LOCAL DISPERSAL OF STINK BUGS (HEMIPTERA: PENTATOMIDAE) IN MIXED

AGRICULTURAL LANDSCAPES OF THE COASTAL PLAIN

by

TA-I HUANG

(Under the Direction of Michael D. Toews)

ABSTRACT

Phytophagous stink bugs (Hemiptera: Pentatomidae) are key agricultural pests around the

world. Among them, the southern green stink bug, Nezara viridula (L.), the brown stink bug,

Euschistus servus (Say), and the green stink bug, Chinavia hilaris (Say) are the three most

abundant species in the southeastern United States and have become serious economic pests of

cotton in the past decade. Stink bugs are sensitive and mobile insects that disperse from crop to

crop according to crop phenology. In addition, scouting stink bugs to determine population size

and treatment threshold is time-consuming. The overall objective of this research was to

investigate the behavior and movement of stink bugs to provide knowledge for developing

ecologically based strategies for stink bug management.

Stink bug feeding preference and movement on individual cotton plants were evaluated in

the laboratory using digital cameras and a video recording system. The author discovered

different feeding preferences between N. viridula and E. servus. N. viridula spent more time on

the two larger boll classes, 2.1-2.5 and 2.6-3.0 cm, while E. servus exhibited a stronger

preference for 2.1-2.5 cm bolls. Movement was greater for both species during photophase than

scotophase.

Intercrop movement of stink bugs was investigated using immunomarking techniques in

cotton-peanut-soybean farmscapes. While there were differences in marking efficiency among

marking proteins applied to a specific crop, there were no differences among stink bug species.

During cotton bloom, data indicated that at least a small number of stink bugs moved between all

possible combinations of adjacent crops; however, the majority of the dispersal was into cotton.

There were distinct differences among movement patterns among stink bug species. There were

no differences in dispersal distance among stink bug species or between sexes. However, stink

bugs moving from cotton to soybean travelled significantly further than bugs travelling between

remaining adjacent crops. Differences in stink bug density, seedcotton yield, gin turnout, and

fiber color were correlated with changes in cotton boll damage.

INDEX WORDS: Stink bug, Nezara viridula, Euschistus servus, Chinavia hilaris, dispersal,

cotton, immunomarking, feeding preference, IPM

LOCAL DISPERSAL OF STINK BUGS (HEMIPTERA: PENTATOMIDAE) IN MIXED

AGRICULTURAL LANDSCAPES OF THE COASTAL PLAIN

by

TA-I HUANG

B.S., Chinese Cultural University, Taiwan, 2003

M.S., University of Florida, 2008

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2012

© 2012

Ta-I Huang

All Rights Reserved

LOCAL DISPERSAL OF STINK BUGS (HEMIPTERA: PENTATOMIDAE) IN MIXED

AGRICULTURAL LANDSCAPES OF THE COASTAL PLAIN

by

TA-I HUANG

Major Professor: Michael D. Toews

Committee: John N. All G. David Buntin John R. Ruberson Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2012

DEDICATION

To my parents, my lovely wife Hsiao Shih Chang, and my daughter Yun-Jen Huang.

iv

ACKNOWLEDGEMENTS

I sincerely thank a number of people for their assistances during my journey through

doctoral studies. This project and my degree could not have been completed without them.

I am thankful to my major advisor, Dr. Michael D. Toews for his guidance, support, and

always being patient from the beginning to the last minute of my student career. I thank you for

introducing the world of stink bugs and field cropping system to me, teaching me the correct way

to do research, and being responsible for every time I needed help from you. I will never forget

about those moments we sank in the cotton field, wet under the pivot, and stuck in the middle of

the dirt road!

I am grateful to all my committee members, Dr. John All, Dr. David Buntin, and Dr. John

Ruberson for your assistance and organizing my research. This dissertation would not have been

completed without your directions and encouragement. I appreciate you all for giving me

support on my research and professional career. I also thank my lab colleagues David Griffin,

Dr. John Herbert, Annie Horak, Barry Luke, Miguel Soria, and Ishakh Pulakkatu thodi for their

friendship, providing suggestions whenever I needed, and helping me collecting samples. As an

international student, I thank the staff members in the Department of Entomology and the Office

of International Education at UGA who helped me and made my life easier.

Finally, special thanks to my wife Hsiao Shih Chang for your company, being supportive

and always patient while I am busy. Thank you for being a wonderful wife and soul mate.

v

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.............................................................................................................v

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW .....................................................1

2 FEEDING PREFERENCE AND MOVEMENT OF NEZARA VIRIDULA (L.) AND

EUSCHISTUS SERVUS (SAY) (HEMIPTERA: PENTATOMIDAE) ON

INDIVIDUAL COTTON PLANTS ............................................................................27

3 INTERCROP MOVEMENT OF STINK BUGS (HEMIPTERA: PENTATOMIDAE)

IN GEORGIA FARMSCAPES ...................................................................................55

4 LOCAL DISPERSAL OF STINK BUGS (HEMIPTERA: PENTATOMIDE) AND

ASSOCIATED CHANGES IN COTTON FIBER QUALITY ...................................89

5 CONCLUSION..........................................................................................................120

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Phytophagous stink bugs (Hemiptera: Pentatomidae) are serious worldwide pests of

agriculture. In the U.S., the boll weevil eradication project and genetically modified (Bt) cotton

has been used to successfully eradicate (boll weevil) or drastically decrease (corn

earworm/tobacco budworm) two former major pests of cotton production. As a result of

decreased insecticide applications to manage the boll weevil and lepidopteran insect pests, stink

bugs are now becoming serious economic pests of cotton production in the southeastern US

(McPherson and McPherson 2000). The most abundant stink bug species in cotton production

include the southern green stink bug, Nezara viridula (L.), the green stink bug, Chinavia hilaris

(Say), and the brown stink bug, Euschistus servus (Say). Although these pests account for nearly

60 million dollars in losses to cotton in Georgia alone (Williams, 2010), a lack of basic

knowledge of stink bug biology and ecology has prevented the development of effective

management methods. To meet these challenges, the rationale behind our study is to provide

ecological-based strategies for stink bug management program.

1.1 History of cotton industry and boll weevil

Cotton is the single most important textile fiber in the world, accounting for nearly 40%

of total world fiber production (Haney et al. 2009). The birth of cotton in America occurred

when seeds imported from the West Indies were first planted in the Jamestown Colony, circa

1620. In Georgia, cotton was first planted in Trustees Garden, Savannah, with seed sent from

1

England in 1733. Cotton production in Georgia increased rapidly from 1000 bales in 1791 to

21,000 bales in 1796, or 21% of the total U.S. production with the invention of cotton gin by Eli

Whitney (Haney et al. 2009). Boll weevil, Anthonomus grandis (Coleoptera: Cuculionidae), is

thought to be native to Central America, and was first described by Boheman in 1843 from

specimens received from Vera Cruz, Mexico. The boll weevil first appeared in the United States

in 1892 near Brownsville, Texas. By 1904 the weevil was still only in Texas, but it had

progressed 500 miles north from Brownsville. By 1911 the boll weevil had advanced to central

Alabama and was first observed in Georgia in Thomasville (Thomas County) on August 25,

1915. Cotton production in Georgia peaked in 1911, with 2,900,000 bales produced on nearly

five million production acres. However, after the boll weevil appeared in 1915, cotton

production declined to just 600,000 bales by 1923, or only 20% of the pre- weevil production. In

1917, every cotton- producing county in Georgia reported the boll weevil and cotton production

had fallen by 30% (2.82 million bales vs. 1.96 million bales). By the end of 1919, the boll

weevil was distributed across the entire Cotton Belt, from south Texas through the Carolinas

(Haney et al. 2009). Total loss in Georgia in 1919 was estimated at $40 million (Campbell

1919).

Aerial applications of calcium arsenate dust began in the early 1920s. These applications

helped preserve some yield, but overall production declined steadily for another 60 years. The

greatest decline in Georgia happened in 1934 when boll weevil damage exceeded $200 million

per year across the Cotton Belt, and cotton acreage in Georgia had dropped to just 45% of the

total farmed in 1910–1914. Brazzel and Newsom (1959) documented the winter diapause

behavior of the boll weevil in 1959 which showed of properly timed fall insecticide applications

combined with cultural practices (stalk plowing) effectively reduced overwintering boll weevil

2

populations. This publication provided a foundation of integrated boll weevil control. The boll

weevil eradication program in Georgia began in 1987, followed by the successful five-year

program in North Carolina. The fall diapause treatment phase of the eradication program began

in September 1987. The insecticide recommendations from USDA in 1980 for boll weevil

control included aldicarb, azinphos-methyl, carbaryl, EPN, EPN plus methyl parathion,

malathion alone, malathion plus methyl parathion, methyl parathion plus methomyl,

monocrotophos, toxaphene alone, and toxaphene plus methyl parathion (Anonymous 1980). The

pheromone trapping phase of the eradication program began in April 1988, with an average of

one trap per acre placed on all 346,548 acres of cotton involved. The total cost of the eradication

program in Georgia from the beginning in 1987 to 1995 season was estimated at $99.3 million.

In 1995, 2.0 million bales were produced on 1.49 million harvested acres (59% more than in

1994), this was the largest yield since 1919, with total revenues of about $720 million (the

highest for cotton in Georgia’s history) (Haney et al. 2009). There are about 80 countries around

the world producing cotton; however, the United States, China, and India together provide over

half the world's cotton. The US is the leading exporter of cotton, accounting for over one-third

of global trade in raw cotton. The cotton industry in the U.S. generates more than $25 billion in

products and services annually (USDA 2009).

1.2 Pests in cotton

A number of insect pests damage cotton in the U.S., these peat are especially in the

family Coleoptera, Lepidoptera, and Hemiptera. The major beetle pest in cotton is the boll

weevil, A. grandis. Lepidopterans such as bollworm, Heliothis zea (Boddie), tobacco budworm,

Heliothis virescens (Fabricius), pink bollworm, Pectinophora gossypiella (Saunders), beet

3

armyworm, Spodoptera exigua (Hübner), fall armyworm, Spodoptera frugiperda (J.E. Smith),

European corn borer, Ostrinia nubilalis (Hübner), southern armyworm, Spodoptera eridania

(Stoll), cutworms, Agrotis spp., saltmarsh caterpillar, Estigmene acrea (Drury), cabbage looper,

Trichoplusia ni, (Hübner) and soybean looper, Chrysodeixis includens, (Walker) are historical

cotton pests but the populations have decreased in U.S. due largely to the adoption of Bt cotton

cultivars.

Hemipteran pests in cotton include cotton fleahopper, Pseudatomoscelis seriatus (Reuter),

tarnished plant bug, Lygus spp. and clouded plant bugs, Neurocolpus nubilus (Say), cotton

leafperforator, Bucculatrix thurberiella (Busck), thrips, Thrips spp. and Frankliniella spp., cotton

aphid, Aphis gossypii (Glover), whiteflies such as banded winged whitefly, Trialeurodes

abutilonea (Haldeman), silverleaf whitefly, Bemisia argentifolii (Bellows & Perring), and sweet

potato whitefly B biotype, Bemisia tabaci (Gennadius), and stink bugs such as C. hilaris, N.

viridula, E. servus, E. quadrator (Rolston), E. tristigmus (Say), Thyanta custator (F.), Oebalus

pugnax (F.), Piezodorus guildinii (Westwood) (Fig. 1).

Other arthropod pests in cotton include spider mites, Tetranychus spp., and grasshoppers

such as lubber grasshopper, Brachystola magna (Girard) and differential grasshopper,

Melanoplus spp., (Williams, 2010).

1.3 Biology of stink bug

Insect pest management requires a basic knowledge of the pest biology and ecology.

Insect behaviors are particularly crucial factors that influence sampling efficiency. Female

southern green stink bugs prefer to oviposit eggs under leaves or pods in the upper portion of

crops (Todd 1989). The number of eggs per egg mass is variable ranging from 30-130 eggs but

4

usually around 80 eggs per mass, and is determined by the crop condition or plant chemicals

(Todd 1989; Panizzi et al. 2004). First instars nymphs of N. viridula do not feed on plant tissue,

but their emergence and survivorship are strongly affected by relative humidity (RH) (Hirose et

al. 2006). Second instars nymphs begin to feed but typically do not leave eggs mass until 3rd

instars. By 5th instars, population distribution is nearly random (Todd 1989), but the feeding site

preferences and resting locations of late instars nymphs are poorly understood.

Feeding damage on cotton bolls caused by stink bugs will result in boll abscission, lint

staining, reduced lint quality, reduced yields and external lesions (Cassidy and Barber 1939,

Toscano and Stern 1976, Barbour et al. 1990, Greene et al. 1999, Turnipseed et al. 2003, Toews

et al. 2009). A study conducted by Greene et al. (1999) reported that 5th instars of N. viridula

cause significantly more damage than adult stink bugs or earlier instars on a 9 d old boll.

However, another study showed no difference in boll abscission, yield, fiber quality, and seed

germination feeding between N. viridula males and females or between adults and fourth to fifth

instars (Bommireddy et al. 2007). Feeding by adult L. lineolaris causes similar damage to 4th

instars of N. viridula, but significantly more damage is caused by N. viridula (Greene et al.

1999).

Feeding by N. viridula can also result in rotten and hard-locked bolls on cotton due to

association with pathogens (Willrich et al. 2004). In addition, recent work discovered that

feeding by N. viridula induces transmission of Pantoea agglomerans strain (Sc 1-R) into cotton

bolls (Medrano et al. 2007). This infection causes rotting of the entire locule and masks internal

carpel wounds. Further research has shown that young developed bolls could be severely

damaged by the transmission of boll rot pathogens, but are immune to the pathogen at 3 weeks

post-anthesis (Medrano et al. 2009). Southern green stink bug infestations not only reduce the

5

value of cotton production but also impact in other crops, such as delayed maturity of soybean

(Boethel et al. 2000) and damaged kernels in macadamia orchards (Wright et al. 2007).

Stink bug feeding damage can be monitored on the husk, shell, and kernel by staining

their feeding probes (Golden et al. 2006). Fluorescence technology and examination of external

feeding lesions further improved the efficiency of detecting cotton boll damage (Xia et al. 2011).

Assessment of feeding injury to bolls is a reliable method for assessing populations of stink bugs

and making treatment thresholds (Greene et al. 2001). Although adult feeding of N. viridula was

more prolonged during scotophase than photophase (Shearer and Jones 1996), other feeding

behavioral preference such as the age of cotton bolls along with duration of time spent on the

boll and movement on individual cotton plant are unknown.

1.4 Communication

Insect communication through chemicals or substrate-borne vibrations has been widely

studied and confirmed in many insect orders (Virant-Doberlet and Cokl 2004). These chemical

or physical signals directly mediate insect behavior and might be potential tools for integrated

pest management. Immature stink bugs of different species may share some common chemical

compounds, such as repellent compound (E)-2-decenal is found in both 1st and 2nd instars of N.

viridula and C. hilaris (Fucarino et al. 2004). The presence of these natural compounds allows

natural enemies such as generalist tachinid flies to use these stink bug pheromones as host-

finding kairomones. One example is that Euclytia flava (Townsend) which is cross attracted by

pheromones of Podisus and Euschistus pentatomids (Aldrich and Zhang 2002). Contact

kairomones from N. viridula were found to elicit foraging behavior of the egg parasitoid

Trissolcus basalis (Wollaston) (Colazza et al. 2007). Defensive secretions such as 2-oxo-(E)-2-

6

hexenal are also found to attract two generalist parasitoids T. basalis and T. podisi (Laumann et

al. 2009). Host-parasitoid relationships mediated by these semiochemicals are important

information to improve the effectiveness of biological control.

Many insects use their host plant as transmission substrates for signals (Henry 1994;

Mcbrien et al. 2002). Mating choice of N. viridula is mediated by substrate-borne vibrational

songs/signals produced by body vibration through plant structures (Harris 1982). Female calling

and courtship songs caused males to approach, but invoked no response from other females

(Cokl et al. 1999). Some pentatomids, such as N. viridula and C. hilaris, share some spectral

characteristics in vibration signals; therefore, although males of N. viridula prefer the female

calling song from their own population, they also respond to the female calling songs of other

sympatric species, such as C. hilaris when tested in a choice experiment (Cokl et al. 2001;

Miklas et al. 2003). The efficiency of substrate-borne communication of stink bugs is based on

optimal tuning between the frequency of vibratory songs and receiving sensory organs, and the

resonant properties of plants (Cokl et al. 2005). Cokl (2008) further reported that plants with low

pass filtering properties transmit signals optimally, and all stink bug species emit similar signals

with dominant narrow frequency peaks around 100 Hz and mostly below 600 Hz.

1.5 Dispersal

Insect population dynamics, seasonal movement, and abundance are greatly affected by

the host plant type and stage (Todd 1989, Smith et al. 2009). Stink bugs are a good model to

study spatial patterns of dispersion because they have a broad host range associated with over

200 known plant species (McPherson and McPherson 2000). ‘Edge effect’ where adults

commonly aggregate in a corner or edge of a field was documented (Nakasuji et al. 1965, Todd

7

1989). Soybean was reported as the most preferred host of stink bug when adjacent to corn,

cotton, and grain sorghum (Smith et al. 2008). Toews & Shurley (2009) further reported that

stink bug damage in cotton plots was highly associated with plot edges when adjacent to

soybean, corn, and peanut. Those data suggested that when making decisions on farmscape

planning, growers should also consider adjacent crops and the timing of planting. The

movement of stink bugs might be driven by environmental conditions such as drought (Toews

and Shurley 2009). Stink bugs are known to be highly mobile and good fliers capable of moving

at least 120 m (Tillman et al. 2009). However, the flight distance associated with farmscape

level is poorly understood and study focusing on stink bug dispersal is needed.

Like other pentatomids, N. viridula overwinters in the adult stage under substrates. The

individuals move to feed and oviposit on host plants when the temperature is warm in spring

(Todd 1989). A dispersal study in Japan reported that crops that N. viridula chose for

oviposition were different from those upon which they feed (Kiritani et al. 1965). The

phenomenon of stink bug species feeding on different plants throughout the growing season is

known as cyclic colonization of ephemeral habitats (Wissinger 1997). Further studies reported

that maturity group IV soybeans planted early can escape heavy stink bug (Gore et al. 2006,

Smith et al. 2009). Although early-season soybean production systems could avoid drought and

reduced late season insect problems (McPherson et al. 2001), they cannot protect full-season

soybean production systems for long term period (Smith et al. 2009). Crop phenology is crucial

for stink bug population growth on soybean, especially when peak oviposition and population

increase occurring during crop development stage (Todd 1989, Smith et al. 2009).

Although stink bug dispersal from host to host has been reported for at least 60 years in

studies of the abundance of stink bugs at the farmscape level (Borden et al. 1952), most studies

8

lacked direct evidence that stink bugs move directly from one crop to another. Recently,

spatiotemporal analysis was developed to monitor stink bug population dynamics. Results

showed a strong aggregating behavior on the interface or common boundary between peanut and

cotton (Tillman et al. 2009); but whether or not those stink bugs found in cotton originated from

peanut remains unclear.

1.6 Mark and recapture techniques

Studies of insect dispersal across the agricultural landscape are important to understand

the role of alternative host plants, to estimate population density, and to develop novel pest

management strategies. The most direct way to ascertain distance and speed of travel is to

externally mark insects, release them from a common location, and then recapture them at a later

date. Historically, scientists have marked insects using paints, tags, fluorescent powders

(Schroeder and Mitchell 1981), trace elements such as rubidium (Berry et al. 1972),

radioisotopes (Service 1993), dyes (Hendricks and Graham 1970), insect mutilation (Murdock

1963), and most recently immunomarking technologies (Hagler 1997). Paints, tags, dyes, and

powders are ineffective for ecological studies because the markers interfere with small insect

dispersal, mating ability, or longevity. Similarly, radioactive tracers may present health and

environmental risks making them undesirable for this use. Trace elements have been used

successfully, but retention of the marker declines rapidly in some insect species and application

has been linked to altered development of some insect pests (Stimmann et al. 1973).

Immunomarkers, such as purified mammalian immunoglobulin G, are invisible to the naked eye

but can be easily detected using an enzyme-linked immunosorbent assay (ELISA). These

methods have been successfully adopted to study movement of small predators and parasitoids in

9

the field, but are prohibitively expensive for large-scale applications because the marking

proteins cost ≈$500 per liter.

A recent breakthrough in this field was the development of inexpensive immunomarking

techniques including diluted chicken egg albumin, bovine casein, and soy protein (Jones et al.

2006). These marking proteins cost ≈$0.12 to $0.26 per liter thereby applying markers in a

larger scale of farmscape ecosystem is feasible. Preliminary trials conducted with pear psylla

and codling moths showed the protein markers can be detectable at extremely low levels (7.8-

31.2 ppb) even after 3 wk in the field. In addition, different protein markers are separable from

each other when applied in the same time. More details are discussed in the chapter 3.

