoreceptors responsible for perceiving glucosinolates, a strong phagostimulant for this lepidopteran. The antifeedant effect of azadirachtin varies among insect species. In this study, D. abbreviatus was deterred by azadirachtin during the choice tests, consuming more control disks than treated ones. But in the nochoice bioassays the terpenoid did not reduce herbivory of the treated leaf disks. These results concurred with the observations of Showler and collaborators (2004), who evaluated three commercial neem -based insecticides (Agroneem, Ecozin and Neemix) as potential feeding and oviposition deterrents against gravid female boll weevils, Anthonomus grandis grandis Boheman on cotton plants. They found that in choice assays, only Ecozin deterred the weevils from feeding, whereas in no -choice tests, none of the products reduced the consumption of cotton le aves after 24 h. It is probable that D. abbreviatus had habituated to azadirachtin due to repeated exposure to the chemical during the 24 h time interval of the experiments. Desensitization to azadirachtin has been reported previously. Fifth instar S. litu ra larvae became desensitized to pure azadirachtin, in both choice and no-choice assays, after being exposed to the terpenoid for 2 h (Bomford and Isman 1996). Similarly, Held and colleagues (2001) observed adults of the Japanese beetle, P. japonica, to ha bituate to a commercially formulated neem extract, applied to linden, Tilia cordata L., in a series of 4 -h choice or no -choice tests over four successive days. Neem oil reduced feeding of treated leaf disks by S. americana and D. abbreviatus in paired choi ce assays but was unable to deter both insects in no -choice tests. D. abbreviatus even consumed significantly more neem oil treated leaf disks than untreated ones. The increased acceptability of neem oil by the insects may be due to desensitization. The bi ological activity of neem oil is closely related to its azadirachtin content (Isman et al. 1990, Isman 2006). Neem

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49 seeds normally contain 0.2% to 0.6% azadirachtin by weight, while azadirachtin formulations contain 10% to 50% (Isman 2006). Desensitization occurs more frequently when an antifeedant exerts weak inhibitory stimuli (Held et al. 2001), in this case lower concentrations of azadirachtin. Although neem oil includes considerable quantities of other triterpenoids such as salannin and nimbin (Schmutte rer 1990, Isman 2006) that can act as antifeedants as well (Koul et al. 2004), the feeding strategy of S. americana and D. abbreviatus may have counteracted their effect. The capacity for habituation in insects may be greater in polyphagous than in oligophagous or monophagous species (Jermy 1990, Held et al. 2002) because the taste sensitivity of insect herbivores to deterrents is lower in generalists than in specialists (Bernays et al. 2000). Rotenone was ineffective as a feeding deterrent of S. americana in this research. There is evidence that some species of grasshoppers are able to detect flavonoids (Bernays and Chapman 2000). Chapman and co-workers (1991) detected that salicin, a phenolic glycoside that stimulates a deterrent neuron in S. americana, h ad a phagostimulatory effect on this grasshopper at low concentrations. But it seemed that the activity of the deterrent cells at higher concentrations of salicin was sufficient to override the phagostimulatory effects of the sucrose -sensitive cells, produ cing an antifeedant effect (Chapman et al. 1991). Therefore, the inefficiency of rotenone in reducing herbivory by S. americana may be a consequence of the imbalance between the weak deterrent stimuli generated by the isoflavonoid and the strong phagostimulatory effect of the carbohydrates present in the lettuce leaf disks. Contrary to the effect observed with the grasshoppers, rotenone applied to lettuce leaf disks effectively deterred D. abbreviatus adults in both choice and nochoice bioassys. This resul t supports previous studies that tested rotenone against curculionids and other coleopterans.

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50 Rotenone showed a strong antifeedent effect against adults and larvae of the wheat weevil, Sitophilus granarius L., adults of the confused flour beetle, Tribolium confusum Jacquelin du Val, and larvae of the khapra beetle, Trogoderma granarium Everts (Nawrot et al. 1989). The grass grub, Costelytra zealandica (White) was also deterred by rotenone in an artificial diet (Lane et al. 1985). Ryanodine was the only chem ical tested other than sabadilla that significantly reduced the feeding behavior of S. americana and D. abbreviatus in both choice and no -choice tests. The antifeedant effect of ryanodine on insects has been proven previously. Ryanodine reduced the food co nsumption of the tobacco budworm, Heliothis virescens (F.), and the tobacco cutworm, Spodoptera litura F., under laboratory conditions (Yoshida and Toscano 1994, Gonzalez Coloma et al. 1996). Larvae of the African cotton leafworm, Spodoptera littoralis Boi sduval, were more sensitive to the deterrent action of ryanodine than adults of the Colorado potato beetle, L decemlineata (Gonzalez Coloma et al. 1999). The observed antifeedant action of the alkaloid ryanodine potentially implicates the involvement of a common ligand -gated ion channel that mediate s the taste response to these compounds in both the grasshoppers and weevils nerve cells. A possible candidate could be a ryanodine receptor. Ryanodine receptors are ryanodine -sensitive intracellular Ca2+ releas e channels (Nauen 2006) Ca2+ is a n intracellular messenger which intercede s in many cellular and physiological activities such as neurotransmitter release, hormone secretion, gene expression and muscle contraction (Nauen 2006). In this study, capsaicin di d not exert a relevant antifeedent effect against S. americana or D. abbreviatus The ineffectiveness of capsaicin as an insect feeding deterrent has been previously documented. Capsaicin did not decrease herbivory of grape leaves by the rose chafer, Macro dactylus subspinosus F. (Isaacs et al. 2004). Potato plots treated with a capsaicin extract