1.7 Scouting procedures in cotton

Scouting methods such as sweep net and drop cloth are more efficient for sampling

hemipterans when compared to other direct sampling methods (Musser et al. 2007). However,

Espino et al. (2008) suggested that visual sampling methods are more cost-reliable than sweep

net when sampling the rice stink bug, Oebalus pugnax (F.) in rice fields. Sweep net sampling

(10-20 sweeps per sample) and drop cloth sampling (each covering six row feet) are most

common methods for scouting stink bug populations. The standard net used for sweeping has an

opening of 15 inches in diameter and is about one meter long. When scouting, the net is swing

from side to side in about a 120 - 180 degree arc and ca. one stroke per step is taken while

walking through the field. Although sweep net is a simple and time saving scouting method, it

usually tends to bias the sample toward adult stink bugs (Toews et al. 2008). This is likely

because nymphs tend to aggregate in the lower part of the plant, while adults tend to active

throughout. Shake cloth or beat cloth samples tend to bias the captures toward nymphs as

10

opposed to adults. Collection of quarter-sized bolls and determination of internal boll injury

takes the longest time but has the highest accuracy among these methods. Internal boll injury

can be determined through dissection of each boll and recording the presence of boll-wall

penetration, callous growth on the internal carpel wall, and lock with lint staining. The time

required for each technician to complete the protocol using a specific sampling method can be

quantified using a stopwatch. Toews et al. (2008) suggested that internal boll damage was 10-

fold more sensitive at detecting positive hits (boll damage compared with detecting individual

insects); however, it requires 4-fold more time than sweep net sampling and six times more time

than beat cloth procedures, respectively. Whole plant visual sampling (including terminals,

leaves, flowers, squares, balls, and stems) is ideally comprehensive but lacks practicality for

growers due to time and labor demanded.

1.8 Pheromone traps

Pheromone-baited traps are widely used for monitoring lepidopteran and coleopteran

agricultural pests but have not been extensively used for stink bugs. This could be a potential

advantage to monitor hemipterans using stink bug pheromone traps. For example, a field study

found that the synthetic aggregation pheromone methyl (2E,4Z)-decadienoate attracted both

males and females of adult Euschistus conspersus Uhler within 24-48 hours (Krupke et al. 2001).

A number of recent studies have focused on identifying stink bug sex pheromones by gas

chromatography (GC) and GC-mass spectrometry. The major sex-specific component produced

by male green stink bug C. hilaris is (4S)-Cis-(Z)-bisabolene epoxide ((4S)-cis-Z-BAE) (Mcbrien

et al. 2001); methyl (E)-6-2,3-dihydrofarnesoate was identified as male-produced pheromone by

Chlorochroa sayi (Ho and Millar, 2001); methyl (2E,4Z,6Z)-decatrienoate (2E,4Z,6Z-

11

10:COOMe) was identified as the major sex pheromone produced by male red-shouldered stink

bug, Thyanta perditor (Mcbrien et al. 2002, Moraes et al. 2005). Two male-specific isomers of

1′S- zingiberenol were identified as male-produced sex pheromone from the rice stalk stink bug,

Tibraca limbativentris (Stål) (Borges et al. 2006); (7R)-(+)-β- sesquiphellandrene was identified

as male-produced pheromone of the neotropical redbanded stink bug, Piezodorus guildinii

(Westwood) (Borges et al. 2007). The aggregation pheromone of the brown-winged green bug,

Plautia stali Scott, methyl (2E,4E,6Z)- decatrienoate, was also reported to attract the invasive

species the brown marmorated stink bug, Halyomorpha halys (Stål) (Aldrich et al. 2007,

Khrimian et al. 2008). In most phytophagous pentatomid bugs, the male-produced sex

pheromones only attract females but not males, and females do not attract either sex (Moraes et

al. 2005). In general, long range pheromone is attractive to males and females but they only land

nearby instead of on the source, short range cues are necessary for further courtship behavior.

Thus, a monitoring trap should include both cues to increase the efficiency.

Advanced technology in recent year trap designs and semiochemical attractants offers the

opportunity for monitoring stink bugs in the field. Brown stink bug aggregation pheromone was

identified in 1991 (Aldrich et al. 1991) and a patented stink bug trap was subsequently developed

by Mizell and Tedders (1995). Previous work in pecans has produced several improvements in

trap design and usability that may improve capture efficiency in cotton (Cottrell et al. 2000,

Cottrell 2001). Despite these advances, very little progress with stink bug trapping in cotton is

reported in the literature. Preliminary investigation of pheromone based trapping for brown stink

bug in Arkansas cotton suggested that this method was useful for following in-field populations

of stink bugs (Green and Capps 2003). Tillman et al. (2007) described preliminary testing of a

putative southern green stink bug sex pheromone.

12

1.9 Stink bug management

Biological control using natural organisms such as bacteria, fungi, viruses, pheromones

or metabolites provides another option of integrated pest management program. Toxins

produced by a new described bacterium Chromobacterium subtsugae (Martin et al.) were

reported to kill 100% of N. viridula within six days in laboratory assays (Martin et al. 2007). A

study conducted from southeast Virginia reported that four species of hymenopteran parasitoids

in the family Scelionidae and one in the family Mymaridae were recovered from stink bug eggs

(Koppel et al. 2009). Among those stink bug species, E. servus eggs were attacked by

Telenomus podisi, whereas C. hilaris eggs were attacked by T. basalis, T. euschisti, and T.

edessae. However, none of them were collected from cotton fields (Koppel et al. 2009). In N.

viridula, the endoparasitoid Trichopoda pennipes (F.) attacks both late-stage nymphs and adults.

However, several insecticides used to target N. viridula such as dicrotophos, indoxacarb, oxamyl,

and thiamethoxam are actually more toxic to T. pennipes (Tillman 2006). Therefore, economic

threshold is crucial when making insecticide application.

Genetically modified cotton cultivars containing Bacillus thuringiensis proteins (e.g.

Bollgard II with Cry1AC and Cry2Ab2 or Widestrike with Cry1AC and Cry1F proteins)

significantly reduced damage from lepidopterans, but provide no protection from stink bugs.

Although some insecticides, such as methyl parathion, λ-cyhalothrin or cyfluthrin effectively

control most stink bug species in both transgenic and conventional cotton, studies showed that

the brown stink bug, E. servus is tolerant to pyrethroid insecticides (Greene et al. 2001;

Snodgrass et al. 2005). Cultivar resistance provides an alternative management strategy which

can reduce insecticide use. For instance, N. viridula showed different responses feeding on

13

soybean breeding lines containing the stink bug resistant ‘IAC-100’ which provides a potential

for soybean resistance development (McPherson et al. 2007). Similar cultivar resistance was

also confirmed in corn inbred and hybrids lines when field screening the combined damage from

E. servus and N. viridula (Ni et al. 2007; Ni et al. 2008).

Current labeled organophosphates for control of stink bugs in Georgia cotton include:

acephate (Orthene), dicrotophos (Bidrin 8), and methyl parathion. And current labeled

pyrethroids for control of stink bugs in Georgia cotton include: beta-cyfluthrin (Baythroid XL),

bifenthrin (Brigade 2EC), esfenvalerate (Asana XL), gamma-cyhalothrin (Prolex 1.25), lambda-

cyhalothrin (Karate with Zeon), and zeta-cypermethrin (Mustang Max 0.8). There are a number

of premixed or co-packaged insecticides also labeled for control of stink bugs in cotton, such as

Bifenthrin + imidacloprid (Brigadier), Dicrotophos + bifenthrin (Bidrin XP), Imidacloprid +

cyfluthrin (Leverage), Lambda-cyhalothrin + thiamethoxam (Endigo), Spinosad + gamma-

cyhalothrin (Consero), Zeta-cypermethrin + bifenthrin (Hero). The treatment threshold is to

apply when 20% of medium sized bolls (the diameter of a quarter) display internal signs of

feeding and stink bugs are observed or when stink bugs exceed 1 bug per 6 row-feet (Roberts et

al. 2009). Very recent recommendations for southeastern cotton growers suggest altering the

treatment threshold by week of bloom (Green et al. 2008, 2009).

Many challenges remain for effective stink bug management programs. First of all,

although the biology and ecology of N. viridula have been intensively studied, movement at the

farmscape scale remains unclear. Second, information on life history, basic biology and ecology

of other stink bug species is very limited. Third, Bt-transformed cotton cultivars offer good

protection from caterpillar pests, but these products have no effect on stink bugs. Fourth,

tolerance of pyrethroid insecticides has been built up in the brown stink bug. Fifth, stink bugs

14

tend to aggregate across fields and can be difficult to sample. To meet these challenges and to

develop more efficacious pest management strategies, a better understanding of stink bug

feeding behavior and movement is needed. Our first objective is described in chapter 2 which

characterizes stink bug feeding preference and movement on individual cotton plants. The

second objective is described in chapter 3 which identifies environmental and host conditions

that trigger stink bug movement into cotton fields from adjacent crops. The third objective is

described in detail in chapter 4 and is to determine the correlation between stink bug local

dispersal and cotton fiber quality. The final chapter is the conclusion of all my findings and

suggestions on the ecologically-based integrated pest management program for stink bugs.

15

References Cited:

Aldrich, J., M. Hoffman, J. Kochansky, W. Lusby, J. Eger and J. Payne. 1991. I

dentification and attractiveness of a major component for nearctic Euschistus spp. stink

bugs (Heteroptera: Pentatomidae). Environ. Entoml. 20: 477-483.

Aldrich, J. R., A. Khrimian, M. J. Camp. 2007. Methyl 2,4,6-decatrienoates attract stink bugs

and tachinid parasitoids. J. Chem. Ecol. 33: 801-815.

Aldrich, J. R. and A. Zhang. 2002. Kairomone strains of Euclytia flava (Townsend), a

parasitoid of stink bugs. J. Chem. Ecol. 28: 1565-82.

Anonymous. 1980. Guidelines for the control of insect and mite pests of foods, fibers, feeds,

ornamentals, livestock, households, forests and forest products. USDA Agr. Handbook

No. 571: 54–55.

Barbour, K. S., J. R. Bradley, Jr., and J. S. Bacheler. 1990. Reduction in yield and quality of

cotton damaged by green stink bug (Hemiptera: Pentatomidae). J. Econ. Entomol. 83:

842-845.

Berry, W. L., M.W. Stimmann, and W.W. Wolf. 1972. Marking of native phytophagous insects

with rubidium: a proposed technique. Ann. Entomol. Soc. Am. 65: 236-238.

Boethel D. J., J. S. Russin, A. T. Wier, M. B. Layton, J. S. Mink, and M. L. Boyd. 2000.

Delayed maturity associated with southern green stink bug (Heteroptera: Pentatomidae)

injury at various soybean phenological stages. J. Econ. Entomol. 93: 707-712.

Bommireddy, P. L., B. R. Leonard, and J. H. Temple. 2007. Influence of Nezara viridula

feeding on cotton yield, fiber quality, and seed germination. J. Econ. Entomol. 100: 1560-

1568.

16

Borges, M., M. Birkett, J. R. Aldrich, J. E. Oliver, M. Chiba, Y. Murata, R. A. Laumann, J.

A. Barrigossi, J. A. Pickett, and M. C. B. Moraes. 2006. Sex attractant pheromone

from the rice stalk stink bug, Tibraca limbativentris Stal. J Chem. Ecol. 32: 2749-2761.

Borges, M., J. G. Millar, R. A. Laumann, M. C. B. Moraes. 2007. A male-produced sex

pheromone from the Neotropical redbanded stink bug, Piezodorus guildinii (W.). J.

Chem. Ecol. 33: 1235-1248.

Brazzel, J. R. and L. D. Newsom. 1959. Diapause in Anthonomus grandis Boh. J. Econ.

Entomol. 52: 603-611.

Campbell, J. P. 1919. Stop the losses in farming. Ga. State Col. of Agr. Ext. Div. Bull. 211: 14-

15.

Cassidy, T. P. and T. C. Barber. 1939. Hemipterous insects of cotton in Arizona: their

economic importance and control. J. Econ. Entomol. 32: 99-104.

Cokl, A. 2008. Stink bug interaction with host plants during communication. J. Insect Physiol.

54: 1113-1124.

Cokl, A., H. L. Mcbrien, and J. G. Millar. 2001. Comparison of substrate-borne vibrational

signals of two stink bug species, Acrosternum hilare and Nezara viridula (Heteroptera:

Pentatomidae). Ann. Entomol. Soc. Am. 94: 471-479.

Cokl, A., M. Virant-Doberlet, A. McDowell. 1999. Vibrational directionality in the southern

green stink bug, Nezara viridula (L.), is mediated by female song. Anim Behav. 58: 1277-

1283.

Cokl, A., M. Zorovic, A. Zunic and M. Virant-Doberlet. 2005. Tuning of host plants with

vibratory songs of Nezara viridula L. (Heteroptera: Pentatomidae). J. Exp. Biol. 208:

1481-1488.

17

Colazza, S., G. Aquila, C. D. Pasquale, E. Peri, J. G. Millar. 2007. The egg parasitoid

Trissolcus basalis uses n-nonadecane, a cuticular hydrocarbon from its stink bug host

Nezara viridula, to discriminate between female and male hosts. J. Chem. Ecol. 33: 1405-

1420.

Cottrell, T. E. 2001. Improved trap capture of Euschistus servus and Euschistus tristigmus

(Hemiptera: Pentatomidae) in pecan orchards. Fla. Entomol. 84: 731-732.

Cottrell, T. E., C. E. Yonce, and B. W. Wood. 2000. Seasonal occurrence and vertical

distribution of Euschistus servus (Say) and Euschistus tristigmus Say) (Hemiptera:

Pentatomidae) in pecan orchards. J. Entomol. Sci. 35: 421-431.

Espino, L., M. O. Way, and L. T. Wilson. 2008. Determination of Oebalus pugnax (Hemiptera:

Pentatomidae) spatial pattern in rice and development of visual sampling methods and

population sampling plans. J. Econ. Entomol. 101: 216-225.

Fucarino, A., J. G. Millar, J. S. Mcelfresh, and S. Colazza. 2004. Chemical and physical

signals mediating conspecific and heterospecific aggregation behavior of first instars

stink bugs. J. Chem. Ecol. 30: 1257-69.

Golden, M., P. A. Follett, and M. G. Wright. 2006. Assessing Nezara viridula (Hemiptera:

Pentatomidae) feeding damage in macadamia nuts by using a biological stain. J. Econ.

Entomol. 99: 822-827.

Gore, J., C. A. Abel, J. J. Adamczyk, and G. Snodgrass. 2006. Influence of soybean planting

date and maturity group on stink bug (Heteroptera: Pentatomidae) Populations. Environ.

Entomol. 35: 531-536.

Green, J. K. and C. D. Capps. 2003. Management considerations for stink bugs. pp. 113-120.

In Proc. 2003 Beltwide Cotton Conf., National Cotton Council. Memphis, TN.

18

Greene, J. K., P. M. Roberts, J. S. Bacheler, J. R. Ruberson, J. W. Van Duyn, M. D. Toews,

E. L. Blinka, D. Robinson, D. W. Mott, T. Walker, C. Davis, S.C. St. Matthews, and

R.B. Reeves. 2008. Refining treatment thresholds for stink bugs in the southeast 2007,

pp. 1204-1211. In Proceedings of the Beltwide Cotton Conferences, January 8-11,

Nashville, TN, National Cotton Council of America.

Greene, J. K., P. M. Roberts, J. S. Bacheler, M. D. Toews, J. R. Ruberson, F.P.F. Reay-

Jones, D. Robinson, D. W. Mott, T. Walker, C. Davis, and D. E. Morrison, T.

Pegram, and R. Reeves. 2009. Continued evaluation of internal boll-injury thresholds

for stink bugs in the southeast, pp. 1092-1101. In Proceedings of the Beltwide Cotton

Conferences, January 5-8, San Antonio, TX. National Cotton Council of America,

Memphis, TN.

Greene, J. K., S. G. Turnipseed, M. J. Sullivan and G. A. Herzog. 1999. Boll damage by

southern green stink bug (Hemiptera: Pentatomidae) and tarnished plant bug (Hemiptera:

Miridae) caged on transgenic Bacillus thuringiensis cotton. J. Econ. Entomol. 92: 941-

944.

Greene, J. K., S. G. Turnipseed, M. J. Sullivan, and O. L. May. 2001. Treatment thresholds

for stink bugs (Hemiptera: Pentatomidae) in Cotton. J. Econ. Entomol. 94: 403-409.

Haggler, J. R. 1997. Field retention of a novel mark-release-recapture method. Environ.

Entomol. 26: 1079-1086.

Haney, P. B., W. J. Lewis, W. R. Lambert. 2009. Cotton production and the boll weevil in

Georgia: history cost of control, and benefits of eradication. UGA Research Bulletin No.

428.

19

Harris, V. E., Todd, J. W., Webb, J. C. & Benner, J. C. 1982. Acoustical and behavioral

analyses of the songs of the southern green stink bug, Nezara viridula. Ann. Entomol.

Soc. Am. 75: 234-249.

Hendricks, D.E., and H. M. Graham. 1970. Oil-soluable dye in larval diet for tagging moths,

eggs, and spermatophores of tobacco budworms. J. Econ. Entomol. 63: 1019-1020.

Henry, C. S. 1994. Singing and cryptic speciation in insects. Trends Ecol. Evol. 9: 388-392.

Hirose, E., A. R. Panizzi, and A. J. Cattelan. 2006. Effect of relative humidity on emergence

and on dispersal and regrouping of first instars Nezara viridula (L.) (Hemiptera:

Pentatomidae). Neotrop. Entomol. 35: 757-61.

Ho, H., and J. G. Millar. 2001. Identification and synthesis of a male-produced sex pheromone

from the stink bug Chlorochroa sayi. J. Chem. Ecol. 27: 1177-1201.

Jones, V. P., J. R. Hagler, J. F. Brunner, C. C. Baker, and T. D. Wilburn. 2006. An

inexpensive immunomarking technique for studying movement patterns of naturally

occurring insect populations. Environ. Entomol. 35: 827-836.

Khrimian, A., P. W. Shearer, A. Zhang, G. C. Hamilton, and J. R. Aldrich. 2008. Field

trapping of the invasive brown marmorated stink bug, Halyomorpha halys, with

geometric isomers of methyl 2,4,6-decatrienoate. J. Agric. Food Chem. 56: 197-203.

Kiritani, K., N. Hokyo, K. Kimura, F. Nakasuji. 1965. Imaginal dispersal of the southern

green stink bug, Nezara viridula, in relation to feeding and oviposition. Jpn. J. Appl.

Entomol. Zool. 9: 291-97.

Koppel, A. L., D. A. Herbert, JR., T. P. Kuhar, and K. Knmminga. 2009. Survey of stink

bug (Hemiptera: Pentatomidae) egg parasitoids in wheat, soybean, and vegetable crops in

southeast Virginia. Environ. Entomol. 38: 375-379.

20

Krupke, C. H. J. F. Brunner, M. D. Doerr, and A. D. Kahn. 2001. Field attraction of the stink

bug Euschistus conspersus (Hemiptera: Pentatomidae) to synthetic pheromone-baited

host plants. J. Econ. Entomol. 94: 1500-1505.

Laumann, R. A., M. F. S. Aquino, M. C. B. Moraes, M. Pareja, M. Borges. 2009. Response

of the egg parasitoids Trissolcus basalis and Telenomus podisi to compounds from

defensive secretions of stink bugs. J. Chem. Ecol. 35: 8-19.

Martin, P. A. W., E. Hirose, and J. R. Aldrich. 2007. Toxicity of Chromobacterium subtsugae

to southern green stink bug (Heteroptera: Pentatomidae) and corn rootworm (Coleoptera:

Chrysomelidae). J. Econ. Entomol. 100: 680-684.

Mcbrien, H. L., J. G. Millar, L. Gottlieb, X. Chen, and R. E. Rice. 2001. Male-produced sex

attractant pheromone of the green stink bug, Acrosternum hilare (Say). J. Chem. Ecol.

27: 1821-1839.

Mcbrien, H. L., J. G. Millar, R. E. Rice, J. S. Mcelfresh, E. Cullen, F. G. Zalom. 2002. Sex

attractant pheromone of the red-shouldered stink bug Thyanta pallidovirens: A

pheromone blend with multiple redundant components. J. Chem. Ecol. 28: 1797-1818.

McPherson, R. M., G. R. Buss, and P. M. Roberts. 2007. Assessing stink bug resistance in

soybean breeding lines containing genes from germplasm IAC-100. J. Econ. Entomol.

100: 1456-1463.

McPherson, J. E. and R. M. McPherson. 2000. Stink bugs of economic importance in America

north of Mexico. CRC Press, Boca Raton, FL, USA. McPherson, R. M., M. L. Wells

McPherson, R. M., M. L. Wells, and C. S. Bundy. 2001. Impact of the early soybean

production system on arthropod pest populations in Georgia. Environ. Entomol. 30: 76-

81.

21

Medrano, E. G., J. F. Esquivel and A. A. Bell. 2007. Transmission of cotton seed and boll

rotting bacteria by the southern green stink bug (Nezara viridula L.). J. Appl. Microbiol.

103: 436-444.

Miklas, N., A. Cokl, M. Renou, M. Virant-Doberlet. 2003. Variability of vibratory signals and

mate choice selectivity in the southern green stink bug. Behav. Processes. 61: 131-142.

Mizell, R. F. and W. L. Tedders. 1995. A new monitoring method for detection of the stink bug

complex in pecan orchards. In proceedings, 1995 Pecan Grow. Assoc. 88: 36-40.

Moraes, M. C. B., J. G. Millar, R. A. Laumann, E. R. Sujii, C. S. S. Pires, and M. Borges.

2005. Sex attractant pheromone from the Neotropical red-shouldered stink bug, Thyanta

perditor (F.). J. Chem. Ecol. 31: 1415-1427.

Murdock, W.W. 1963. A method for marking Carabidae (Col.). Entomol. Monthly 99: 22-24.

Musser, F., S. Stewart, R. Bagwell, G. Lorenz, A. Catchot, E. Burris, D. Cook, J. Robbins,

J. Greene, G. Studebaker, and J. Gore. 2007. Comparison of direct and indirect

sampling methods for tarnished plant bug (Hemiptera: Miridae) in flowering cotton. J.

Econ. Entomol. 100: 1916-1923.

Nakasuji, F., N. Hokyo, K. Kiritani. 1965. Spatial distribution o f three plant bugs in relation to

their behavior. Res. Popul. Ecol. 4: 99-108.

Ni, X., M. D. Krakowsky, G. D. Buntin, B. G. Rector, B. Guo, and M. E. Snook. 2008.

Identification of multiple ear-colonizing insect and disease resistance in CIMMYT maize

inbred lines with varying levels of silk maysin. J. Econ. Entomol. 101: 1455-1465.

Ni, X., W. Xu, M. D. Krakowsky, G. D. Buntin, S. Brown, R. D. Lee, and A. E. Coy. 2007.

Field screening of experimental corn hybrids and inbred lines for multiple ear-feeding

insect resistance. J. Econ. Entomol. 100: 1704-1713.

22

Panizzi, A. R., M. Berhow, and R. J. Bartelt. 2004. Artificial substrate bioassay for testing

oviposition of southern green stink bug conditioned by soybean plant chemical extracts.

Environ. Entomol. 33: 1217-1222.

Roberts, P. M., J. R. Ruberson, M. D. Toews, 2009. Cotton insect control. Georgia Pest

Management Handbook.

Schroeder, W. J. and W.C. Mitchell. 1981. Marking Tephritidae fruit fly adults in Hawaii for

release-recovery studies. Proc. Hawaiian Entomol. Soc. 23: 437-440.

Service, M.W. 1993. Mosquito ecology: field sampling methods. Elsevier, New York.

Shearer, P. W., V. P. Jones. 1996. Diel feeding pattern of adult female southern green stink bug

(Hemiptera: Pentatomidae). Environ. Entomol. 25: 599-602.

Smith, J. F., R. G. Luttrell, and J. K. Greene. 2009. Seasonal abundance, species composition,

and population dynamics of stink bugs in production fields of early and late soybean in

south Arkansas. J. Econ. Entomol. 102: 229-236.

Smith, J. F., R. G. Luttrell, and J. K. Greene, and C. Tingle. 2009. Early-season soybean as a

trap crop for stink bugs (Heteroptera: Pentatomidae) in Arkansas’ changing system of

soybean production. Environ. Entomol. 38: 450-458.

Snodgrass, G. L., J. J. Adamczyk, Jr., and J. Gore. 2005. Toxicity of insecticides in a glass-

vial bioassay to adult brown, green, and southern green stink bugs (Heteroptera:

Pentatomidae). J. Econ. Entomol. 98: 177-181.

Stimmann, M. W., W. W. Wolf and W. L. Berry. 1973. Cabbage loopers: biological effects of

rubidium in the larval diet. J. Econ. Entomol. 66: 324-326.

23

Tillman, P. G. 2006. Susceptibility of pest Nezara viridula (Heteroptera: Pentatomidae) and

parasitoid Trichopoda pennipes (Diptera: Tachinidae) to selected insecticides. J. Econ.

Entomol. 99: 648-657.

Tillman, G. J. Aldrich, and A. Khrimian. 2007. New compounds for capturing southern green

stink bugs in baited traps. In Proceedings of the Beltwide Cotton Conferences. January 9-

12, New Orleans.

Tillman, P. G., Northfield, T. D., Mizell, R. F. and Riddle, T. C. 2009. Spatiotemporal

patterns and dispersal of stink bugs (Heteroptera: Pentatomidae) in peanut-cotton

farmscapes. Environ. Entomol. 38: 1038-1052.

Toews, M. D., E. L. Blinka, J. W. Van Duyn, D. A. Herbert, JR., J. S. Bacheler, P. M.

Roberts, and J. K. Greene. 2009. Fidelity of External Boll Feeding Lesions to Internal

Damage for Assessing Stink Bug Damage in Cotton. J. Econ. Entomol. 102: 1344-1351.

Toews, M. D., J. Greene, F. P. F. Reay-Jones, and R. B. Reeves. 2008. A comparison of

sampling techniques for stink bugs in cotton, pp. 1193D1203. In Proceedings of the

Beltwide Cotton Conferences, Nashville, TN, 8D11 January 2008. National Cotton

Council of America, Memphis, TN.

Toews, M. D. and W. D. Shurley. 2009. Crop juxtaposition affects cotton fiber quality in

Georgia farmscapes. J. Econ Entomol. 102: 1515-1522.

Todd, J. W., 1989. Ecology and behavior of Nezara viridula. Ann. Rev. Entomol. 34: 273-292.

Toscano, N. C. and V. M. Stern. 1976. Cotton yield and quality loss caused by various levels of

stink bug infestations. J. Econ. Entomol. 69: 53-56.

Turnispseed, S. G., M. J. Sullivan, A. M. Hagerty, and A. L. Gibson. 2003. Boll damage by

the stinkbug/plant bug complex in South Carolina cotton. Proceedings of the Beltwide

24

Cotton Conferences, January 2003, Nashville, TN, National Cotton Council of America,

Memphis, TN.

Virant-Doberlet, M. and A. Čokl . 2004. Vibrational communication in insects. Neotrop.

Entomol. 33: 121-134.

Williams, M. 2010. Cotton insect losses-2009. In Proceedings of the Beltwide Cotton

Conferences, January 4-7, New Orleans, LA. National Cotton Council of America,

Memphis, TN (in press).

Willrich, M. M., B. R. Leonard, and G. B. Padgett. 2004. Influence of southern green stink

bug, Nezara viridula L., on late-season yield losses in cotton, Gossypium hirsutum L.

Environ. Entomol. 33: 1095-1101.

Wissinger, S. A. 1997. Cyclic colonization in predictably ephermeral habitats: a template for

biological control in annual crop systems. Biol. Control. 10: 4-15.

Wright, M. G., P. A. Follett and M. Golden. 2007. Long-term patterns and feeding sites of

southern green stink bug (Hemiptera: Pentatomidae) in Hawaii macadamia orchards, and

sampling for management decisions. Bull. Entomol. Res. 97: 569-575.

Xia, J., A. Mustafic, M. D. Toews, and M. A. Haidekker. 2011. Stink bug feeding induces

fluorescence in developing cotton bolls. J. Biol. Eng. 5: 11.

25

Figure 1. Common phytophagus stink bugs (Hemiptera: Pentatomidae) of Georgia cotton system.

26

CHAPTER 2

FEEDING PREFERENCE AND MOVEMENT OF NEZARA VIRIDULA (L.) AND

EUSCHISTUS SERVUS (SAY) (HEMIPTERA: PENTATOMIDAE) ON INDIVIDUAL

COTTON PLANTS

_______________________

T-I Huang and M. D. Toews. 2012. Journal of Economic Entomology, Accepted

27

ABSTRACT

Experiments were conducted in an environmental growth chamber to determine the

movement and feeding preferences of Nezara viridula (L.) and Euschistus servus (Say) on

individual cotton plants. Fifth instars were caged by species on a single cotton plant (FM 9063

B2RF) containing four discrete boll sizes, ranging from 1.1 to 3.0 cm in diameter, over a period

of 5 d per replication. Two digital video cameras were simultaneously focused on each of the

four bolls per plant to visually confirm stink bug resting and movement. During the study, a

total of 4080 h of video footage was recorded and analyzed. Results showed that N. viridula and

E. servus did not prefer the exact same boll sizes. In a trial with eight stink bugs per plant, N.

viridula spent more time on the three larger boll classes, 1.6-2.0, 2.1-2.5, and 2.6-3.0 cm. In a

separate trial with one stink bug per plant, N. viridula spent more time on the larger boll classes

while E. servus exhibited the strongest preference for 1.1-1.5 and 2.1-2.5 cm bolls. Nezara

viridula moved more often than E. servus and both species moved more often during photophase

compared to scotophase. Regardless of species or number of bugs released, bolls in the smallest

boll size class fell off the plant about three days after the bugs were released. These results

confirm that scouts who are estimating stink bug damage should select bolls in the 2.1 - 2.5 cm

diameter boll size class.

KEY WORDS: feeding preference, intraplant movement, stink bug, sampling, pest management

28

Introduction

Stink bugs (Hemiptera: Pentatomidae) are key cotton pests in the southeastern and mid-

southern United States. The southern green stink bug, Nezara viridula (L.) (Hemiptera:

Pentatomidae), is considered one of the most important agricultural pests in the world (Todd

1989). The brown stink bug, Euschistus servus (Say) (Hemiptera: Pentatomidae) is the most

widespread member of the Pentatomidae in North America (Munyaneza and McPherson 1994),

and has received a great deal of recent attention (Siebert et al. 2005, Tillman 2010, Tillman et al.

2010, Herbert and Toews 2011). Both species are polyphagous and cause economic damage to a

wide-range of agricultural crops such as corn, cotton, peanut, soybean, and wheat (McPherson

and McPherson 2000, Reay-Jones 2010, Olson et al. 2011, Herbert and Toews 2011). Stink bug

feeding on developing cotton bolls can result in boll abscission, lint staining, reduced lint quality,

and reduced yields (Cassidy and Barber 1939, Willrich et al. 2004, Toews et al. 2009, Xia et al.

2011). Further, N. viridula is reported to acquire and transmit Pantoea agglomerans strain Sc 1-

R, an opportunistic boll rot bacterium, into cotton bolls. Boll rot pathogens are transmitted by

both nymphs and adults of N. viridula (Esquivel 2011).

Although N. viridula biology and ecology are extensively documented (DeWitt and

Godfrey 1972, Todd 1989), little is known concerning temporal feeding patterns of N. viridula

and E. servus. A previous study showed that adults and nymphs of N. viridula were observed to

feed during early morning and late afternoon in the field (Corpuz 1969, Lockwood and Story

1986); however, both of those studies were conducted only during photophase. A previous study

showed that adult female N. viridula could be seen feeding throughout the day, but fed for longer

durations during the scotophase (~12 min/h) than the photophase (~4 min/h) (Shearer and Jones

1996).

29

Recently, a video-based assay was developed to monitor Lygus hesperus Knight

(Hemiptera: Miridae) movement, stylet-probing durations, feeding location on squares, and

oviposition behaviors on cotton (Cooper and Spurgeon 2011). They found that prereproductive

adults of L. hesperus were less active and feed more compared with reproductive adults, but

behaviors varied by gender and mating states. Due to the recent awareness of stink bugs as a

vector of boll rot pathogens (Esquivel 2011, Medrano et al. 2007, 2009), the authors anticipate

that future stink bug feeding studies will be conducted to show how these pathogens are

transmitted. The role of light condition on feeding will be necessary for these studies.

Common scouting methods for stink bugs in cotton include the sweep net, drop cloth and

internal boll injury. Although it requires comparatively less time, estimating population density

with the sweep net and drop cloth have documented age biases; more adults tend be captured in

the sweep net, while more nymphs tend to be captured on the drop cloth (Toews et al. 2008).

Assessment of boll injury, determined by evaluating bolls for the presence of internal warty

growths or yellow stained lint, requires more time but is much more sensitive (Wene and Sheets

1964, Greene and Herzog 1999). For example, assessing internal boll injury was 10-fold more

sensitive for detecting boll injury than use of a sweep net or drop cloth to detect insect presence;

however, assessing internal boll injury required 4 and 6-fold more time than sweep net and drop

cloth samples, respectively (Toews et al. 2008). A new technique based on external boll feeding

lesions was proposed to improve time consuming internal boll injury method for assessing stink

bug damage in cotton (Toews et al. 2009); however, only a moderately strong correlation was

detected between external stink bug feeding symptoms and internal feeding damage (Toews et al.

2009, Blinka et al. 2010).

30

Since detection of internal boll damage remains the standard for stink bug scouting,

choosing the correct sized boll is critical. Siebert et al. (2005) showed that 7 to 27 d old bolls

were more frequently injured by E. servus, which corresponded to bolls with a diameter of 1.2 to

3.6 cm. The current Extension recommended practice is to collect quarter-sized (2.4 cm)

diameter bolls and then visually inspect the internal tissues for signs of feeding (Bacheler 2007).

However, feeding behavior on bolls may vary among different stink bug species and movement

of stink bugs within plants is poorly understood. The objectives of this study were to determine

the boll size that was most frequently injured by N. viridula and E. servus and to examine

differences in the rate of stink bug movement as a function of photoperiod.

Materials and Methods

Stink bug rearing. The N. viridula colony was founded with ~50 adults collected from

Tift County, Georgia, in April 2007. Feral individuals were introduced annually to prevent

decline in colony vigor and viability. The colony was maintained in the lab following the

methods of Harris and Todd (1981) on pesticide-free green beans, field corn, or okra pods,

depending on seasonal availability. Adults diets were supplemented with free water, shelled

green peanuts and raw shelled sunflower seeds. Eggs through 3rd instars were held in ventilated

Petri dishes, while 4th and 5th instars were held in 0.95 liter clear plastic jars (part no. JSS32-

120PP, Olcott Plastics, St. Chas, IL). Adults were held in 37.9 liter glass aquaria with paper

towels serving as oviposition substrate. All life stages were held in environmental chambers that

maintained conditions at temperature between 21 and 26° C, 65% RH, and a 14:10 (light dark)

photoperiod.

31

The E. servus colony was founded from ~50 adults collected in May 2009 from corn in

Tifton County, Georgia. The colony was maintained under a 16:10 photoperiod at 21.0 to 28.0°

C and 65% RH. Individuals were fed a diet of pesticide-free green beans, field corn, or okra

pods. Adults were supplemented with shelled green peanuts and raw shelled sunflower seeds.

Eggs through 3rd instars were held in ventilated Petri dishes, while 4th and 5th instars were held

in 0.95 liter clear plastic jars. Adults were held as individual mating pairs in 0.47-liter clear

plastic jars; each jar was provisioned with a 3 cm wide by 8 cm long piece of cheesecloth

suspended from the lid for the oviposition substrate.

Plant cultivation. Individual potted cotton plants (FM 9063 B2F, Bayer CropScience)

were grown year round in a greenhouse maintained at 21 to 35° C with a 14:10 (L:D)

photoperiod. Individual seeds (1 seed per pot) were sown in 11.35-liter plastic pots filled with

Metro Mix 300 growing medium (Sun Gro Horticulture, Bellevue, WA) and fertilized bimonthly

with 6.5 g of Osmocote 14-14-14 and a 5.5 g of Micromax 90505 (The Scotts Co. LLC,

Marsville, OH), following the methodology of Bundy et al. (2000). Plants were watered twice

daily using a timer system. A weak solution (6 ml per liter of water) of mepiquat pentaborate

(Pentia, BASF Corporation, Research Triangle Park, NC) was applied weekly during fruiting to

prevent rank vegetative growth.

Once the plants began flowering, individual white flowers were tagged daily with a piece

of vinyl flagging tape that was loosely wrapped around the boll petiole with a short piece of 0.51

mm diameter steel wire. Cotton bolls were allowed to continue developing for a period of 3 to

14 d. Individual plants were randomly chosen and transferred to an experimental chamber at the

time of the experiment had first position bolls that fit into the following four external size

classes: 1.1-1.5 cm, 1.6-2.0 cm, 2.1-2.5 cm, and 2.6-3.0 cm. The diameter of each boll was

32

measured with a vernier caliper to tenths of a cm and the height above ground of each boll was

measured with a meter stick on the first day of the experiment. Boll age was calculated before

each experiment by tagging (bloom). Any additional bolls that were not part of the experiment

(typically second position bolls at the bottom of the plant) were removed with scissors before

starting the trial.

Environmental conditions. All experiments were conducted by caging stink bugs on an

individual cotton plant in an upright growth chamber (model I-36 LLVL, Percival Scientific,

Perry, IA) that maintained a 14:10 (L:D) photoperiod and temperature at 25° C during the day

and 20° C at night. Environmental conditions were confirmed and monitored using a data logger

(Hobo H8, Onset Computer Corp., Cape Cod, MA). Lighting was provided by four F32T8 4100

K fluorescent light bulbs. Light intensity was measured in the middle of the chamber using a

digital light meter (model 401025, Extech Instruments, Nashua, NH). Two shelves, one

positioned as far toward top of the chamber as possible and a second positioned as far toward the

bottom of the chamber as possible, were left in the chamber so that video camera flex mounts

could be attached. The potted plant was positioned on the bottom shelf with the plant canopy

extending approximately 75 to 90% of the way to the top of the chamber.

Video camera positioning. Two high-resolution miniature color video cameras

(HCCM474M, Honeywell, Morristown, NJ) were focused on each of the four cotton bolls to

record stink bug activity. Cameras were mounted on flex mounts (model 817-13, PanaVise

Products Inc., Reno, NV) and linked to a digital video recorder (EDR920 Powerplex, EverFocus

Electronics Corp., Taipei, Taiwan) via cables that passed through a port in the side of the

chamber. Each camera was outfitted with a vari-focal CCTV lens (model YV5x2.7R4B-SA2L,

Fujinon Corp., Tokyo, Japan). A liquid crystal display color monitor was connected to the

33

digital video recorder for focusing and observation from outside the closed growth chamber. A

light-emitting diode infrared illuminator (IR-200, ProVideo, Amityville, NY) was mounted on a

tripod and directed toward each of the four bolls to facilitate night vision. The cameras

automatically switched from color to black and white mode when the growth chamber lights

were off; in black and white mode, the camera had sufficient light sensitivity to the infrared

illumination that the stink bugs could be observed in the absence of visual light. Power to the

illuminators was controlled by a separate automated light sensor that activated when the chamber

lights went off.

Experiment types. Two separate types of experiments were conducted. Using N.

viridula only, the first experiment was conducted by releasing eight stink bugs simultaneously on

the same cotton plant. Results from that experiment suggested that the insect density could be

reduced. In the second type of experiment, a single N. viridula or E. servus was released on a

single cotton plant.

Protocol with eight stink bugs per plant. This experiment was conducted in an upright

growth chamber at the Riverbend Entomology greenhouse in Athens, GA, in 2009. Trials were

conducted with 4th or 5th instars of N. viridula that were shipped overnight from Tifton with

moist tissues and fresh green beans in a plastic container. To increase visibility of the stink bugs,

the dorsal side of the abdomen was lightly brushed with orange fluorescent dust (DayGlo Color

Corp., Cleveland, OH). In each replicate, four N. viridula nymphs were released on the bottom

leaf of the plant while the other four nymphs were released from the top leaf of the plant. After

each 5 d observation period, the cotton plant was removed from the chamber and the bolls were

excised from the plant and internally examined for the presence of punctures, warts, and stained

lint. Resulting video files were examined and the total time each stink bug spent on a particular

34

boll was recorded. Ten replicates (individual plants) were conducted from August to December

in 2009.

The treatments were analyzed as a one way ANOVA arranged in a randomized complete

bock design. The experimental unit for the trial was each boll on a living plant throughout the 5

d recording period. Measurements of feeding behavior and movement were taken from the video

footage. During each 5 d recording period, investigators scored the number of times a stink bug

moved onto a specific boll and the length of time that the stink bugs spent on each boll class.

The response variable, time on bolls, was subjected to a logarithmic transformation (Zar 1999)

and then compared among boll classes using a one-way ANOVA (PROC GLIMMIX, SAS

Institute 2008). Boll class was modeled as a fixed effect and ten replicates (plants) were

modeled as a random effect. Treatment means were separated using LSMEANS test (P < 0.05).

Regardless of transformations, actual time (h) stink bugs spent on bolls are presented in the text

and figures.

Protocol of one stink bug per plant. Experiments were conducted in an upright growth

chamber during 2010 and 2011 at the Coastal Plain Experiment Station located at Tifton, GA.

All replicates were conducted with newly molted fifth instars. The dorsal side of the abdomen

was lightly brushed with orange fluorescent dust (DayGlo Color Corp., Cleveland, OH) to

increase visibility of the stink bugs. In each replicate, a single 5th-instar N. viridula or E. servus

nymph was released on the bottom leaf of the cotton plant located in the environmental growth

chamber. At the conclusion of each 5 d replicate, the plant was removed and bolls were excised

from the plant and internally examined for evidence of stink bug feeding, denoted by the

presence of punctures, warts, or stained lint. Measurements of feeding behavior and movement

were taken from the video footage. Video files were examined and the total time each stink bug

35

spent on a particular boll was recorded. Twelve replicates (plants) each with N. viridula or E.

servus were conducted.

The experiment was organized as a two-way factorial arrangement of treatments in a

randomized complete block design. Response variables, time spent on bolls and movement

frequency (bugs moving on or off a given boll), were log transformed to correct for

heteroscedastic variances. A two-way ANOVA (PROC GLIMMIX, SAS Institute 2008) was

performed to compare the length of time that stink bugs spent on each boll class where species

and boll class were fixed effects and replicates (individual plants) were random effects; treatment

means were separated using the LSMEANS test (P < 0.05) and the slice option (SAS Institute

2008) if there was a significant interaction. Movement frequency was also analyzed as a two-

way ANOVA (SAS Institute 2008) to detect differences between species and light condition.

Species and light condition were fixed effects and replicates were modeled as random effects.