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51 did not decrease the incidence of the Colorado potato beetle, Leptinotarsa decemlineata Say (Moreau et al. 2006). Baumler and Potter (2007) also reported the ineffi cacy of capsaicin in reducing defoliation of linden ( Tilia cordata L.) by the Japanese beetle. Kaolin clay, an aluminosilicate material, did not effectively deter any of the insects tested in this study. Schistocerca americana consumed less kaolin-treated leaf disks than untreated ones in choice bioassays, but in a no choice scenario the presence of the aluminosilicate did not prevent the entire consumption of the treated disks. Against D. abbreviatus kaolin clay did not even exert a significant protection for treated leaf disks, in choice tests. Other studies have observed successful suppression of arthropod pests on fruit plants coated with kaolin (Glenn et al. 1999, Puterka et al. 2000). According to these studies, a plant coated with a hydrophobic parti cle film barrier, such as kaolin clay, can repel arthropods not only by making the plant visually or tactilely unrecognizable as a potential host but also by restraining mobility and snaring the arthropods mouthparts. But kaolin clay as a plant protector is maybe only effective against small arthropods with piercing-sucking mouthparts, such as the ones tested in these studies (pear psylla, Cacopsylla pyricola Foerster, spirea aphid, Aphis spirecola Patch, potato leaf hopper, Empoasca fabae Harris, twospott ed spider mite, Tetramychus urticae Koch and pear rust mite, Epitrimerus pyri Nalepa). For large insects with chewing mouthparts, such as D. abbreviatus or S. americana, kaolin clay does not represent a physical barrier. Our results also contrast with thos e of Lapointe (2000), who observed a reduction in feeding by D abbreviatus adults on citrus plants treated with kaolin clay under laboratory conditions. Lapointe suggested that the antifeedant effect was caused by interference with tactile recognition of citrus plants as hosts, using the research of Puterka and colleagues (2000) as a reference. This mode of action is probably correct against specialist arthropods, like the pear psylla and the pear rust mite, which

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52 depend on very specific visual, olfactory, gustatory and mechano -sensory stimuli in order to select a host plant, but this may not apply to generalist insects that rely more, at short distances, on tasting most plants and eating those that lack strong feeding deterrent compounds in order to select a host plant (Schoonhoven et al. 2005). The contrasting results suggest a necessity for further studies of the antifeedant effect of kaolin clay on D. abbreviatus Field Trial Residual activity of the tested antifeedants under field conditions proved to be quite brief. All the chemicals tested did not significantly reduce herbivory by S. americana or D. abbreviatus after 4 h of exposure to sunlight. The only exception was sabadilla, which showed an antifeedant effect against nymphs of S. americana after 4 h, 8 h and 12 h of exposure. Photodegradation of botanical pesticides under sunlight has been reported previously (Liang et al. 2003, Showler et al. 2004, Capinera and Froeba 2007) and represents one of several problems affecting plant based insecticides u nder field conditions. Behavioral Bioassays: Plant Extracts In this study, both methanol and methylene chloride extracts from the Florida rosemary, C. ericoides deterred D. abbreviatus adults; whereas only the methylene chloride extracts from this plant s pecies exerted an antifeedant effect on S. americana nymphs. Chemical analysis of C. ericoides has previously demonstrated the presence of several classes of flavonoids including, dihydrochalcones, flavones, catechins and epicatechins (Tanrisever et al. 19 87). Flavonoids may affect the feeding behavior of insects by stimulation of a specialized deterrent receptor (Simmonds 2001, Koul 2008). Flavonoids are compounds (Ding 1998) that are only soluble in a polar solvent such as methanol. Therefore, these phenolic compounds may be the basis of the antifeedant effect of C. ericoides methanol extracts on D. abbreviatus The feeding inhibition that the C. ericoides methylene chloride extracts observed against S. americana and D.