Treatment means were separated using LSMEANS test (P < 0.05). Regardless of

transformations for data analyses, actual means and standard errors are presented in the text,

table, and figures.

Results

A total of 2,880 h of video footage was recorded for the single stink bug per plant

experiment and an additional 1,200 h was logged for the 8 stink bugs per plant experiment.

During those experiments, the mean temperature in the growth chamber was 23.65 ± 0.02° C

during photophase and 20.48 ± 0.02° C during scotophase. Mean relative humidity was 87.0%

during photophase and 89.6% during scotophase. Similarly, light intensity was 2628.5 ± 3.5 lux

during photophase and 12.6 ± 0.5 lux during scotophase. Mean ± SEM boll size in each class

36

was 1.22 ± 0.02, 1.74 ± 0.02, 2.27 ± 0.02, and 2.78 ± 0.02 cm, respectively (n=24). This

corresponded with a mean ± SEM boll age in each class of 3.4 ± 0.2, 6.9 ± 0.3, 9.9 ± 0.3, and

13.0 ± 0.5 d, respectively (n=17). The mean ± SEM distance above ground level for each boll

class from the smallest to the largest boll size class was 100.0 ± 2.5, 94.1 ± 2.4, 85.6 ± 2.3, and

77.6 ± 2.5 cm, respectively (n=17). The mean ± SEM age of N. viridula used for this experiment

was 24.7 ± 0.9 d, whereas the mean ± SEM age of E. servus was 20.3 ± 1.1 d.

Protocol with eight stink bugs per plant. In the experiments with 8 N. viridula released

on a single cotton plant, internal feeding symptoms were evident on 9, 10, 10, and 10 (smallest to

largest boll classes) of the 10 total bolls in each boll size class. The mean time spent on bolls

was significantly greater on the 3 larger boll classes compared to the smallest boll class (F =

19.70; df = 3, 27; P < 0.0001) (Fig. 1). The smallest boll abscised from the plant before the end

of the 5 d period in eight of the ten replicates. Considering replicates where the bolls fell off

during the experiment, abscission occurred 72.8 ± 4.7 h after the infestation was initiated.

Protocol of one stink bug per plant. Regardless of species, stink bugs generally fed on

all boll classes. In experiments with a single N. viridula per cotton plant, feeding symptoms

were evident on 9, 7, 9, and 12 (smallest to largest boll classes) of the 12 total bolls in each boll

size class at the end of the 5 d period. In ten out of the twelve replicates, bolls of the smallest

boll class abscised from the plant during the experiment. Abscised bolls dropped an average of

71.55 ± 8.22 h after f N. viridula infestation. In replicates with a single E. servus individual per

cotton plant, feeding symptoms were evident on 12, 7, 10, and 6 (smallest to largest boll classes)

of the 12 total bolls in each boll size class at the end of 5 d replicate. The smallest boll abscised

from the plant in six out of the twelve replicates. Abscised bolls fell off during the experiment,

mean time for the smallest boll to abscise was 77.6 ± 14.2 h.

37

In experiments with one bug per plant, statistical analyses of time spent on bolls showed

that there was a significant interaction between insect species and boll size class (F = 7.57; df =

3, 77; P = 0.0002) (Fig. 2). Further analyses of the interaction (sliced by species) showed that

there were differences among boll classes by species (E. servus F = 5.81; df = 3, 77; P = 0.0012)

(N. viridula F = 3.86; df = 3, 77; P = 0.0125). Further analyses of the interaction (sliced by boll

class) showed that there were no differences between species on bolls in the three smallest boll

classes (F = 0.00 to 3.01, df = 1, 77; P = 0.0868 to 0.9976), but there were differences between

species for the largest boll class (F = 18.86; df = 1, 77; P < 0.0001).

There were no interactions between species and light condition when examining

statistical analyses for movement frequency during each experiment (F = 0.03; df = 1, 33; P =

0.8549). Conversely, there were significant differences in movement between fixed effects for

species (F = 4.49; df = 1, 33; P = 0.0418) and between photophase and scotophase (F = 49.39; df

= 1, 33; P < 0.0001) (Table 1). Nezara viridula moved more often than E. servus, and both

species were more active during photophase than scotophase.

Discussion

Data obtained from the digital video-based system clearly demonstrated that this system

was a useful tool for insect behavior studies. In the protocol with one bug per plant, a small

amount of fluorescent dust and the infrared illuminators facilitated tracking of each stink bug’s

movement on the bolls. Unfortunately, movement of individual stink bugs could not be tracked

when 8 stink bugs were simultaneously released because the bugs were not uniquely identified.

Future attempts with multiple bugs should be conducted with different dust colors or other

unique marking technologies. The authors were unable to differentiate between stink bugs

38

resting on bolls and stink bugs actively feeding on bolls in the video records. This occurred

because of the relatively large area that had to be imaged on the boll and the fact that there was

no way to predict where the stink bug(s) would insert their stylets. In all cases, more than one-

half of the bolls in a particular boll size class exhibited symptoms of feeding damage. This

observation suggests that bugs actively move among bolls, regardless of the number of

individuals on each plant. While it is unknown if the stink bugs were actually feeding during the

entire duration that they were observed on the bolls, observed damaged (>50% per boll in every

replicate) strongly suggests that the bugs generally took a meal each time they were observed on

a boll.

These data suggest that stink bug feeding preference was significantly influenced by stink

bug species and cotton boll size. While individual N. viridula fifth instars exhibited feeding

preference for the two larger boll size classes (2.1-2.5 cm and 2.6-3.0 cm), E. servus showed a

preference for the 2.1-2.5 and 1.1 to 1.5 cm boll size classes. These observations clearly

indicated that current Extension recommended practice, collection and examination of quarter-

sized (2.4 cm) bolls, is highly appropriate for detecting stink bug damage by both species in the

field (Greene and Herzog 1999). Since the bugs preferred quarter-sized bolls (2.1-2.5 cm), it is

logical that those same sized bolls may be more susceptible to boll-rot pathogens introduced

during feeding (Esquivel 2011, Medrano et al. 2007, Medrano et al. 2009). Feeding preference

influenced by cotton boll age has previously been documented for E. servus (Siebert el at. 2005).

In their observations, 7-27-d-old bolls (boll diameter of 1.161-3.586 cm with a mid-range of

2.375 cm) were the most frequently injured by E. servus. Our data provide a more precise boll

size range.

39

One drawback to the stated experimental design is that the position of the bolls (boll

location) could not be randomized on the plant. It was hypothesized that in the one bug per plant

experiment, the largest bolls would garner more visits since those bugs were released at the

bottom of the plant would have to pass by the largest boll to get to a smaller boll. Numerically

speaking this held true for N. viridula, but that hypothesis was revisited when stink bugs were

simultaneously released at the top and bottom (8 bugs per plant trial) and the same trends were

evident. Conversely, E. servus spent considerably less time on the 2.6 to 3.0 cm bolls. Since

boll size and location could not be randomized, they are confounded in these data. Although not

examined as part of this experiment or any other experiments with stink bugs feeding on cotton,

it is possible that preferential feeding on a specific boll size could be influenced by agronomic or

environmental factors that affect boll growth characteristics (Chen et al. 2005, Chen et al. 2007,

Adhikari and Parajulee 2008). However, that hypothesis is tempered by the fact that more than

50% of all bolls in this experiment exhibited feeding damage, thereby suggesting that the bugs

were active among all bolls on the plant.

Stink bugs feed on the developing seed and associated fibers of the immature cotton boll

(Barbour et al. 1988). Siebert et al. (2005) hypothesized that fewer injured bolls in the smallest

boll class might be due to the absence of internal seed development, therefore these bolls would

have decreased nutritional value. Our results also show that bolls from the smallest boll size

class were not highly preferred by N. viridula. A more obvious explanation for the disparity is

that there were fewer opportunities for feeding on the smallest bolls because they generally fell

off the plant about 3 d after the experiments were initiated. For example, with N. viridula 83%

of the smallest bolls abscised in the one bug per plant experiment; a similar number of bolls

40

(80%) fell from the plant in the eight bugs per plant trial. Abscission of the smallest bolls is a

known indicator of stress in cotton plants and stink bug feeding induces stress.

Euschistus servus exhibited a strong avoidance of the largest bolls. This is interesting

because the largest bolls would be the first ones encountered when released from the bottom of

the plant. It is worth noting that all of the smallest bolls (1.1 to 1.5 cm) exhibited internal

damage, while only 58% of the next smallest boll class (1.6 to 2.0 cm) exhibited feeding damage.

In the E. servus infested plants, only 50% of the smallest bolls abscised compared with 83% boll

abscission when infested by the same number of N. viridula. These data may suggest that boll

injury from E. servus feeding is less damaging than feeding injury from N. viridula. In addition,

greater movement frequency in N. viridula suggests that this species visits more bolls per unit of

time and therefore causes more damage than E. servus. Clearly, additional data on probing

intensity and feeding duration would be helpful to evaluate this hypothesis.

Movement frequency on an individual cotton plant during photophase was at least five

times greater than scotophase for both N. viridula and E. servus. Generally speaking, stink bugs

moved on and off the bolls during photophase and became sessile during scotophase. While it is

true that there were four more hours (16.7%) of light than dark in the experimental photoperiod,

this relatively small discrepancy does not compensate for a fivefold increase in activity. The

authors selected the experimental conditions based on typical environmental conditions, such as

photoperiod, when cotton is flowering in the field. Previous studies with adult L. hesperus and

adult female N. viridula demonstrated that both species fed significantly more during scotophase

than photophase (Sevacherian 1975, Shearer and Jones 1996). Prolonged feeding during

scotophase would explain why both N. viridula and E. servus showed less movement frequency

during scotophase.

41

Methods described here provide insight and increased knowledge on behavioral ecology

of two important stink bug species. To summarize, both species preferred resting on 2.1-2.5 cm

bolls and moved less (stayed on bolls longer) during scotophase. Pest management programs for

stink bugs would be improved by utilizing quarter-sized bolls for sampling boll damage.

Furthermore, regardless of photoperiod, feeding and movement studies need to be conducted for

a minimum of 24 hours as there are obvious differences attributed to photo condition.

42

Acknowledgments

The authors gratefully acknowledge Dean Kemp, Anne Horak, and David Griffin for

their assistance growing potted cotton plants and maintaining the stink bug colonies. Xinzhi Ni

provided a helpful review of an earlier manuscript draft. This research was funded by the

Georgia Cotton Commission under project number 11-827GA and by the USDA-NIFA-Special

Research Grants Program under award number 2009-34566-20100. Mention of trade names or

commercial products in this publication is solely for the purpose of providing specific

information and does not imply recommendation or endorsement by the University of Georgia.

43

References Cited

Adhikari, M. B. and M. N. Parajulee. 2008. Relationship between cotton boll age and Lygus

injury. ESA Southwestern Branch Meeting Abstract, February 23-26, Ft. Worth, TX.

Bacheler, J. S. 2007. Managing insects on cotton. pp. 136-160. In 2007 Cotton Information.

North Carolina Cooperative Extension: AG-417. North Carolina State University,

Raleigh, NC.

Barber, K. S., J. R. Bradley, Jr., and J.S. Bacheler. 1988. Phytophagous stink bugs in North

Carolina cotton: an evaluation of damage potential. pp. 280. In Proceedings of the

Beltwide Cotton Conference, 5-8 January 1988, New Orleans, LA. National Cotton

Council, Memphis, TN.

Blinka, E. L., A. Herbert, S. Malone, J. W. Van Duyn, P. Roberts, J. R. Bradley, and J. S.

Bacheler. 2010. Relationship between external stink bug (Hemiptera: Pentatomidae)

boll-feeding symptoms and internal boll damage with respect to cotton lint gin-out and

fiber quality. J. Econ. Entomol. 103: 2236-2241.

Bundy, C. S., R. M. McPherson, and G. A. Herzog. 2000. An examination of the external and

internal signs of cotton boll damage by stink bugs (Hemiptera: Pentatomidae). J.

Entomol. Sci. 35: 402-410.

Cassidy, T. P., and T. C. Barber. 1939. Hemipterous insects of cotton in Arizona: their

economic importance and control. J. Econ. Entomol. 32: 99-104.

Chen, F., G. Wu, F. Ge, M. N. Parajulee, and R. B. Shrestha. 2005. Effects of elevated CO2

and transgenic Bt cotton on plant chemistry, performance, and feeding of an insect

herbivore, the cotton bollworm. Entomol. Exp. Appl. 115: 341-350.

44

Chen, F., G. Wu, M. N. Parajulee, and F. Ge. 2007. Long-term impacts of elevated CO2 and

transgenic Bt cotton on performance and feeding of three generations of cotton bollworm.

Entomol. Exp. Appl. 124: 27-35.

Cooper, W. R., and D. W. Spurgeon. 2011. Laboratory studies of variations in feeding

behaviors among Lygus hesperus (Hemiptera: Miridae) of different gender and

reproductive states. Environ. Entomol. 40: 367-373.

Corpuz, L. R. 1969. The biology, host range, and natural enemies of Nezara viridula L.

(Pentatomidae: Hemiptera). Phillip. Entomol. 1:225-231.

DeWitt, N. B., and G. L. Godfrey. 1972. A bibliography of the southern green stink bug,

Nezara viridula (L.) (Hemiptera: Pentatomidae). Ill. Nat. Hist. Surv. Biol. Notes. 78:23.

Esquivel, J. F. 2011. Estimating potential stylet penetration of southern green stink bug - a

mathematical modeling approach. Entomol. Exp. Appl. 140: 163–170.

Greene, J. K., and G. A. Herzog. 1999. Management of stink bugs using symptoms of boll

injury as a monitoring tool, pp. 1041-1044. In Proceedings of the Beltwide Cotton

Conference, 3-7 January 1999, Orlando, FL. National Cotton Council, Memphis, TN.

Harris, V. E., and J. W. Todd. 1981. Rearing the southern green stink bug, Nezara viridula,

with relevant aspects of its biology. J. Georgia Entomol. Soc. 16: 203-209.

Herbert, J. J., and M. D. Toews. 2011. Seasonal abundance and population structure of brown

stink bug (Hemiptera: Pentatomidae) in farmscapes containing corn, cotton, peanut, and

soybean. Ann. Entomol. Soc. Am. 104: 909-918.

Lockwood, J. A., and R. N. Story. 1986. The diurnal ethology of the southern green stink bug,

Nezara viridula (L.), in cowpeas. J. Georgia Entomol. Soc. 21:175-184.

45

Medrano, E. G., J. F. Esquivel, and A. A. Bell. 2007. Transmission of cotton seed and boll

rotting bacteria by the southern green stink bug (Nezara viridula L.). J. Appl. Microbiol.

103: 436-444.

Medrano, E. G., J. F. Esquivel, R. L. Nichols, and A. A. Bell. 2009. Temporal analysis of

cotton boll symptoms resulting from southern green stink bug feeding and transmission

of a bacterial pathogen. J. Econ. Entomol. 102: 36-42.

McPherson, J. E., and R. M. McPherson. 2000. Stink bugs of economic importance in

America North of Mexico. CRC Press, Boca Raton, FL, USA.

Munyaneza, J., and J. E. McPherson. 1994. Comparative study of life histories, laboratory

rearing, and immature stages of Euschistus servus and Euschistus variolarius (Hemiptera,

Pentatomidae). Great Lakes Entomol. 26: 263–274.

Olson, D. M., J. R. Ruberson, A. R. Zeilinger, and D. A. Andow. 2011. Colonization

preference of Euschistus servus and Nezara viridula in transgenic cotton varieties,

peanut, and soybean. Entomol. Exp. Appl. 139: 161-169.

Reay-Jones, F. P. F. 2010. Spatial and temporal patterns of stink bugs (Hemiptera:

Pentatomidae) in wheat. Environ. Entomol. 39: 944-955.

SAS Institute. 2008. SAS/STAT 9.2 user’s guide. SAS Institute, Cary, NC.

Sevacherian, V. 1975. Activity and probing behavior of Lygus hesperus in the laboratory. Ann.

Entomolo. Soc. Am. 63: 557-558.

Shearer, P. W., and V. P. Jones. 1996. Diel feeding pattern of adult female southern green stink

bug (Hemiptera: Pentatomidae). Environ. Entomol. 25: 599-602.

46

Siebert, M. W., B. R. Leonard, R. H. Gable, and L. R. LaMotte. 2005. Cotton boll age

influences feeding preference by brown stink bug (Heteroptera: Pentatomidae). J. Econ.

Entomol. 98: 82–87.

Tillman, P. G. 2010. Composition and abundance of stink bugs (Heteroptera: Pentatomidae) in

corn. Environ. Entomol. 39: 1765-1774.

Tillman, P. G., J. R. Aldrich, A. Khrimian, and T. E. Cottrell. 2010. Pheromone attraction

and cross-attraction of Nezara, Acrosternum, and Euschistus spp. Stink bug (Heteroptera:

Pentatomidae) in the field. Chem. Ecol. 39: 610-617.

Todd, J. W. 1989. Ecology and behavior of Nezara viridula. Ann. Rev. Entomol. 34: 273–292.

Toews, M. D., J. K. Greene, F. P. F. Reay-Jones, and R. B. Reeves. 2008. A comparison of

sampling techniques for stink bugs in cotton, pp. 1193-1203. In Proceedings of the

Beltwide Cotton Conference, 8-11 January 2008, Nashville, TN. National Cotton

Council, Memphis, TN.

Toews, M. D., E. L. Blinka, J. W. Van Duyn, D. A. Herbert, Jr., J. S. Bacheler, P. M.

Roberts, and J. K. Greene. 2009. Fidelity of external feeding lesions to internal damage

for assessing stink bug damage in cotton. J. Econ. Entomol. 102: 1344-1351.

Wene, G. P., and L. W. Sheets. 1964. Notes on and control of stink bugs affecting cotton in

Arizona. J. Econ. Entomol. 57: 60-62.

Willrich, M. M., B. R. Leonard, and G. B. Padgett. 2004. Influence of southern green stink

bug, Nezara viridula L., on late-season yield losses in cotton, Gossypium hirsutum L.

Environ. Entomol. 33: 1095-1101.

47

Xia, J., A. Mustafic, M. D. Toews, M. A. Haidekker. 2011. Stink bug feeding induces

fluorescence in developing cotton bolls. J. Biol. Eng. 5:11, available online

http://www.jbioleng.org.content/5/1/11.

Zar, J. H. 1999. Biostatistical Analysis, 4th ed. Prentice Hall. Upper Saddle River, NJ.

48

Table 1. Mean ± SEM movement frequency (moved off boll or moved on boll) by lighting

condition and species during 5 day trials

Species Lighting condition 1Mean ± SE

Euschistus servus Photophase 2.1 ± 0.2a

Scotophase 0.3 ± 0.3b

Nezara viridula Photophase 3.3 ± 0.6a

Scotophase 0.8 ± 0.3b

1Means followed by the same letter, within species, are not significantly different (P < 0.05;

LSMEANS test).

49

Figure legends

Fig. 1. Mean ± SEM time spent by late instars (8 individuals released concurrently) of Nezara

viridula on bolls of four boll size classes ranging from 1.1 to 3.0 cm in diameter. Means

followed by the same letter are not significantly different (P < 0.05; LSMEANS test).

Fig. 2. Mean ± SEM time spent by a single 5th instars Euschistus servus or Nezara viridula on

bolls of four boll size classes ranging from 1.1 to 3.0 cm in diameter. Means followed by

the same letter are not significantly different (P < 0.05; LSMEANS test).

Fig 3. (A) (B) Stink bug excreted fluids while resting on bolls; (C) (D) Feeding on young cotton

bolls (1.1~1.5 cm in diameter) during photophase; (E) Feeding on cotton boll during

scotophase; (F) (G) (H) Aggregation feeding on larger bolls (2.6~3.0 cm in diameter).

Fig 4. Eclosion process of N. viridula on a cotton leaf (from left to right, up to bottom).

50

Boll diameter (cm)

1.1-1.5 1.6-2.0 2.1-2.5 2.6-3.0

Tim

e on

bol

ls (h

)

0

50

100

150

200

AA

A

B

F = 19.70; df = 3. 27; P < 0.0001n = 10

Figure 1.

51

Boll diameter (cm)

1.1-1.5 1.6-2.0 2.1-2.5 2.6-3.0

Tim

e on

bol

ls (h

)

0

15

30

45

60Euschistus servusNezara viridula

A

A

AB

ABBCBC

BC

C

Figure 2.

52

Figure 3.

53

Figure 4.

54

CHAPTER 3

INTERCROP MOVEMENT OF STINK BUGS (HEMIPTERA: PENTATOMIDAE) IN

GEORGIA FARMSCAPES

___________________________________

T-I Huang, R. Srinivasan, and M. D. Toews. 2012 Environmental Entomology, submitted

55

ABSTRACT

A 2-year field study was conducted using an immunomarking technique to detect local

dispersal of naturally occurring stink bug (Hemiptera: Pentatomidae) populations in Georgia

agroecosystems. Replicated plots were established with approximately 0.6 ha of cotton planted

between similar-sized plots of peanut and soybean. Starting when the cotton began flowering,

protein markers including egg whites (chicken egg albumin), cow’s milk (bovine casein), and

soymilk (soy protein) were applied using standard spray equipment to peanut, cotton, and

soybean, respectively. Stink bug density was estimated with sweep nets at 24 and 48 h after

proteins were sprayed each week. Captured insects were individually transferred into 1.5 ml

microcentrifuge tubes and then assessed for the presence of protein markers on the outer cuticle

using an antigen coated plate sandwich enzyme-linked immunosorbent assay (ACP-ELISA).

Results showed that 52.7% of stink bugs captured within the 2-ha study areas tested positive for

at least one of the three proteins. While there were differences in marking efficiency among

marking proteins applied to a specific crop, there were no differences in marking efficiency

among stink bug species. During cotton bloom, data indicated that at least a small number of

stink bugs moved between all possible combinations of adjacent crops; however, the majority of

the dispersal was into cotton. There were distinct differences in movement patterns among stink

bug species. These observations suggest that general movement trends within species are

predictable and this knowledge would allow managers to target their control efforts in

accordance with movement patterns.

KEY WORDS: immunomarking, marker-capture, stink bug, insect dispersal

56

Introduction

Polyphagous stink bugs (Hemiptera: Pentatomidae) are among the most damaging and

widespread agricultural pests worldwide (Todd 1989). The southern green stink bug, Nezara

viridula (L.), the brown stink bug, Euschistus servus (Say), and the green stink bug, Chinavia

hilaris (Say), are the three most abundant species in the southeastern United States and have

become economic pests of cotton (Gossypium hirsutum L.) (McPherson and McPherson 2000,

Greene et al. 2001, Reay-Jones et al. 2009). Stink bugs utilize a wide variety of agronomic row

crops in Georgia, including corn (Zea mays L.), cotton, peanut (Arachis hypogaea L.), and

soybean [Glycine max (L.) Merr.] (McPherson and McPherson 2000, Tillman 2010, Olson et al.

2011, Herbert and Toews 2011). In general, stink bugs feed on crops during fruit or pod

formation and move to other hosts according to crop phenology (Ehler 2000). Therefore,

management practices focused solely on a specific crop may have little effect on the general

stink bug population. In order to develop new strategies to manage these pests at the landscape

level, knowledge of stink bug movement among crops is vital.

Local dispersal in the field is an essential element for understanding insect ecology and

developing pest management strategies. Surveys on local insect movement have been conducted

using a wide variety of marking techniques. For example, honeybees, Apis mellifera (L.), can be

marked with commercial plastic color tags, platinum-iridium tags, and more recently with

harmonic radar (Gary 1971, Nelson and Baldwin 1977, Capaldi et al. 2000). The major

advantage of marking insects with tags is that they are inexpensive and durable. Conversely,

marking with tags is time-consuming and tedious (Hagler and Jackson 2001). Larvae of gypsy

moth, Lymantria dispar (L.), were marked by mutilating different prolegs or numbers of prolegs

to study their movement and population dynamics (Weseloh 1985). The advantage of mutilation

57

is that the marks are persistent and can be recognized quickly in the field, but it is also time-

consuming and might alter the insect’s movement and behavior (Hagler and Jackson 2001). Red

imported fire ants, Solenopis invicta (Buren), can be marked with ballpoint paint pens (Wojcik et

al. 2000); similar marking methods using paints or inks can be inexpensive and easy to apply, but

some chemicals in the markers can be toxic to insects (Hagler and Jackson 2001). Stink bugs in

the laboratory were marked with inexpensive and quick to apply Day-Glo fluorescent dust (Day-

Glo Color Corp., Cleveland, OH) which can be detected under UV light (Huang and Toews

2012); however, excessive dust can kill or produce adverse behavioral effects (Hagler and

Jackson 2001).

Insects in the laboratory can also be marked using an ingested label. For example, insects

can be marked with trace elements, such as rubidium, after feeding on marked food sources

(Johnson and Reeves 1995). The limitation of marking with trace elements is that it requires

expensive detection equipment and technical expertise to analyze the results (Akey et al. 1991).

Certain oil-soluble dyes such as Calco red N-1700 can be accumulated in insect body fluids or

tissues after oral ingestion (Gast and Landin 1966); for example, lepidopteran pests have been

marked by adding dye directly into their larval diets (Graham and Mangum 1971). However,

dyes may not be persistent in the field and can be harmful to insects (Naranjo 1990, Su et al.

1991).

Self-marking and genetic approaches have also been used for mark and recapture studies.

Hendrix et al. (1987) showed that Helicoverpa zea (Boddie) moths collected in Arkansas

possessed pollen from hosts in Texas, a sure indicator of movement by the moths. Self-mark

with pollen is advantageous for mark-capture studies, but pollen analysis is costly, time-

consuming, and can be influenced by the time of the year when sampling occurred (Hagler and

58

Jackson 2001). Mosquito dispersal was studied using various visible genetic markers such as

body and eye colors (Fay and Craig 1969, Hausermann et al. 1971). Marker genes were

successfully transferred using transposable elements in a Sterile Insect Release (SIR) program

for pink bollworm, Pectinophora gossypiella (Saunders), but analysis of such samples could be

costly and may not be suitable for a wide range of insects (Thibault et al. 1999).

External protein-based methods overcome many of the drawbacks associated with

previously mentioned marking techniques (Hagler et al. 1992, Hagler 1997, Hagler and Jackson

1998). This technique utilizes commercially available vertebrate-specific antibodies to test for

sprayed protein presence on insects using antigen coated plate sandwich enzyme-linked

immunosorbent assay (ACP-ELISA). Protein markers are effective externally as a spray or

internally by incorporation into diet (Hagler 1997). When proteins are applied externally,

external surfaces of stink bugs could get contaminated with them by direct contact during an

application or when insects walk on hosts’ surface previously marked with proteins (Hagler et al.

1992, Jones et al. 2006, Boina et al. 2009). Previous authors have documented the use of

inexpensive and commonly available food proteins such as chicken egg albumin (egg white),

bovine casein (cow’s milk), and soy protein (soymilk) to conduct mass mark-capture studies in

the field (Jones et al. 2006, Boina et al. 2009).

Here, mass mark and capture was used to quantify local movement of stink bugs in pilot

scale farmscapes. The objectives of this study were to: 1) quantify the local dispersal of stink

bugs in time by sex, species, and growth stage in the cotton-peanut-soybean system; 2) determine

the effect of different stink bug species and crops on acquisition of protein marker residues.

59

Materials and Methods

Plot Layout. Research was conducted in 2010 and 2011 in Georgia at the Coastal Plain

Experiment Station (31° 31´26´´ N, 83° 32´52´´ W) operated by the University of Georgia. A

1.8 ha field, measuring approximately 135 m by 133 m, was subdivided into 3 equal sized

subplots of approximately 0.6 ha. Cotton (DP1050 B2RF) was planted in the center subplot

while peanut (TiftGuard) was planted on one side of the cotton, and soybean (AG 7502) on the

opposite side of the cotton. Row spacing for all crops was 0.9 m and all crops were grown using

conventional tillage practices. Each crop was 48 rows across with 2.7 meters of bare soil

between adjacent rows of neighboring crops. Fertilization, irrigation, tillage, and crop

management followed Georgia Cooperative Extension recommendations for each crop, except

no insecticides were applied on any of the fields after planting. In 2010 and 2011, all crops were

planted on 20 May and 16 May, respectively.

Protein Marker Application. Protein markers were applied using a self-propelled

insecticide sprayer at a rate of 187.4 liter/ha. The sprayer was equipped with flat fan tips (model

8003VS, TeeJet® Technologies, Wheaton, IL) installed on a 5.5 m long boom that was

positioned approximately 45 cm above the canopy. Rate control was governed by an automated

spray and rate controller (model 844-E, TeeJet Technologies) with a proximity speed sensor.

The protein applied to the cotton fields was 20% nonfat instant cow’s milk diluted in water.

Peanut fields received 10% egg whites, while soybean fields were treated with 20% soymilk.

The spray tank was filled with water and drained three times and then clean water was sprayed

out the boom for three minutes between protein applications to mitigate potential cross

contamination among proteins. Weekly protein applications began the 3rd week of cotton bloom

in 2010 and 2nd week of cotton bloom in 2011 (both in late July), and ended after the 8th week of

60

cotton bloom when cotton bolls at the bottom of the plant naturally opened (early September) in

both years.

Data Collection. A regularly spaced sampling grid was created in each of the three

subplots (18 locations per subplot). First, designated sampling rows were marked. Sampling

rows in cotton began from row 1 (adjacent to peanut on one side and soybean on the opposite

side), and continued every 6 rows until converging at the center (row 24). Sampling rows in

peanut and soybean began at row 1 (adjacent to cotton) and continued every 6 rows until the

outer edge of the field at row 48. Each sampling row was subdivided into three equal lengths

and grid points were marked at the location of the two interior intersecting points using vinyl

flags mounted on a 2.4-m-long fiberglass pole (Agri Drain Corp., Adair, IA). Stink bugs were

collected weekly using 38.1-cm diameter sweep nets from the 2rd to 9th wk of bloom. During

each week, sampling was conducted 24 and 48 h after proteins solutions were sprayed. Twenty

sweeps were conducted at each sample point with 10 sweeps before the flag and 10 sweeps after

the flag at 24 h after protein application. Fifty sweeps were conducted at each sample point 48 h

after proteins were sprayed in order to obtain more individuals for assessing movement. After

collection, individual stink bugs from sweep nets were immediately transferred into labeled 1.5-

ml microcentrifuge tubes and stored at -20 °C. Separate sweep nets were used in each crop to

avoid cross contamination of proteins. Immediately following each sample date, nets were hand

washed three times with dishwashing detergent and air dried.

ACP-ELISA. Immunoassays were performed by ACP-ELISA (Bandla, et al. 1994,

Crowther 2001, Srinivasan et al. 2012) generally following the methodology of Jones et al.

(2006). All incubations were performed at 37°C in an incubator (Thermo Fisher Scientific,

Waltham, MA). One ml of buffer [distilled water with tris-buffered saline (TBS, pH 8.0; T-664;

61

Sigma-Aldrich)] and 0.3 g/L sodium ethylenediamine tetra acetate (EDTA; S657; Sigma-

Aldrich) were added to microcentrifuge tubes containing stink bugs and the tubes were vortexed

for 1 min. An 80-µl aliquot of the supernatant was pipetted into individual wells of a 96-well

microtiter plate (Nunc Polysorp; Thermo Scientific, Pittsburgh, PA). Euschistus servus and N.

viridula adults obtained from a laboratory colony [rearing protocols described in Huang and

Toews (2012)] served as unmarked controls. Eighty μl aliquots of sample buffer from the

control treatment insects served as negative controls.

Stink bug washings in microtiter plates were incubated for 2 h at 37°C. For milk and egg

white protein assays, wells were washed five times with 300 µl of phosphate buffered saline

(PBS; P3813,Sigma-Aldrich) + 0.09% Triton-X100 (37426; Sigma-Aldrich). For the soymilk

protein assays, wells were washed three times with 300 µl PBS + 2.3 g/liter sodium dodecyl

sulfate (SDS; Sigma-Aldrich) (PBS-SDS) followed by two times of 300 µl PBS. After washing,

300 µl of StartingBlock (37538; Pierce Biotechnology, Rockford, IL) was added to each well for

egg white and soymilk assays. For the cow’s milk assays, 300 µl of PBS + 10% ethanolamine

(Sigma-Aldrich) was added per well. These samples were then incubated for 1 h at 37°C. Plates

were then washed once with 300 µl of PBST per well. Eighty ml of the primary antibodies,

diluted appropriately, were added. After 30 min of incubation, the antibodies were discarded and

plates were washed five times with 300 µl of PBST per well. After washing, 80 µl of the

secondary antibodies, diluted appropriately as determined by checkerboard titration assay were

added (Crowther 2001). A comprehensive list of antibodies, dilution buffers, and dilution ratios

is shown in Table 1. After 2 h of incubation, all secondary antibodies were discarded and plates

were washed three times with 300 µl of PBS-SDS per well followed by two times of 300 µl of

62

PBS. After washing, 80 µl of TMB (ImmunoPure, Ultra TMB substrate kit 34028; Pierce

Biotechnology) sources was added to each well.

Thereafter, the plates were placed on a digital microtiter shaker (IKA Works, Inc.

Wilmington, NC) and incubated in the dark at room temperature for 10 min. After 10 min of

incubation, 80 µl of 2 N H2SO4 was added to each well to stop the reaction. Optical density

(OD) from each well was read with a dual wavelength plate reader (EMax microplate reader;

Molecular Devices, Sunnyvale, CA) at 450 nm using 490 nm as the reference standard. All

readings were corrected (blanked) using wells with TBS + EDTA extraction buffer. If the OD

value detected from the treatment was higher than the mean plus 4 SD of the negative control,

that stink bug was considered positive for that protein (Jones et al. 2006).

Statistical Analyses. The marking efficiency (# marked / # total captured) of each

protein was compared among stink bug species. Year and sampling week were modeled as

random effects, and species was a fixed effect. The response variable, marking efficiency, was

subjected to the arcsine transformation (Zar 1999) and compared using a one-way ANOVA

(PROC GLIMMIX, SAS Institute 2008); treatment means were separated using LSMEANS test

(P < 0.05).

The marking efficiency (Mef) in a given crop was determined by the total number of stink

bugs marked with the specific protein applied in that crop (tMsp) divided by total stink bugs

captured in that crop [tCp (crop)] plus those individuals that were marked with the same protein

but recovered in other crops [InMsp (other crops)].

Mef = tMsp / [tCp(crop) + InMsp (other crops)]

63

For example, in peanut this calculation would be (tMeg) / ([tcp (peanut) + InMeg (cotton

& soybean). To determine the function of crop affects marking efficiency in the field, the

percentage of marking efficiency in each crop was compared using PROC GLIIMMIX (SAS

Institute 2008). Year and sampling week were modeled as random effects, and crop was a fixed

effect. The response variable, marking efficiency, was subjected to arcsine transformation prior

to analysis; treatment means were separated using LSMEANS test (P < 0.05).

Intercrop movement of stink bugs was inferred when a stink bug collected in one crop

was marked with a protein marker from another crop. Intercrop movement of four major adult

stink bug species C. hilaris, E. servus, Euschistus quadrator, and N. viridula was evaluated by

species, sex, and life stage. Data are presented as sum total by crop in each week. Differences in

the attractiveness of one crop vs. another were examined by comparing the average number of

stink bugs that moved into a specific crop (i.e., cotton → peanut, peanut → cotton, cotton →

soybean, soybean → cotton, peanut → soybean, and soybean → peanut) within each species

using PROC GLIMMIX (SAS Institute 2008), where movement direction was a fixed effect, and

year and sampling week were random effects; prior to these analyses, the response variable,

mean number of stink bugs, was subjected to a square-root transformation (Zar 1999) and

treatment means were separated using the LSMEANS test (P < 0.05). To determine whether

there were differences in movement attributed to sex, the mean numbers of males and females

that moved into cotton were subjected to a square-root transformation (Zar 1999) and compared

within each species using a t-test (PROC TTEST, SAS Institute 2008).

Investigation of Potential Contamination from Sweep Nets. While separate sweep

nets were used in each crop to avoid cross contamination of proteins, that practice would not

ameliorate the situation whereby unmarked individuals could get contaminated with protein

64

residues in the net. The authors conducted a simple experiment to address this issue. After all

sampling was completed for the day (18 sample locations per crop), E. servus and N. viridula

adults (20 individuals for each species) were obtained from laboratory colonies and placed in the

nets, shaken vigorously, and then processed using the ACP-ELISA assay described above.

Results

Species Composition. A total of 539 and 327 stink bugs were captured during the

experiment period in 2010 and 2011, respectively. Of those 539 stink bugs collected in 2010, E.

servus constituted 36.9% of the total, followed by C. hilaris (30.8%), E. quadrator (16.9%), N.

viridula (9.8%), Piezodorus guildinii (Westwood) (4.3%), and a few (1.3%) unidentified species.

In 2011, C. hilaris constituted 41.6% of the total, followed by E. servus (37.6%), E. quadrator

(8.6%), N. viridula (8.3%), P. guildinii (0.9%), and several (3.0%) unidentified specimens. In

2010, 45.5% of the total stink bugs were collected in cotton, 46.2% were collected in soybean,

and 8.3% were collected in peanut. In 2011, 51.7% were collected in cotton, 43.1% were

collected in soybean, and 5.2% were collected in peanut.

Effect of Stink Bug Species and Crop on Protein Marker Acquisition. In 2010,

57.3% (309/539) of stink bugs were marked with at least one of the protein markers. Of the 309

marked individuals, 23.6% (73/309) were marked with two markers and one insect (0.3%) was

marked with all three markers. In 2011, 45% (147/327) were marked with at least one of the

markers. Of these 147 marked individuals, 24.5% (36/147) were marked with two markers and

none was marked with all three markers. In 2011 alone, the percentage of E. quadrator that

scored positive for any markers was higher than the other four major species (F = 6.11; df = 4,

65

20; P = 0.0022) (Table 2). Across crop and sampling dates, the ranges of OD value of each stink

bug species scored positive for any proteins are provided in Table 3.

Analyses of marking efficiency by crop showed that the marking efficiency in peanut was

significantly greater than in cotton and soybean in both years (2010: F = 6.23; df = 2, 10; P =

0.0175) (2011: F = 68.62; df = 2, 10; P < 0.0001) (Table 4). Across species and sampling dates,

the ranges of OD values that scored positive in each crop (obtained from insects) were provided

by comparing between 24 h and 48 h after protein sources were sprayed (Table 5).

Intercrop Movement of Stink Bugs in Peanut-Cotton-Soybean Farmscapes.

Intercrop movement of adults of four major stink bug species (C. hilaris, E. servus, E. quadrator,

and N. viridula) among crops was documented. Although marking proteins were sprayed at the

2nd wk of bloom in 2011 only, no movement was detected. Regardless of year, movement of

stink bugs was detected beginning the 3rd wk of cotton bloom and ending the 8th wk of bloom.

Although protein markers were not sprayed the 9th wk of bloom, sampling was conducted and no

stink bugs were captured in cotton.

C. hilaris. Movement of C. hilaris into cotton from soybean began the 3rd wk of bloom

and ended the 8th wk of bloom (Fig. 1). The number of C. hilaris that moved into cotton from

soybean generally increased through the 5th week of bloom, and then movement in the opposite

direction was observed during the 7th and 8th wks of bloom. Interestingly, only two C. hilaris

(one at 5th wk and the other at 8th wk of bloom) moved into cotton from peanut and none moved

into peanut from cotton. Across all sampling dates, the mean number of C. hilaris that moved

from soybean into cotton was significantly greater than other movement directions (F = 14.62;

df = 4, 20; P < 0.0001). Of the population that moved into cotton, there were no differences

between the average number of males and females (T = 1.85; df = 10; P = 0.0939).

66

E. servus. Movement of E. servus into cotton from both peanut and soybean began the

3rd wk of bloom and ended the 8th wk of bloom (Fig. 2). Some individuals moved from cotton to

soybean starting the 6th wk of bloom and this phenomenon lasted until our last sampling date.

Few E. servus moved to peanut from cotton (two at 5th wk and one at 7th wk). Across all

sampling dates, the mean numbers of E. servus that moved into cotton from both peanut and

soybean were significantly greater than other movement directions (F = 15.72; df = 4, 20; P <

0.0001). However, the mean number was not significantly different between peanut → cotton

and soybean → cotton. Of those individuals that moved into cotton, significantly more females

were collected than males (T =2.58; df = 10; P = 0.0273).

E. quadrator. Movement of E. quadrator into cotton from peanut began the 3rd wk of

bloom and ended the 8th wk of bloom (Fig. 3). In stark contrast to E. servus, movement into

cotton from soybean was only observed the 3rd wk, 6th wk and 8th wk of bloom and in very low

numbers. Some individuals began to move into soybean from cotton at 7th wk and 8th wk of

bloom. Similar to E. servus, only a few E. quadrator moved to peanut from cotton (three at 5th

wk and one at 7th wk). Across all sampling dates, the mean number of E. quadrator that moved

into cotton from peanut was significantly greater than other movement directions (F = 12.99; df

= 4, 20; P < 0.0001). Of those individuals that moved into cotton, significantly more females

than males were collected (T = 2.63; df = 10; P = 0.0252).

N. viridula. Movement of N. viridula into cotton began the 3rd wk of bloom from

soybean and 4th wk of bloom from peanut (Fig. 4). Only one N. viridula was documented to

move to peanut from cotton; this occurred during the 7th wk of bloom. Two N. viridula were

detected to move to soybean from cotton, one at 6th wk and the other at 8th wk of bloom. Across

all sampling dates, the mean numbers of N. viridula among movement directions were not

67

significantly different (F = 2.68; df = 4, 20; P = 0.0613). Of those individuals that moved into

cotton, no differences were observed between males and females (T = 2.14; df = 10; P =

0.0579).

Immatures. In addition to adult stink bugs, immatures were also captured and analyzed

to detect intercrop movement. In 2010, 45.9% (28/61) of immatures were marked with at least

one protein marker. Of that population, one E. servus moved from peanut to cotton the 3rd wk of

bloom, one C. hilaris moved from cotton to soybean the 7th wk of cotton bloom, and two N.

viridula moved from peanut to cotton the 7th wk of cotton bloom. No intercrop movement was

detected for the other 24 marked immatures. In 2011, 33.3% (19/57) of immatures were marked

with at least one protein. Of these, one C. hilaris moved from soybean to cotton the 7th wk of

bloom and four C. hilaris moved from cotton to soybean the 8th wk of bloom. No intercrop

movement was detected for the remaining 14 marked immatures.