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53 abbreviatus suggests the presence of a non -polar chemical that interacts with the chemosensory system of the two insect species. A possible candidate would be a class of terpenoid, due to the relative non -polar character of these plant chemicals (Fischer et al. 1994) and their potential as feeding deterrents (Koul 2008). Methanol extracts from A. crenata functioned as a feeding deterrent against both S. americana nymphs and D. abbreviatus adults. The latter insect species was also deterred by methylene chloride extracts from A. crenata Trite rpenoid saponins have been isolated from A. crenata plants (Liu et al. 2007). These non-polar terpenoids are able to modify the feeding behavior of insects. Larvae of the diamondback moth, Plutella xylostella L., were deterred by a triterpenoid saponin ext racted using chloroform from wintercress, Barbarea vulgaris W. T. Aiton (Shinoda et al. 2002). Terpenoids can inhibit insect feeding by distortion of the normal function of phagostimulant receptors, excitation of deterrent receptors and/or stimulation of b road spectrum receptors, among others (Koul 2008). Isocoumarins are another type of phytochemical that has been identified in members of the genus Ardisia (Kobayashi and de Mejia 2004). Isocoumarins are phenolic compounds that can produce a feeding deterre nt effect aminobutyric acid (GABA) (Ozoe et al. 2004). GABA and related aminobutyric acids have been shown to stimulate feeding and induce taste cell responses among herbivorous insects of four orders including Orthoptera and Coleoptera (Mullin et al. 1994). GABA -gated chloride channels in the peripheral nervous system of insects participate in chemoreception. In excitable cells, binding by a ligand (GABA, in this case) changes channel conformation, which leads to an opening of the ion p ore and an inhibitory inward Clmovement, due to high external Cl-. But, according to Mullin et al. (1994), opening of these channels under low Clconcentrations of plant tissues, in the absence of a mechanism to maintain high

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54 extracellular Clin the se nsillar fluid (which is a mucopolysaccharide material surrounding the tip of the dendrite s of the contact chemoreceptors), could result in an outward Clmovement leading to depolarization of the sensory neuron. Thus, if a phytochemical (i.e. isocoumarins) antagonizes GABA at the binding site of a ligand -gated chloride channel in a gustatory cell, feeding deterrence will be induced. In summary, of the ten chemicals tested only sabadilla, azadirachtin and ryanodine deterred S. americana under laboratory conditions. Sabadilla was the only compound that maintained its remarkable antifeedant properties against the grasshoppers after 12 h of exposure to sunlight. Sabadillas deterrent effect and relative durability under field conditions makes it a potential too l for integrated management of the American bird grasshopper. Against D. abbreviatus ryanodine, rotenone and sabadilla acted as feeding deterrents, but only in the laboratory bioassays. The stability of these chemicals in the field must be improved if eff ective protection against the sugarcane rootstock borer weevil is desired. The effectiveness of the extracts obtained from C. ericoides and A. crenata in reducing herbivory of the two insect species tested is an indication that many plants contain phytoche micals that could potentially be developed as antifeedants, and examination of these compounds is a logical next step for this research.

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63 BIOGRAPHICAL SKETCH Andres Felipe Sandoval Mojica was born on 1981 in Bogota, Colombia. The youngest of four children, he grew up in Tunja, Boyaca, Colombia graduating from Colegio de Boyac in 1997. He obtained his undergraduate degree in biology from the Pontificia Universidad Javeriana, where he developed an interest in entomology af ter taking the course Biology of Arthropods. Due to an excellent academic performance in this subject, he became the teaching assistant for the same course in the year 2001. His undergraduate thesis identified the pattern of altitudinal variation in rich ness, abundance, diversity and composition of the O rthoptera community in an altitud inal gradient between 2,000 m and 3,000 m. in an Andean cloud forest. This research contributed to understand the controversial relationship that exists between altitude an d species richness. It also provided information about factors affecting insect distribution in environmental gradients. Due to the significance of this work, he received financial support, as a scholarship, from the Colombian Society of Entomology (SOCOLEN) The research was nominated as the best student paper at the XXXII meeting of the same institution in 2005 and the results published in the 32nd volume of Revista Colombiana de Entomologia in 2006. In 2005, he was hired by Fundacin OMACHA (O MACH A Foundation, an organization committed to the sustainable development, research and conservation of the Colombian natural resources, with emphasis in aquatic ecosystems), where he studied the entomological fauna that exist in the Bojonawi Natural Reserv e in the Colombian Orinoquia. He proposed and developed a research subject that compared the structure and composition of dung beetles, ants and butterflies communities in three land units tha t are present at the reserve : savannah, galleria forest and palm trees. This study which was presented at the XXXIII meeting of the Colombian Society of Entomology was nominated as the best study presented by a professional in 2006. A new report of Phanaeus haroldi Kirsch (Coleoptera: Scarabaeidae) for the Department of Vichada

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64 (Colombia) was also obtained. During 2006, he work ed as a volunteer at the Colombian Corporation of Agricultural Research (CORPOICA ).