Investigation of Potential Contamination from Sweep Nets. Results showed that

although the OD reading from those stink bugs was slightly greater than the control insects from

the colony, none of them scored positive based on our criterion of mean OD plus 4 SD of the

negative control. Across all stink bugs captured during the study, only 52.7% tested positive for

any protein. If insects were truly being contaminated through sweep nets, one would expect that

nearly 100% of the captured insects would be positively marked. Regardless, assessments of

movement direction would not be affected at all since use of separate nets precluded protein

cross contamination.

68

Discussion

The mass mark and capture technique applied in this research was successful in marking

the naturally occurring stink bug populations in the field, allowing stink bug intercrop movement

to be tracked in the cotton-peanut-soybean farmscape. In this study, cotton was intentionally

planted between peanut and soybean so that movement from adjacent crops to cotton could be

quantified. Although dispersal of stink bug populations from one host to another has been

reported for the past six decades, direct movement in those studies was simply inferred based on

temporal sequences of captures (Borden et al. 1952, Tillman et al. 2009, Herbert and Toews

2011). Mark and recapture studies have been previously conducted to investigate dispersal of

stink bugs from crop to crop (Iwao et al. 1966, Kiritani et al. 1966, Jones and Sullivan 1982,

James 1990, and Tillman 2009). However, the recapture rate using traditional mark and

recapture techniques was generally extremely low and releasing all of the marked individuals at

one site likely biased dispersal behavior. Mass marking of naturally occurring stink bugs in the

field provides a promising and less biased method, which is the strongest evidence to date of

direct movement among different hosts by the same individuals.

The marking efficiency observed in these trials was similar to rates shown with protein

markers in other model systems. In this study, 52.7% of stink bugs were marked with at least

one marker, 23.9% with two markers and 0.2% were marked with three markers. These rates are

similar to marking rates reported by Jones et al. (2006) with the codling moth, Cydia pomonella

(L.). They observed that 46.5% of moths were marked with at least one marker, 32.2% were

marked with two markers, and 2.3% were marked with three markers. Boina et al. (2009)

reported that approximately 70% of captured Diaphorina citri (Kuwayama) were marked with a

protein 3 d after protein source application using a hand gun sprayer.

69

Interestingly, a greater percentage of E. quadrator was marked compared to other species

in 2011, which might suggest that this species might have a unique behavioral quality that lends

itself to a higher rate of marking. However, these results were not confirmed in the 2010 data

and the 2011 marked population consisted of only 27 marked individuals. Marking efficiency

varied by crop; a greater percentage of the general population acquired the mark in peanut in

both years compared to cotton and soybean. Crop and individual protein marker were

confounded in this experiment, so this result carries an important assumption. Assuming that

there are no differences among different markers, the authors hypothesized that the observed

differences are attributed to contrasting crop architecture. Soybean and cotton plants have

relatively large leaves and tall upright plants, while peanut has very small leaves and grows

prostrate on the ground. Perhaps the underside of the large leaves provided a shelter from the

residues or the liquid marker did not cover the entire canopy of the taller plants.

Protein marker durability in the field was not investigated here. Boina et al. (2009)

reported egg whites, milk, and soymilk proteins could be detected up to 30, 30, and 20 d after

application, respectively. Rainfall is known to affect residual longevity of protein markers in the

field (Jones et al. 2006, Boina et al. 2009), but no rain occurred between protein application and

sample dates within the same week in this study. However, our cotton plots received overhead

irrigation 72 h prior to protein application each week. Our assumption was that the irrigation

likely washed away the water soluble proteins. Jones et al. (2006) reported that the formation of

dew on top of a highly concentrated water-soluble residue of any protein markers may increase

marking efficiency. While this was not evaluated here, the authors note that the high relative

humidity in South Georgia favors the formation of heavy dew every night.

70

Intercrop movement of stink bugs depicted in Figs 2-5 clearly shows that during bloom,

cotton served as a stink bug sink, while peanut and soybean acted as stink bug sources.

Although the four key stink bug species moved into cotton, there were unique differences in the

process of invasion. The vast majority of C. hilaris captured in cotton immigrated from soybean

(> 96%) but not peanut. This suggests that cotton and soybean are more suitable hosts than

peanut for C. hilaris, which is consistent with the findings from other studies (Tillman et al.

2009, Herbert and Toews 2012). In contrast, E. quadrator captures in cotton were primarily

marked in peanut (> 89%) rather than soybean, which indicates that cotton and peanut are a more

suitable host than soybean for E. quadrator. Mean numbers of E. servus and N. viridula

captured in cotton indicated no difference between the number originating from peanut or

soybean, which suggests that both species can utilize cotton, peanut, and soybean as hosts.

However, during the period between the 3rd and 8th wk of bloom, cotton appeared to be a more

suitable host than peanut and soybean for both species.

Generally, more females than males were marked in one crop and then recovered in

cotton. This bias was statistically significant for E. quadrator and E. servus. This is interesting

because males of Euschistus obscurus (Palisot) and N. viridula produce aggregation pheromones

(Harris and Todd 1980, Aldrich et al. 1994). One hypothesis is that developing cotton bolls

provided a better nutrient source for females that were getting ready to produce eggs for the fall

generation (Herbert and Toews 2011, 2012). Although limited, intercrop movement of immature

stink bugs was also detected, especially in the 7th and 8th wks of bloom. A limited number of

nymphs of E. servus and N. viridula moved from peanut to cotton and nymphs of C. hilaris

moved between cotton and soybean, suggesting that cotton, peanut, and soybean are reproductive

hosts for specific species.

71

In summary, movement of stink bugs into flowering cotton was detected beginning the

3rd wk of bloom and lasting until the 8th wk of bloom. After the 9th wk of bloom, more than 50%

of bolls were open, and the number of soft bolls was greatly reduced due to cotton senescence.

At the same time, the soybean plants were producing seeds in pods (stages R5 to R6) that were

apparently highly attractive to all four stink bug species. Cotton changed from being a stink bug

sink to a source for stink bugs leaving toward peanut or soybean. Similar trends for movement

out of cotton and with population build up in fall soybean were reported by Hebert and Toews

(2011, 2012).

Findings presented in this study will benefit growers and managers who want to target

stink bug populations in time and space. For example, these data suggest that spraying cotton for

stink bug control prior to the 3rd week of bloom would not likely yield economic returns.

Similarly, identification of the primary stink bug species in a particular region may dictate how

crops should be spatially arranged to minimize intercrop movement. Knowledge of farmscape

ecology of stink bugs is important for understanding the temporal role of agronomic hosts in the

stink bug life history; armed with new knowledge of the sequence of host utilization, growers

should begin planning farmscapes that decrease or avoid contiguous crop borders where stink

bugs easily disperse.

72

Acknowledgements

The authors gratefully acknowledge John Herbert, Anne Horak, and Ishakh Pulakkatu

thodi for their assistance sampling stink bugs in the field. We also thank David Griffin and

Wesley Stephens for managing the plots. Francis Reay-Jones and Eric Blinka provided a helpful

review of an earlier manuscript draft. This research was funded by the Georgia Cotton

Commission under project number 11-827GA and by the USDA-NIFA-Special Research Grants

Program under award number 2009-34566-20100. Mention of trade names or commercial

products in this publication is solely for the purpose of providing specific information and does

not imply recommendation or endorsement by the University of Georgia.

73

References Cited

Akey, D. H., J. L. Hayes, S. J. Fleischer. 1991. Use of elemental markers in the study of

arthropod movement and trophic interactions. Southwest. Entomol. 14: 1-87.

Aldrich, J. R., J. E. Oliver, W. R. Lusby, J. P. Kochansky, and M. Borges. 1994.

Identification of male-specific volatiles from nearctic and neotropical stink bugs

(Heteroptera: Pentatomidae). J. Chem. Ecol. 20: 1103-1111.

Bandla, M. D., D. M. Westcot, K. D. Chenault, D. E. Ullman, T. L. German, and J. L.

Sherwood. 1994. Use of monoclonal antibody to the nonstructural protein encoded by

the small RNA of Tomato spotted wilt Tospovirus. Phytopathology. 84: 1427-1431.

Boina, D. R., W. L. Meyer, E. O. Onagbola, and L. L. Stelinski. 2009. Quantifying dispersal

of Diaphorina citri (Hemiptera: Psyllidae) by immunomarking and potential impact of

unmanaged groves on commercial citrus management. Environ. Entomol. 38: 1250-1258.

Borden, A. D., H. F. Madsen, and A. H. Retan. 1952. A stink bug, Euschistus conspersus,

destructive to deciduous fruits in California. J. Econ. Entomol. 45: 254-257.

Capaldi, E. A., A. D. Smith, J. L. Osborne, S. E. Fahrbach, S. M. Farris, D. R. Reynolds, A.

S. Edwards, A. Martin, G. E. Robinson, G. M. Poppy, J. R. Riley. 2000. Ontogeny of

orientation flight in the honeybee revealed by harmonic radar. Nature. 403: 537-540.

Crowther, J. R. 2001. The ELISA guidebook. Humana Press, Totowa, NJ.

Ehler, L. E. 2000. Farmscape ecology of stink bugs in Northern California. Entomological

Society of America, Lanhan, MD.

Fay, R. W., and G. B. Craig. 1969. Genetically marked Aedes aegypti in studies of field

populations. Mosq. News 29: 121-27.

74

Gary, N. E. 1971. Magnetic retrieval of ferrous labels in a capture-recapture system for honey

bees and other insects. J. Econ. Entomol. 64: 961-965.

Gast, R. T., and M. Landin. 1966. Adult boll weevils and eggs marked with dye fed in larval

diets. J. Econ. Entomol. 59: 474-475.

Graham, H. M., and G. L. Mangum. 1971. Larval diets containing dyes for tagging pink

bollworm moths internally. J. Econ. Entomol. 64: 377-379.

Greene, J. K., M. J. Turnipseed, M. J. Sullivan, and O. L. May. 2001. Treatment thresholds

for stink bugs (Hemiptera: Pentatomidae) in cotton. J. Econ. Entomol. 94: 403-409.

Hagler, J. R. 1997. Field retention of a novel mark-release-recapture method. Environ. Entomol.

26: 1079-1086.

Hagler, J. R., and C. G. Jackson. 1998. An immunomarking technique for labeling minute

parasitoids. Environ. Entomol. 27: 1010-1016.

Hagler, J. R., and C. G. Jackson. 2001. Methods for marking insects: current techniques and

future prospects. Ann. Rev. Entomol. 46: 511-543.

Hagler, J. R., A. C. Cohen, D. Dradley-Dunlop, and F. J. Enriquez. 1992. New approach to

mark insects for feeding and dispersal studies. Environ. Entomol. 21: 20-25.

Harris, V. and J. Todd. 1980. Male-mediated aggregation of male, female and 5th-instar

southern green stink bugs and concomitant attraction of a tachinid parasite, Trichopodia

pennipes, Entomol. Exp. Appl. 27: 117-126.

Hausermann, W., R. W. Fay, and C. S. Hacker. 1971. Dispersal of genetically marked female

Aedes aegypti in Mississippi. Mosq. News 31: 37-51.

75

Hendrix, W. H. III, T. F. Mueller, J. R. Phillips, and O. K. Davis. 1987. Pollen as an

indicator of long-distance movement of Heliothis zea (Lepidoptera: Noctuidae). Environ.

Entomol. 16: 1148-1151.

Herbert, J. J., and M. D. Toews. 2011. Seasonal abundance and population structure of brown

stink bug (Hemiptera: Pentatomidae) in farmscapes containing corn, cotton, peanut, and

soybean. Ann. Entomol. Soc. Am. 104: 909-918.

Herbert, J. J., and M. D. Toews. 2012. Seasonal abundance and population structure of

Chinavia hilaris (Say) and Nezara viridula (L.) (Hemiptera: Pentatomidae) in Georgia

farmscapes containing corn, cotton, peanut, and soybean. Ann. Entomol. Soc. Am. (In

Press).

Huang, T-I, and M. D. Toews. 2012. Feeding preference and movement of Nezara viridula (L.)

and Euschistus servus (Say) (Hemiptera: Pentatomidae) on individual cotton plants. J.

Econ. Entomol. (Accepted).

Iwao, S., K. Kiritani, and N. Hokyo. 1966. Application of a marking-and-recapture method for

the analysis of larval-adult populations of an insect, Nezara viridula (Hemiptera:

Pentatomidae). Res. Popul. Ecol. 8: 147-160.

James, D. G. 1990. Energy reserves, reproductive status, population biology of overwintering

Biprorulus bibax (Hemiptera: Pentatomidae) in southern New South Wales citrus groves.

Aust. J. Zool. 38: 415-22.

Johnson, P. C., and M. R. Reeves. 1995. Incorporation of the biological marker rubidium in

gypsy moth (Lepidoptera: Lymantriidae) and its transfer to the predator Carabus

nemoralis (Coleoptera: Carabidae). Environ. Entomol. 24: 46-51.

76

Jones, W. A., and M. J. Sullivan. 1982. Role of host plants in population dynamic of stink bug

pest of soybean in South Carolina. Environ. Entomol. 11: 867-875.

Jones, V. P., J. R. Hagler, J. F. Brunner, C. C. Baker, and T. D. Wilburn. 2006. An

inexpensive immunomarking technique for studying movement patterns of naturally

occurring insect populations. Environ. Entomol. 35: 827-836.

Kiritani, K., N. Hokyo, and S. Iwao. 1966. Population behavior of the southern green stink

bug, Nezara viridula, with special reference to the developmental stages of early-planted

paddy. Res. Popul. Ecol. 8: 133-146.

McPherson, J. E., and R. M. McPherson. 2000. Stink bugs of economic importance in

America North of Mexico. CRC Press, Boca Raton, FL, USA.

Naranjo, S. E. 1990. Influence of two mass-marking techniques on survival and flight behavior

of Diabrotica virgifera (Coleptera: Chrysomelidae). J. Econ. Entomol. 83: 1360-1364.

Nelson, D. L., and W. F. Baldwin. 1977. The use of radioactive platinum-iridium (IP-192) tags

for locating honeybee queens in colonies (Hymenoptera: Apidae). Can. Entomol. 109:

319-320.

Olson, D. M., J. R. Ruberson, A. R. Zeilinger, and D. A. Andow. 2011. Colonization

preference of Euschistus servus and Nezara viridula in transgenic cotton varieties,

peanut, and soybean Entomol. Exp. Appl. 139: 161-169.

Reay-Jones, F. P. F., J. K. Greene, M. D. Toews, and R. B. Reeves. 2009. Sampling stink

bugs (Hemiptera: Pentatomidae) for population estimation and pest management in

southeastern cotton production. J. Econ. Entomol. 102: 2360-2370.

SAS Institute. 2008. SAS/STAT 9.2 user’s guide. SAS Institute, Cary, NC.

77

78

Srinivasan, R., S. Sundaraj, H. R. Pappu, S. Diffie, D. G. Riley, and R. D. Gitaitis. 2012.

Transmission of Iris yellow spot virus by Frankliniella fusca and Thrips tabaci

(Thysanoptera: Thripidae). J. Econ. Entomol. 105: 40-47.

Su, N-Y., P. M. Ban, and R. H. Scheffrahn. 1991. Evaluation of twelve dye markers for

population studies of the eastern and Formosan subterranean termite (Isoptera:

Rhinotermitidae). Sociobiology 19: 349-362.

Thibault, S. T., H. T. Luu, N. Vann, and T. A. Miller. 1999. Precise excision and transposition

of piggyBac in pink bollworm embryos. Insect Mol. Biol. 8: 119-123.

Tillman, P. G., T. D. Northfield, R. F. Mizell, and T. C. Riddle. 2009. Spatiotemporal patterns

and dispersal of stink bugs (Heteroptera: Pentatomidae) in peanut-cotton farmscapes.

Environ. Entomol. 38: 1038-1052.

Tillman, P. G. 2010. Composition and abundance of stink bugs (Heteroptera: Pentatomidae) in

corn. Environ. Entomol. 39: 1765-1774.

Todd, J. W. 1989. Ecology and behavior of Nezara viridula. Ann. Rev. Entomol. 34: 273-292.

Weseloh, R. M. 1985. Dispersal, survival, and population abundance of gypsy moth, Lymantria

dispar (Lepidoptera: Lymantriidae), larvae determined by releases and mark-recapture

studies. Ann. Entomol. Soc. Am. 78: 728-35.

Wojcik, D. P., B. J. Burges, C. M. Blanton, and D. A. Focks. 2000. An improved and

quantified technique for marking individual fire ants (Hymenoptera: Formicidae). Fla.

Entomol. 83: 74-78.

Zar, J. H. 1999. Biostatistical Analysis, 4th ed. Prentice Hall. Upper Saddle River, NJ.

Table 1. Summary of primary and secondary antibodies used in ACP-ELISA

Antigen

source

Antigen Primary

antibody

Dilution

buffer

Dilution

ratio

Secondary

antibody

Dilution

buffer

Dilution

ratio

Egg

White

Chick egg

albumin

Rabbit anti-

chicken egg

albumin

StartingBlock + 1300

ppm Silweet L-77

1:4,000 Donkey anti-rabbit

IgG (H + L)

StartingBlock 1:6,000

Cow’s

milk

Bovine

casein

Sheep anti-

casein

20% bovine serum

albumin mixed with PBS

+ 1300 ppm Silwet L-77

1:500 Donkey anti-sheep

IgG

StartingBlock 1:3,000

Soymilk Soy

protein

Rabbit anti-soy StartingBlock 1:4,000 Donkey anti-rabbit

IgG (H + L)

StartingBlock 1:8,000

Rabbit anti-chicken egg albumin (Sigma-Aldrich)

Sheep anti-casein (Biodesign International, Saco, ME)

Rabbit anti-soy (R-Biopharm, South Marshall, MI)

Bovine serum albumin (HyClone, Logan, UT)

Donkey anti-rabbit IgG (H + L) (Pierce Biotechnology, Rockford, IL)

Donkey anti-sheep IgG (Sigma-Aldrich)

79

Table 2. Comparisons by species for number of stink bugs collected and differences in the

marking efficiency by year

Percentage of marked among species followed by the same letter, within year, are not

significantly different (P < 0.05; LSMEANS test). Percentages of positive values from stink bug

indicate the percentage of each species detected positive for any one of the three proteins.

Year Species Collected % Positive

2011 C. hilaris 199 53.6a

E. servus 166 52.8a

E. quadrator 53 61.5a

N. viridula 91 73.6a

P. guildinii 23 68.2a

2010 C. hilaris 123 42.6b

E. servus 136 44.7b

E. quadrator 27 78.6a

N. viridula 28 36.0b

P. guildinii 3 N/A

80

Table 3. Ranges of absorbance values at 450 nm (mean ± SD) by stink bug species Range of OD values at 450 nm (mean ± SD)

Species Bovine casein Chick egg albumin Soy protein

C. hilaris 0.19 ± 0.20 – 1.15 ± 0.06 0.18 ± 0.06 – 1.37 ± 1.20 0.12 ± 0.07 – 0.28 ± 0.17

E. servus 0.17 ± 0.11 – 1.75 ± 1.28 0.10 ± 0.06 – 1.78 ± 1.21 0.10 ± 0.02 – 0.15 ± 0.14

E. quadrator 0.15 ± 0.14 – 1.16 ± 0.84 0.15 ± 0.04 – 0.98 ± 0.75 0.11 ± 0.02 – 0.15 ± 0.06

N. viridula 0.12 ± 0.08 – 0.60 ± 0.17 0.13 ± 0.07 – 1.32 ± 1.40 0.10 ± 0.01 – 0.15 ± 0.10

P. guildinii 0.30 ± 0.24 – 0.40 ± 0.28 0.24 ± 0.15 – 0.68 ± 0.81 0.18 ± 0.02 – 0.47 ± 0.34

E. servus

(negative)

0.01 ± 0.01 – 0.06 ± 0.03 0.01 ± 0.00 – 0.04 ± 0.02 0.01 ± 0.00 – 0.04 ± 0.01

N. viridula 0.01 ± 0.01 – 0.05 ± 0.02 0.01 ± 0.01 – 0.05 ± 0.01 0.02 ± 0.01 – 0.06 ± 0.01

(negative)

At least 15 microtiter plates were used for each species. Positive and negative thresholds were

determined for each microtiter plate and the ranges in OD values are provided. Provided are: 1)

Ranges of OD values for ELISA positive stink bug species, and 2) ranges of OD values for

ELISA negative stink bug species.

81

Table 4. Comparisons by crop for number of stink bugs collected and differences in the marking efficiency by year

Year Crop Collected % Positive

2010 Cotton 245 47.1b

Peanut 45 76.1a

Soybean 249 47.5b

2011 Cotton 169 34.8b

Peanut 17 98.3a

Soybean 141 48.3b

Percentage of marked among crops followed by the same letter, within year, are not significantly

different (P < 0.05; LSMEANS test). Percentage of positive values from crop indicate the

percentage of stink bugs collected from the specific crop that detected positive across species.

82

Table 5. Ranges of absorbance values at 450 nm (mean ± SD) by crop

Crop 24 h 48 h

Cotton 0.18 ± 0.04 – 1.68 ± 0.44 0.15 ± 0.03 – 1.57 ± 0.55

Peanut 0.15 ± 0.11 – 2.34 ± 0.45 0.16 ± 0.23 – 2.05 ± 0.75

Soybean 0.11 ± 0.04 – 0.18 ± 0.07 0.10 ± 0.05 – 0.21 ± 0.23

At least 15 microtiter plates were used for each species. Positive thresholds were determined for

each microtiter plate and the ranges in OD values are provided. Ranges of OD values for ELISA

positive stink bug species collected from various crops at two time intervals.

83

Figure legends Fig. 1. Cumulative number of C. hilaris adults marked in one crop and recovered in adjacent

crop, by week of cotton bloom.

Fig. 2. Cumulative number of E. servus adults marked in one crop and recovered in adjacent

crop, by week of cotton bloom.

Fig. 3. Cumulative number of E. quadrator adults marked in one crop and recovered in adjacent

crop, by week of cotton bloom.

Fig. 4. Cumulative number of N.viridula adults marked in one crop and recovered in adjacent

crop, by week of cotton bloom.

84

Stink bugs moving to adjacent crop

wk 3

wk 6

wk 7

wk 8

wk 4wk 5

Peanut Cotton Soybean

010 1020 20 010 10 20

Wee

k of

blo

om

Figure 1.

85

Stink bugs moving to adjacent crop

wk 3

wk 6

wk 7

wk 8

wk 4wk 5

Peanut Cotton Soybean

010 1020 20 010 10 20

Wee

k of

blo

om

Figure 2.

86

Stink bugs moving to adjacent crop

wk 3

wk 6

wk 7

wk 8

wk 4wk 5

Peanut Cotton Soybean

010 1020 20 010 10 20

Wee

k of

blo

om

Figure 3.

87

Stink bugs moving to adjacent crop

wk 3

wk 6

wk 7

wk 8

wk 4wk 5

Peanut Cotton Soybean

010 1020 20 010 10 20

Wee

k of

blo

om

Figure 4.

88

CHAPTER 4

LOCAL DISPERSAL OF STINK BUGS (HEMIPTERA: PENTATOMIDAE) AND

ASSOCIATED CHANGES IN COTTON FIBER QUALITY

________________________

T-I Huang, and M. D. Toews. 2012 Bulletin of Entomological Research, submitted

89

Abstract

A two-year field study was conducted to quantify local dispersal of stink bugs among

cotton, peanut, and soybean fields. Replicated plots were established with approximately 0.6 ha

of cotton planted between similar sized plots of peanut and soybean. Starting when the cotton

began flowering, protein markers including egg whites, cow’s milk, and soymilk were applied

weekly to peanut, cotton, and soybean, respectively. Stink bugs were sampled weekly with

sweep nets in the three crops at distances of 0, 5.5, 11, 16.5, and 22 m from the edge of each

field and then stink bug surface washes were analyzed for the presence of the protein markers.

In addition, internal boll damage in the cotton plots was sampled weekly during week 3 through

8 of bloom. Representative cotton lint samples were mechanically harvested, weighed, ginned,

and classed. Generally speaking, the majority of adult stink bugs were recovered in the same

crop where they were marked, or captured within 8.2 m of where they were marked.

Considering only the bugs that dispersed between crops, there were no differences in dispersal

distance among stink bug species or between sexes. However, stink bugs moving from cotton to

soybean travelled significantly further (24.2 m) than bugs travelling between remaining adjacent

crops. Differences in stink bug density, seedcotton yield, gin turnout, and fiber color were

correlated with changes in cotton boll damage. This knowledge is important for developing new

integrated pest management strategies for managing the stink bug complex in cotton at the

farmscape level.

Keywords: protein marker, mark and capture, integrated pest management, crop phenology

90

Introduction

Polyphagous stink bugs (Hemiptera: Pentatomidae) were historically considered minor

pests of cotton (Gossypium hirsutum L.) production in the southeastern United States (US). The

pest status changed as a result of reduced insecticide applications following boll weevil

eradication and widespread adoption of transgenic cotton cultivars. The stink bug species

complex changes with latitude, but in the southeastern US is composed of Nezara viridula (L.),

Euschistus servus (Say), E. quadrator (Rolston), and Chinavia hilaris (Say) (McPherson &

McPherson 2000, Greene et al. 2001, Reay-Jones et al. 2009). These pests utilize a wide variety

of agronomic hosts over the growing season. Those hosts include row crops such as corn (Zea

mays L.), grain sorghum [Sorghum bicolor (L.) Moench], peanut (Arachis hypogaea L.), and

soybean [Glycine max (L.) Merr.] (McPherson & McPherson 2000, Tillman 2010, Olson et al.

2011, Herbert & Toews 2011). Studies in cotton have shown that feeding by stink bugs on

developing bolls will cause boll abscission, lint staining, reduced yield, and reduced lint quality

(Cassidy & Barber 1939, Toscano & Stern 1976, Barbour et al. 1990, Greene et al. 1999,

Turnipseed et al. 2003, Huang & Toews 2012). In addition, stink bugs can transmit boll rot

pathogens that reduce lint yield (Medrano et al. 2007, Esquivel 2011).

Due to eradication of the Anthonomus grandis grandis (Boheman) and widespread

adoption of Bt transgenic cotton to manage the heliothine pest complex, the annual number of

insecticide applications to cotton in Georgia has decreased from 14 to less than 3 in recent years

(Roof 1994, William 2007, 2008). Unfortunately, the adoption of Bt cotton and reduced

spraying contributed to outbreaks of stink bugs, which were formerly considered minor pests. In

the US, cotton losses attributed to stink bugs rose from US $10 M in 1998 to > US $60 M in

2001, and nearly US $70 M in 2005 (Williams 2002, 2006). Despite a relatively light infestation

91

in 2007 and 2008, stink bug losses were estimated at US $22.5 M and US $31 M nationwide

(Williams 2008, 2009). Significant yield losses from this pest complex are also frequent in

soybean, with up to $60 M in losses annually in the United States (McPherson and McPherson

2000). Young et al. (2008) showed that yield loss in soybean ranged from 13.4 to 60.5 kg/ha.

Stink bugs can also be serious pests in corn (Negron and Riley 1987), particularly when fields

are located adjacent to wheat (Blinka 2008).

To prevent continuing losses from stink bugs, studies have focused on the development

of economic thresholds and estimation of population density. For estimating density, researchers

and scouts rely on sweep nets, drop cloths, and direct observation of internal boll injury (Greene

et al. 2001, Reay-Jones et al. 2009, Toews et al. 2009, Reay-Jones et al. 2009). Research showed

that examination of ~2.4 cm diameter bolls for internal warty growths or yellow stained lint

requires more time, but is much more sensitive than other scouting methods (Wene & Sheets

1964, Greene & Herzog 1999). Although it requires comparatively less time, estimating

population density with the sweep net and drop cloth have documented biases; more adults tend

be captured in the sweep net, while more nymphs tend to be captured on the drop cloth (Toews et

al. 2008). New technologies such as volatile emissions and fluorescence spectroscopy have been

proposed for investigating stink bug damage in developing cotton bolls to facilitate sampling

(Degenhardt et al. 2011, Xia et al. 2011).

The term farmscape, as defined by Ehler (2000), refers to habitat patches composed of

cultivated and naturally occurring host plants. This within-farm configuration of resource

patches provides the best scale for studying mobile insect populations at the local ecosystem

level. Stink bug populations and boll damage were documented at higher density on the edge, or

common boundary of the peanut-cotton and soybean-cotton farmscapes (Tillman et al. 2009,

92

Toews & Shurley 2009). Although cotton and soybean have been documented as major food

sources (Greene et al. 1999, Boethel et al. 2000, Willrich et al. 2004), it is less clear if peanut is

an important resource for stink bugs. Recently, all life stages of N. viridula, E. servus,

E.quadrator, Oebalus pugnax (F.) and C. hilaris were reported by observed in peanut (Tillman

2008). However, the ecological role of peanut at the farmscape level for stink bug population

development remains poorly documented.

The authors previously published a report on the temporal dynamics and intercrop

movement of stink bugs in Georgia peanut-cotton-soybean farmscapes. This study focuses on

observed dispersal distance and the impact of local dispersal on cotton fiber quality. The

objectives of this project were to: 1) determine the intercrop movement direction and distance

that individual stink bugs move in a farmscape containing cotton, peanut, and soybean; 2)

evaluate stink bug-induced boll damage, seedcotton yield, fiber quality, and gin turnout in cotton

fields positioned adjacent to peanut and soybean; and 3) determine how far boll damage

extended into cotton fields associated with stink bug travel distance from intercrop margins

between cotton and peanut or cotton and soybean.

Materials and Methods

Plot Layout

Research was conducted in 2010 and 2011 on two University of Georgia experiment

stations. Plots were established at Midville (32° 52´22.90´´ N, 82° 12´52.70´´ W), and Tifton

(31° 31´12.27´´ N, 83° 32´56.84´´ W), Georgia, United States. Overall plot size ranged between

1.8 and 2.0 ha in area depending on available land. Subplot layout and sampling protocols

followed the procedures outlined in Huang et al. (2012). Briefly, each main plot was subdivided

93

into three subplots (approximately 135 by 133 m) that were rectangular in shape and equal in

area. Bt cotton (DP1050 B2RF) was planted centrally between peanut (TiftGuard) and soybean

(AG 7502) on 20 May 2010, and 16 May 2011 at Tifton, and on 11 May 2010, and 10 May 2011

at Midville. The area of each individual crop was approximately 0.6 ha. Each crop was 48 rows

across with 1.8 meters of bare soil between adjacent rows of neighboring crops. Row spacing for

all crops was 0.91 m and all crops were grown using conventional tillage practices. Fertilization,

irrigation, cultivation, and crop management followed Georgia Extension recommendations for

each crop, except no insecticides were applied after planting. Crop maturity was recorded at

each sampling date.

Stink bug sampling

A regularly spaced sampling grid was created on each of the three subplots. First,

designated sampling rows and areas within rows were delineated. Sampling rows in cotton

began from row 1 (adjacent to peanut on one side and soybean on the opposite side), and

continued every six rows until meeting at the centre, row 24. Sampling rows in peanut and

soybean began at row 1 (adjacent to cotton) and continued each 6 rows until the outer edge of the

field at row 48. Each sampling row was subdivided into three equal lengths and grid points were

marked at the location of the two interior intersecting points using vinyl flags mounted on a 2.4-

m-long fiberglass pole (Agri Drain Corp., Adair, IA). Stink bugs were sampled weekly

beginning at the 1st wk of cotton bloom (mid-July) and continuing until cotton bolls at the bottom

of the plants were open (early September). Twenty sweeps with a 38.1-cm sweep net were

conducted at each sample point (10 sweeps before the flag and 10 sweeps after the flag). Adult

stink bugs were identified to species and sex in the field.

94

Stink bug mark and capture studies were conducted at the Tifton field site only each year.

Details on protein application and later detection on captured stink bugs are described in Huang

et al. (2012). Briefly, unique protein markers (10% egg whites applied to peanut, 20% cow’s

milk applied to cotton, and 20% soymilk applied to soybean) were applied to each crop at 187.4

liter/ha. Stink bug sampling occurred 24 and 48 h after protein application. Bugs captured at

Tifton were immediately transferred into individual labeled 1.5-ml centrifuge tubes and stored at

-20 °C in the laboratory. An ACP-ELISA (Bandla, et al. 1994, Crowther 2001, Srinivasan et al.

2012) was conducted on each individual captured at Tifton to determine intercrop movement.

Cotton boll sampling

Boll damage, seedcotton yield, and fiber quality assessment were conducted in each

cotton plot. Assessment of boll damage followed the methods of Greene et al. (1999). Ten

cotton bolls (~2.4-cm diameter) were randomly collected from each sampling point during wk 3

through wk 8 after initiation of anthesis. Bolls were pooled by collection location into labeled

plastic bags and then transported to the laboratory where they were manually examined for

internal damage. Stink bug feeding damage was defined as presence of feeding punctures on the

inner carpel wall, warty growths of any size, lint staining, or rotten locks (Greene &

Herzog1999).

Representative cotton samples were harvested, ginned, and classed. Seedcotton was

picked with a mechanized two-row spindle picker on 5 October in Midville and 7 October in

Tifton in 2010, and 7 October in Midville and 4 November in Tifton in 2011. In total, 36.6 m

(two rows by 18.3 m) were picked into separate bags at distance of 0, 5.5, 11, 16.5, and 22 m

from each edge of the cotton field. Individual bags of seedcotton were weighted and then ginned

95

at the University of Georgia Microgin (Tifton, GA). Ginning the cotton removes the seed and

trash from the lint. Representative ginned fiber samples were sent to the USDA Cotton Classing

Office at Macon, GA, where they were classed following USDA’s official grade standards for

American Upland cotton (USDA-AMS 2001). Response variables for fiber quality included gin

turnout (lint weight divided by seedcotton weight), color Rd (fiber reflectance or brightness),

color +b (fiber yellowness), leaf grade (estimate of cotton plant leaf particles), fiber length

(upper half mean length), micronaire (fiber fineness and maturity), fiber strength, length

uniformity index (ration between the mean length and the upper half mean length), and percent

trash.

Experimental design and statistical analyses

Stink bug travel distances from crop to crop were calculated as the absolute minimum

distance from the crop where the stink bug was marked to the sampling point where the stink bug

was recovered. For example, if a stink bug marked with egg whites (applied in peanut) was

recovered in sample row 6 of cotton, the shortest linear distance from the point of capture to the

crop where that protein was recorded. Experimental design was a randomized complete block

where dispersal distance was the response variable, species and sex were fixed effects, and week

within yr was the random effect. Dispersal distance was log transformed for analyses to correct

for unequal variance (Zar 1999). Analyses were performed using PROC GLIMMIX (SAS

Institute, 2008) with means separation procedures using the LSMEANS test (P < 0.05). Data

were analyzed independently by species. A second statistical analysis was conducted on adults

(all species) that moved between adjacent crops (for example, peanut to cotton movement was

analyzed separately from soybean to cotton movement). The experimental design for these

inquiries was a randomized complete block. This analysis was conducted using PROC

96

GLIMMIX, whereby the log of dispersal distance was the response variable, movement direction

(e.g., peanut to cotton) was a fixed effect, and wk within yr was modeled as a random effect.

Treatment means were separated using the LSMEANS test (P < 0.05).

A two-way ANOVA was conducted to determine how the adjacent crops and distance

into the cotton plot affected response variables including: mean stink bug density, boll damage,

seedcotton yield, gin turnout, color Rd, and color +b. Data were organized in a randomized

complete block with experiment location and year as random effects so that we could generalize

the main effects of adjacent crop and distance. Prior to analyses mean stink bug density was log

transformed while percent boll damage and gin turnout were arcsine transformed (Zar 1999).

Actual values are presented in the figures, text, and tables. Trend analyses (linear, quadratic, or

cubic effects) were used to analyze distance because it is a continuous variable, not suitable for

means separation procedures (Neter et al. 1996). Data were analyzed using PROC GLIMMIX

(SAS Institute, 2008), with the trend analyses being programmed using contrast statements.

Coefficients for orthogonal contrasts were generated using PROC IML (SAS Institute, 2008).

Differences among individual treatment variables and trend analysis were considered

significantly different at the α = 0.05.

A previous report documented that internal boll damage caused by stink bugs is a less

variable method of estimating stink bug activity than capture of actual stink bugs in a sweep net

(Toews et al. 2008). Therefore, Pearson correlation analyses were performed to detect

relationships between mean percent boll damage and the following variables: mean number of

stink bugs captured in sweep net, seedcotton yield, gin turnout, color Rd and color +b.

97

Results

In general, the majority of adult stink bugs were recovered in the same crop where they

were marked, or captured within 8.2 m of where they were marked. A frequency histogram of

dispersal distances within the same crop or from one crop to an adjacent crop is shown in Fig. 1.

In addition, there were four adults that travelled between peanut and soybean between the time

they were marked and captured; two E. servus and one N. viridula travelled at least 49.4 m,

while one C. hilaris travelled at least 87.8 m.

Considering only the individuals that moved from one crop to another, the mean intercrop

dispersal distance ranged from 7 to 20 m (Table 1). There were no significant differences in

intercrop movement distance between males (13.8 ± 2.0 m) and females (10.9 ± 1.2 m) (F1, 236 =

0.06, P = 0.8120) or among stink bug species (F4, 236 = 1.35, P = 0.2529). Analyses of movement

direction showed that individuals dispersing from cotton to soybean travelled further than

individuals travelling between the remaining adjacent crops (F3, 245 = 13.08, P < 0.0001 (Table

2).

Mean stink bug captures differed significantly as a function of distance from the edge of

the cotton plot (F4, 18 = 72.17, P < 0.0001), but not between adjacent crops (F1, 18 = 2.63, P =

0.1223); there was no interaction between these main effects (F4, 18 = 1.62, P = 0.2119). The

mean number of stink bugs captured per 20 sweeps on the edge was at least 5-fold higher than

any rows after 5.5 m away from the edge of cotton plot. Trend analyses by individual adjacent

crop indicated strong cubic trends (Fig. 2). Similarly, cotton boll damage differed significantly

as a function of distance from the edge of the cotton plot (F4, 18 = 34.20, P < 0.0001), but not

between adjacent crops (F4, 18 = 0.12, P < 0.7299), and there was no interaction (F4, 18 = 0.47, P <

0.7599). Greater than 60% of the cotton bolls located immediately adjacent to peanut and

98

soybean were damaged by stink bugs. The percentage of cotton boll damage on the edge was at

least double that observed at any rows 5.5 m away from the edge of cotton plot. Trend analyses

by individual adjacent crops show that there was a cubic trend when located adjacent to peanut

but a quadratic trend when cotton was adjacent to soybean (Fig. 3). The Pearson Correlation

analysis showed a strong correlation between the percentage of boll damage and the average of

stink bug captured (coefficient = 0.78790; P < 0.0001; n = 30).

Seedcotton yields differed significantly as a function of adjacent crop (F1, 18 = 12.13, P =

0.0027) and distance from the edge of the cotton plot (F4, 18 = 6.39, P = 0.0022). No interaction

was detected between these two factors (F4, 18 = 1.18, P = 0.3521). Subsequent trend analyses

showed quadratic trends on seedcotton yield when cotton was positioned adjacent to peanut and

soybean (Fig. 4). The observed suppression in seedcotton yield occurred primarily on the edge

of the plot bordering adjacent peanut or soybean. There was an inverse correlation between

seedcotton yield and the season-long mean boll damage (coefficient= -0.56265; P = 0.0012;

n=30). Percentage gin turnout differed significantly as a function of distance from the edge of

cotton plot (F4, 18 = 14.37, P < 0.0001), but not of adjacent crop (F1, 18 = 3.26, P = 0.0879), and

there was no interaction (F4, 18 = 0.93, P = 0.04711) (Fig. 5). Subsequent trend analyses showed

quadratic trends suggesting that most of the decreased turnout occurred in rows adjacent to

peanut and soybean. A strong correlation was detected between the percentage of gin turnout

and the percentage of boll damage (coefficient = -0.90873; P < 0.0001; n = 30).

There were also differences in the color analyses of the cotton fiber as a function of

distance from the edge of the field. Color Rd was different as a function of adjacent crop (F1, 18 =

24.64, P = 0.0001) and distance from the edge of cotton plot (F4, 18 = 28.92, P < 0.0001).

However, there was an interaction between these two factors (F4, 18 = 3.47, P = 0.0285). Further

99

analyses showed a quadratic trend when cotton was located adjacent to peanut and a cubic trend

when cotton was adjacent to soybean (Fig. 6). The inverse correlation between color Rd and the

percentage of boll damage was significant (coefficient = -0.69636; P < 0.0001; n = 30).

Differences in color +b were a function of distance from the edges of the cotton plot (F4, 16 =

13.47, P < 0.0001) but not of adjacent crop (F1, 16 = 2.38, P < 0.1422), and there was no

interaction (F4, 16 = 0.36, P = 0.8350). Trend analyses showed a quadratic trend when cotton was

adjacent to peanut and a cubic trend when cotton was adjacent to soybean (Fig. 6). A significant

correlation was detected between color +b and the percentage of boll damage (coefficient=

0.77019; P < 0.0001; n = 28). There were no differences in fiber length, micronaire, strength,

length uniformity index, or trash as a function of adjacent crop or distance into the cotton plots

(P > 0.05).

Discussion

This study demonstrated that cotton boll damage was positively correlated with stink bug

density. The data further showed that increasing boll damage resulted in decreased yield,

deceased gin turnout, decreased color Rd, and increased color +b. The direction of change as a

result of increasing boll damage would negatively affect lint value. Toews & Shurley (2009)

also reported that cotton damage was more significant when planted adjacent to peanut and

soybean than when planted adjacent to corn. Greater than 60% of cotton bolls located

immediately adjacent to peanut and soybean were damaged, thus confirming the findings of

Toews & Shurley (2009). More recent studies have shown that seedcotton yield and fiber quality

can be negatively affected by transmission of boll rotting pathogens during stink bug feeding

(Medrano et al. 2007, 2009, Esquivel 2011). Losses to direct stink bug feeding or pathogen

100

transmission may have been confounded in this study since we did not measure pathogen load in

bolls. There were generally very clear trends with potential economic impacts when comparing

the edge of the cotton plot with samples collected several meters into the plot. Stink bug

dispersal into cotton starting from the edge of the field was significant; a similar result was

documented using spatiotemporal analysis to monitor of stink bug populations in cotton-peanut

farmscapes (Tillman et al. 2009).

In this experiment, we directly assessed intercrop stink bug dispersal and calculated the

minimum intercrop travel distance. Lack of significant differences in the dispersal distance

among species and between sexes suggests that most stink bug species have similar dispersal

abilities, at least in this crop arrangement. Clearly, cotton was a suitable host for the five stink

bug species we observed. In addition, similar intercrop dispersal distances when comparing

movement of stink bugs from peanut into cotton (~10.3 m) or from soybean into cotton (~8.5 m)

suggests that the edges of the cotton field are likely to be heavily damaged. Stink bugs can be

strong fliers as evidenced by the E. servus that dispersed 87.8 m in this study; Tillman et al.

(2009) detected dispersal by E. servus as far as 120 m from the point of marking. Here, the vast

majority of individuals dispersed only a few meters between crops indicating that when food is

plentiful, long distance dispersal is unusual. Dispersal into cotton from peanut or soybean

suggests that growers need to be aware of adjacent crops that harbor stink bug populations when

selecting cotton fields.

Previous researchers reported that seedcotton yield, gin turnout (lint yield), color Rd, and

overall lint value were all negatively affected by stink bug feeding (Cassidy & Barber 1939,

Toews & Shurley 2009). Our data also suggest that the number of stink bugs captured, boll

damage, seedcotton yield, gin turnout, color Rd and color +b changed as a function of distance

101

from the edge of cotton plot (Fig. 1-5). However, only seedcotton yield and color Rd were

affected by adjacent crop. Of those factors affected by adjacent crop, lower yield and color Rd

were observed on the edge of cotton adjacent to soybean. This result corresponds with the

findings that soybean apparently serves a more suitable host plant for population growth than

peanut (Herbert & Toews 2012, Huang et al 2012). Color grade could be affected by differences

in the local production environment, such as defoliation timing, harvest timing, climate, or soil

type (Daniel et al. 1999, Johnson et al. 2002). However, stink bug feeding is also known to

produce yellow tinged spots on the lint and this would be detected in the measurement of color

+b strongly suggesting that stink bug damage caused the color change.

The strong edge effects suggest that new treatment strategies could be developed. The

aggregation behavior of stink bugs on the margin of cotton plots was significant as has been

previously reported (Tillman et al. 2009, Reay-Jones 2010). First, crop scouts and pest managers

may want to prioritize monitoring on the edges of peanut-cotton and soybean-cotton early in the

season to determine if stink bug populations are present. If there is no damage along the edges, it

is unlikely that damage would be observed further in the field. Second, border-only spraying of

insecticides during early bloom of cotton adjacent to peanut and soybean may protect cotton

more efficiently and economically than spraying the whole field. This strategy would also

preserve natural enemies in the remainder of the field to improve the function of biological

control (Toews et al. 2011). Finally, the risk of secondary pest outbreaks would be decreased

with border-only applications as opposed to whole-field applications. Further research in this

area is needed.

In conclusion, this study demonstrates that at least five species of stink bugs disperse

directly into cotton fields from peanut and soybean planted adjacent to cotton. Stink bugs will

102

disperse a relatively short distance between crops and this dispersal contributes to the “edge

effect” that has negative impact on the cotton fiber quality and production. Therefore, early

scouting and crop arrangement appear to be critical factors for grower cognizance. Stakeholders

should consider the new knowledge of farmscape ecology, timing of infestation, and initial

points of invasion when developing ecologically based management tactics.

103

Acknowledgements

The authors gratefully acknowledge John Herbert, Anne Horak, and Ishakh Pulakkatu

thodi for their assistance sampling stink bugs in the field. We also thank David Griffin and

Wesley Stephens for managing the plots. John All, David Buntin, and John Ruberson provided

helpful reviews of an earlier manuscript draft. This research was funded by the Georgia Cotton

Commission under project number 11-827GA and by the USDA-NIFA-Special Research Grants

Program under award number 2009-34566-20100. Mention of trade names or commercial

products in this publication is solely for the purpose of providing specific information and does

not imply recommendation or endorsement by the University of Georgia.

104

References

Barbour, K.S., Bradley, Jr., J.R., & Bacheler, J.S. (1990) Reduction in yield and quality of

cotton damaged by green stink bug (Hemiptera: Pentatomidae). Journal of Economic

Entomology 83, 842-845.

Boethel, D.J., Russin, J.S. Wier, A.T., Layton, M.B., Mink, J.S. & Boyd, M.L. (2000)

Delayed maturity associated with southern green stink bug (Heteroptera: Pentatomidae)

injury at various soybean phenological Stages. Journal of Economic Entomology 93, 707-

712.

Blinka, E.L. (2008) Biological and ecological studies on green stink bug, Acrosternum hilare,

and brown stink bug, Euschistus servus (Hemiptera: Pentatomidae), in eastern North

Carolina cropping systems. Ph.D. dissertation, North Carolina State University, Raleigh,

NC.

Cassidy, T.P. & Barber, T.C. (1939) Hemipterous insects of cotton in Arizona: their economic

importance and control. Journal of Economic Entomology 32, 99-104.

Dagenhardt, D.C., Green, J.K., Khalilian, A.K. & Reeves, R.B. (2011) Volatile emissions

from developing cotton bolls in response to hemipteran feeding damage. Journal of

Entomological Science 46, 177-190.

Daniel, J.B., Abaye, A.O., Alley, M.M., Adcock, C.W. & Maitland, J.C. (1999) Winter

annual cover crops in a Virginia no-till cotton production system: II. Cover crop and

tillage effects on soil moisture, cotton yield, and cotton quality. The Journal of Cotton

Science 3, 84-91.

Ehler, L.E. (2000) Farmscape ecology of stink bugs in Northern California. Entomological

Society of America, Lanhan, MD.

105

Esquivel, J.F. (2011) Estimating potential stylet penetration of southern green stink bug - a

mathematical modeling approach. Entomologia Experimentalis et Applicata 140, 163-

170.

Greene, J.K. & Herzog, G.A. (1999) Management of stink bugs using symptoms of boll injury

as a monitoring tool, pp. 1041-1047. In Proceedings, Beltwide Cotton Conferences, 3-7

January 1999, Orlando, FL. National Cotton Council of America, Memphis, TN.

Greene, J.K., Turnipseed, S.G., Sullivan, M.J. & Herzog, G.A. (1999) Boll damage by

southern green stink bug (Hemiptera: Pentatomidae) and tarnished plant bug (Hemiptera:

Miridae) caged on transgenic Bacillus thuringiensis cotton. Journal of Economic

Entomology 92, 941-944.

Greene, J.K., Turnipseed, M.J., Sullivan, M.J. & May, O.L. (2001) Treatment thresholds for

stink bugs (Hemiptera: Pentatomidae) in cotton. Journal of Economic Entomology 94,

403-409.

Herbert, J.J. & Toews, M.D. (2011) Seasonal abundance and population structure of brown

stink bug (Hemiptera: Pentatomidae) in farmscapes containing corn, cotton, peanut, and

soybean. Annals of Entomological Society of America 104, 909-918.

Huang, T-I. & Toews, M.D. (2012) Feeding preference and movement of Nezara viridula (L.)

and Euschistus servus (Say) (Hemiptera: Pentatomidae) on Individual Cotton Plants.

Journal of Economic Entomology (Accepted).

Huang, T-I., Srinivasan R. & Toews, M.D. (2012) Intercrop movement of stink bugs

(Hemiptera: Pentatomidae) in Georgia farmscapes. Environmental Entomology

(submitted 2012).

106

Johnson, R.M., Downer, R.G., Bradow, J.M., Bauer, P.J. & Sadler, E.J. (2002) Variability in

cotton fiber yield, fiber quality and soil properties in a Southeastern Coastal Plain field.

Agronomy Journal 94, 1305-1316.

McPherson, J.E. & McPherson, R.M. (2000) Stink bugs of economic importance in America

North of Mexico CRC Press, Boca Raton, FL, USA.

Medrano, E.G., Esquivel, J.F. & Bell, A.A. (2007) Transmission of cotton seed and boll rotting

bacterial by the southern green stink bug (Nezara viridula L.). Journal of Applied

Microbiology 103, 436-444.

Negron, J. F., and T. J. Riley. (1987) Southern green stink bug, Nezara viridula (Heteroptera:

Pentatomidae), feeding in corn. Journal of Economic Entomology 80, 666-669.

Neter, J., Kutner, M.H., Nachtsheim, C.J. & Wasserman, W. (1996) Applied linear

regression models, 3rd ed. Irwin, Chicago, IL.

Olson, D.M., Ruberson, J.R., Zeilinger, A.R. & Andow, D.A. (2011) Colonization preference

of Euschistus servus and Nezara viridula in transgenic cotton varieties, peanut, and

soybean. Entomologia Experimentalis et Applicata 139, 161-169.

Reay-Jones, F.P.F. (2010) Spatial and temporal patterns of stink bugs (Hemiptera:

Pentatomidae) in wheat. Environmental Entomology 39, 944-955.

Reay-Jones, F.P.F., Greene, J.K. Toews, M.D. & Reeves, R.B. (2009). Sampling stink bugs

(Hemiptera: Pentatomidae) for population estimation and pest management in

southeastern cotton production. Journal of Economic Entomology 102, 2360-2370.

Roof, M.E. (1994) Control of bollworm/budworm and other insect before and after boll weevil

eradication in South Carolina, pp. 798-799. In Proceedings of the Beltwide Cotton

107

Conferences, 5-8 January 1994, San Diego, CA. National Cotton Council of America,

Memphis, TN.

SAS Institute (2008) SAS/STAT 9.2 user’s guide. SAS Institute, Cary, NC.

Tillman, P.G. (2008) Observations of stink bugs (Heteroptera: Pentatomidae) ovipositing and

feeding on peanut. Journal of Entomological Science 43, 447-452.

Tillman, P.G., Northfield, T.D., Mizell, R.F. & Riddle, T.C. (2009) Spatiotemporal patterns

and dispersal of stink bugs (Heteroptera: Pentatomidae) in peanut-cotton farmscapes.

Environmental Entomology 38, 1038-1052.

Tillman, P.G. (2010) Composition and abundance of stink bugs (Heteroptera: Pentatomidae) in

corn. Environmental Entomology 39, 1765-1774.

Toews, M.D., Blinka, E.L., Van Duyn, J.W., Herbert, Jr., D.A., Bacheler, J. S., Roberts,

P.M. & Greene, J.K. (2009) Fidelity of external feeding lesions to internal damage for

assessing stink bug damage in cotton. Journal of Economic Entomology 102, 1344-1351.

Toews, M.D., Greene, J.K., Reay-Jones, F.P.F. & Reeves, R.B. (2008) A comparison of

sampling techniques for stink bugs in cotton, pp. 1193-1203. In Proceedings of the

Beltwide Cotton Conference, 8-11 January 2008, Nashville, TN. National Cotton

Council, Memphis, TN.

Toews, M.D., Reay-Jones, F.P.F. & Greene, J.K. (2011) Application of border

sprays to mitigate stink bugs in cotton. In A. Smith & G. Collins (eds.) 2010 Georgia

Cotton Research and Extension Report. UGA/CPES Research Extension Publication 7,

Georgia Cooperative Extension, University of Georgia College of Agricultural and

Environmental Sciences, Athens.

108

Toews, M.D. & Shurley, D.W. (2009) Crop juxtaposition affects cotton fiber quality in Georgia

farmscapes. Journal of Economic Entomology 102, 1515-1522.

Toscano, N.C. & Stern, V.M. (1976) Cotton yield and quality loss caused by various levels of

stink bug infestation. Journal of Economic Entomology 69, 53-56.

Turnipseed, S.G., Sullivan, M.J., Hagerty, A.M. & Gibson, A.L. (2003) Boll damage by the

stinkbug/plant bug complex in South Carolina cotton, pp. 1015. In Proceedings of the

Beltwide Cotton Conferences, 6-10 January 2003, Nashville, TN. National Cotton

Council of America, Memphis, TN.

[USDA-AMS] United Stated Department of Agriculture-Agriculture Marketing Service.

(2001) The classification of cotton. Agricultural Handbook 566. U.S. Dept. Ag.,

Washington, DC.

Wene, G.P. & Sheets, L.W. (1964) Notes on and control of stink bugs affecting cotton in

Arizona. Journal of Economic Entomology 57, 60-62.

Williams, M.R. (2002) Cotton insect losses-2001, pp. 1-48. In Proceedings of the Beltwide

Cotton Conferences, 8-12 January 2002, Atlanta, GA. National Cotton Council of

America, Memphis, TN.

Williams, M.R. (2006) Cotton insect losses-2005, pp. 1151- 1204. In Proceedings of the

Beltwide Cotton Conferences, 3-6 January 2005, San Antonio, TX. National Cotton

Council of America, Memphis, TN.

Williams, M.R. (2007) Cotton insect losses-2006, pp. 974-1026. In Proceedings of the Beltwide

Cotton Conferences, 9-12 January 2006, New Orleans, LA. National Cotton Council of

America, Memphis, TN.

109

Williams, M.R. (2008) Cotton insect losses-2007, pp. 927-979. In Proceedings of the Beltwide

Cotton Conferences, 8-11 January 2007, Nashville, TN. National Cotton Council of

America, Memphis, TN.

Williams, M.R. (2009) Cotton insect losses-2008, pp. 897- 940. In Proceedings of the Beltwide

Cotton Conferences, 5-8 January 2009, San Antonio, TX. National Cotton Council of

America, Memphis, TN.

Willrich, M.M., Leonard, B.R. & Padgett, G.B. (2004) Influence of southern green stink bug,

Nezara viridula L., on late-season yield losses in cotton, Gossypium hirsutum L.

Environmental Entomology 33, 1095-1101.

Xia, J., Mustafic, A., Toews, M.D. & Haidekker, M.A. (2011) Stink bug feeding induces

fluorescence in developing cotton bolls. Journal of Biological Engineering 5, 11,

available online http://www.jbioleng.org.content/5/1/11.

Young, S.Y., J.K. Greene, and G.M. Lorenz. (2008) Damage to soybean by Acrosternum hilare

(Say) (Heteroptera: Pentatomidae). Journal of Entomological Science 43, 257-267.

Zar, J.H. (1999) Biostatistical Analysis, 4th ed. Prentice Hall. Upper Saddle River, NJ.

110

Table 1. Comparison of mean ±SE intercrop dispersal distances of stink bug adults by species

and sex across two years collected from Tifton, GA.

Species Sex # collected (n) Mean ± SE distance (m)

C. hilaris Male 22 14.2 ± 3.7

Female 53 10.1 ± 2.2

E. servus Male 29 16.0 ± 3.7

Female 59 11.5 ± 2.0

E. quadrator Male 16 8.9 ± 3.7

Female 39 7.8 ± 2.1

N. viridula Male 3 17.3 ± 14.6

Female 14 19.4 ± 5.2

Piezodorus guildinii Male 9 13.7 ± 6.3

Female 4 13.7 ± 11.0

No significant difference was observed across species and sex (P > 0.05; LSMEANS test).

111

Table 2. Mean ± SE intercrop dispersal distances of stink bugs traveling between adjacent crops.

Movement direction No. collected (n) Mean ± SE distance (m)

Cotton → Peanut

Cotton → Soybean

Peanut → Cotton

8

33

107

5.5 ± 1.5b

24.2 ± 2.8a

10.3 ± 1.5b

Soybean → Cotton 112 8.5 ± 1.3b

Mean distances followed by the same letter are not significantly different

(P < 0.05; LSMEANS test).

112

Figure legends

Fig. 1. Frequency distribution of distance traveled by captured marked bugs by species. All

distances were measured as the absolute minimum distance from the crop where the

insect was marked to the sampling point where the stink bug was captured. Distance at 0

m indicates individuals that were marked and subsequently captured in the same crop.

Fig. 2. Mean number of stink bugs captured in cotton as a function of adjacent crop and distance

from the edge of the cotton plot. Distance at 0 indicates the first row of cotton on the

edge of the plot.

Fig. 3. Mean percent cotton boll damage as a function of adjacent crop and distance from the

edge of the cotton plot. Distance at 0 indicates the first row of cotton on the edge of the

plot.

Fig. 4. Mean seedcotton yield as a function of adjacent crop and distance from the edge of the

cotton plot. Distance at 0 indicates the first row of cotton on the edge of the plot.

Fig. 5. Mean percent gin turnout as a function of adjacent crop and distance from the edge of the

cotton plot. Distance at 0 indicates the first row of cotton on the edge of the plot.

Fig. 6. Mean color Rd (A) and color +b (B) by crop and distance from edge of the cotton plot.

Distance at 0 indicates the first row of cotton on the edge of the plot.

113

Euschistus servus

Freq

uenc

y

0

20

40

60

Chinavia hilaris

0

20

40

60

Euschistus quadrator

0

20

40

60

Nezara viridula

Travel distance (m)

0.0 2.7 8.2 13.7 19.2 24.7 30.2 35.7 41.1 46.60

20

40

60

Figure 1.

114

Distance from edge of cotton plot (m)

0.0 5.5 11.0 16.5 22.0

Num

ber o

f stin

k bu

gs c

aptu

red

0

5

10

15

20PeanutSoybean

Cubic trend peanut (F1, 8 = 6.78, P = 0.0314)Cubic trend soybean (F1, 8 = 8.71, P = 0.0184)

Figure 2.

115

Distance from edge of cotton plot (m)

0.0 5.5 11.0 16.5 22.0

Boll

dam

age

(%)

0

15

30

45

60

75

PeanutSoybean

Cubic trend peanut (F1, 8 = 7.06, P = 0.0289)Quadratic trend soybean (F1, 8 = 10.52, P = 0.0118)

Figure 3.

116

Distance from edge of cotton plot (m)

0.0 5.5 11.0 16.5 22.0

Seed

cotto

n yi

eld

(kg/

36.6

m)

5

7

9

11

13

PeanutSoybean

Quadratic trend peanut (F1, 8 = 7.90, P = 0.0228)Quadratic trend soybean (F1, 8 = 7.05, P = 0.0290)

Figure 4.

117

Distance from edge of cotton plot (m)

0.0 5.5 11.0 16.5 22.0

Gin

turn

out (

%)

36

38

40

42

44

PeanutSoybean

Quadratic trend peanut (F1, 8 = 5.72, P = 0.0437)Quadratic trend soybean (F1, 8 = 6.58, P = 0.0334)

Figure 5.

118

C

olor

Rd

73

74

75

76

77

PeanutSoybean

Quadratic trend peanut (F1, 8 = 13.07, P = 0.0068)Cubic trend soybean (F1, 8 = 6.17, P 0.0378)

Distance from edge of cotton plot (m)

0.0 5.5 11.0 16.5 22.0

Col

or +

b

84

86

88

90

92

94 Quadratic trend peanut (F1, 7 = 6.06, P < 0.0434)Cubic trend soybean (F1, 7 = 19.96 P < 0.0029)

A

B

Figure 6.

119

CHAPTER 5

CONCLUSIONS

We adopted a novel immunomarking technique to study the local dispersal of stink bugs

in a mixed agricultural landscape of the southeastern Coastal Plain. Stink bug dispersal from

crop to crop has been inferred for many years by repeated sampling and spatiotemporal analyses.

However, this is the first project to utilize mass marking of naturally occurring populations to

provide strong evidence of direct intercrop movement by these phytophagous pests.

In chapter 2, we concentrated on the feeding preference and intraplant movement of stink

bugs. Utilizing time lapse videography to record stink bug feeding behavior and movement, we

showed that N. viridula and E. servus preferred quarter-sized cotton bolls. Euschistus servus was

very specific in its preferred boll size, whereas N. viridula was more variable. Nezara viridula

showed more frequent movement from one boll to another than E. servus and both species were

more active during photophase than scotophase.

In chapter 3, we focused on the intercrop movement of different stink bug species and the

conditions that triggered movement. The protein-based mass mark-and-capture analyses yielded

significant evidence that stink bugs dispersed into cotton fields from peanut and soybean during

the 3rd to 8th week of cotton bloom. Therefore, it is critical for growers to protect their cotton

from stink bugs during this period. In addition, we discovered that different stink bug species

have distinct habits and preferences. For example, C. hilaris generally invaded cotton from

soybean, whereas populations of E. quadrator found in cotton originated in peanut. Dispersal of

N. viridula and E. servus into cotton occurred from both peanut and soybean.

120

121

Significant correlations between stink bug dispersal and cotton fiber quality were

described in chapter 4. We concentrated on the relationship of distance from the edge of the

cotton plot and associated parameters such as stink bug density, boll injury, seedcotton yield, gin

turnout, color Rd, and color +b. All factors were strongly correlated with distance from the edge

of cotton plot showing a pronounced edge effect. This edge effect is primarily attributed to the

short flight distance of stink bugs between peanut-cotton and soybean-cotton interfaces.

To summarize all findings, stink bug species identification is very important since

different species have unique feeding behavior and dispersal tendencies. Sweep net sampling

and examination of internal boll damage on quarter-sized bolls are sufficient to estimate stink

bug density and damage. Growers need to be aware that stink bug damage is more likely to

occur on the edges of fields where stink bugs start the invasion process. Particular attention

should be paid to cotton fields located adjacent to peanut and soybean. Crop arrangement at the

farmscape level could be manipulated to minimize stink bug activity. If necessary, insecticide

treatments could be targeted early in the bloom cycle to prevent stink bugs from moving into

cotton. Knowledge provided from our research provides important insight about timing of

movement. Growers could use this knowledge to precision target stink bug populations in time

and space, which ultimately supports ecologically as well as chemically based pest management

strategies.